consumerism

this entire passage is just great. I'm very passionate about this because i have storiesss on how people have given me backhanded compliments. alsooo aishwarya rai is a goddess, she's in a bunch of bollywood films that i've watched. but this highlighted piece here, i just wanted to say that this is basically like if you're not white, then you're automatically less beautiful. :(

Track all of your experiments in organized, shareable documents rather than silo'd in your head. This will benefit you and your team. You get to brain dump, your team gets to know your learnings.

From the perspective of a student reading an essay, I was caught by this sentence and found it by far the least persuasive of an otherwise very persuasive essay. Hughes acknowledges, with a tone that I can't help but construe as begrudging, the importance of artistic freedom. Then in the very same sentence, he uses the contrasting conjunction "but" to contradict his minimalistic support for artistic freedom and claims that the artist can't "be afraid to do what he must choose." If Hughes wants to allow that an "artist must be free to choose what he does," how can he also argue that an artist "must choose" anything?

I comprehend his greater point, that the "colored artist" who chooses to paint sunsets over "Negro faces" is actually being subconsciously influenced by his internalized racism, and that this is an endemic problem among his people.

However, this sentence is just so blatantly weak from an argumentative standpoint, especially being at the end of a paragraph. Why does Hughes think the "colored artist" in his example "must choose" to paint "Negro faces" over sunsets? I can think of a number of perfectly valid reasons, but Hughes doesn't present any of them, where I feel there is a great opportunity to have done so. It's even one of the shortest paragraphs in the essay: there was plenty of room to develop his point as expertly as he did in the rest of his powerful essay, instead of ending the paragraph with what I perceive as a self-contradicting sentence.

Annotation #2 (a comment or question). As I was doing this reading, I often found it hard to keep the idea of these exhibits being staged rather hard. I kept looking at pictures and thinking, this was a picture that someone took, but then I had to remind myself that these were all staged. Maybe it's the fact that they're in black and white. But I just found it interesting how easy it is to perceiving something a certain way based on presentation. It reminded me how important it is to be mindful of what your looking at, and how much preconceived notions impact our understanding of what we see. It says earlier in the text that these exhibitions were racist and a way of making other cultures familiar. This obsession with familiarity mentioned throughout the text makes for an interesting conversation about how closed minded many colonial powers were during periods of colonization.

In many ways, I wonder about the subtle ways these ethnographic exhibitions functioned in their depictions of Non-European people, as racist and problematic as they were, I have a vain sense of optimism that maybe just maybe someone looked at them and saw someone on display as a person and reconsidered their perspective. It's easy to disregard something read or heard about because it isn't tangible but seeing - seeing is real.

Author Response:

Reviewer #1 (Public Review):

This work presents a new Bayesian method for detecting those patterns of neural responses that are connected to behavioral output and are worth investigating further. The manuscript contains the derivation of the approach and its test on synthetic and real neural data.

The derivation should be improved by providing additional steps. For example, it was not clear how Eq. 5 was derived and why the double derivative with respect to parameters theta_mu and theta-nu are present ( these terms appear to be missing in the definition of log-likelihood).

Thank you for the suggestion. We have added steps in the derivation leading to the Ising equation for the indicator variables, now in Eq. (8). These intermediate steps corresponds to the two main approximations of the BIA method, namely, the saddle point approximation for the posterior (Eq. (5)) and the Taylor expansion in the inverse regularization strength (Eq. (6)). We hope that these changes improved the readability of the derivation.

Parameter M should be more clearly defined as the number of samples. It is briefly mentioned on line 170, but it was difficult to connect this to equation (7) and those following that use M explicitly.

We thank the Reviewer for the suggestion. We have clarified the definition of M just after Eq. 11.

Is it possible to include multiple binary quantifications of behavior, similarly to how words are constructed from neural spike trains? For example, one can envision describing a particular song segment with respect to multiple binary features simultaneously.

We explicitly examine this question in “Dictionaries for exploratory vs. typical behaviors” and the corresponding Figure 6, which repeats our analysis for different binary discretizations of our behavioral data.

Reviewer #2 (Public Review):

Summary:

Hernandez et al propose a new statistical tool for identifying codeword in multivariate binary data (for instance neural activity patterns), with a small number of measurements. It demonstrates the utility of the approach on neural responses to analyzing the statistical structure of songbird responses and how they change in different contexts (during exploration vs typical song production).

Strengths:

• The approach is innovative, in that it takes advantage of clever tools from sparse linear regression, in particular a method termed Bayesian Ising Approximation (BIA), to be able to identify codewords individually, rather than directly estimating a model of their joint statistics, by comparing to a null model that assumes independence across dimensions. This approach has the advantage of resulting in a very flexible model, with very few assumptions about the statistical structure of the data, that is applicable for a range of datasets sizes; the more data is available, more of the structure underlying it can be revealed .
• The strong mathematical foundations provides clear bounds on data regimes in which the approximation is theoretically well justified and reasons to expect that the estimated models are minimal and interpretable.
• The numerical estimation procedures are fast, and computationally efficient (for a reasonably sized neural dataset, can be run on a regular laptop).
• The code is available on github for quick community dissemination.
• Application to identification of behaviorally relevant patterns of co-activity goes beyond previous Ising-based models used in neuroscience.
• When applied to songbird data, it reveals that the variability in neural responses during exploration has much more structure than previously thought.

Weaknesses:

• Although the paper is written as a methods paper, emphasizing the technical contributions and promising wide applicability to a range of different types of datasets, the numerical validation of the method is very much restricted to the statistical regime of the songbird dataset. From the perspective of a potential future user of the tool it's less clear how the method would behave on different datasets, and what needs to happen in practice for adopting the tool to data with different statistics.

We have edited second half of the abstract and a few sentences in the Introduction (see latexdiff file) to make it clear that our main applications to date have been to songbird data.

• The numerical comparison to other existing methods is minimal.

We have argued in our previous submission that there really are no other methods to compare to, designed to work in the regime similar to uBIA. It seemed to us that it would be unfair to run other methods on our datasets, see them not work well (as expected – because they make assumptions that are invalid in our regime), and then claim success. However, since the concern has been raised again, we really have to address it. To do this, we added a section in the Online Methods “Direct application of MaxEnt methods to synthetic and experimental data”, in which we compare uBIA to the relevant interactions model of Ganmor et al., with which uBIA has the highest similarity. The results are as expected – a method not designed for our data regime fails. We emphasize here again that the relative superiority of uBIA on these data should not be taken as a slight directed at other methods, but rather as an indication than, to cover different data regimes, multiple methods should be combined. We emphasized this in the “Overview of prior related methods in the literature” supplemental section.

• The songbird analysis already reveals some challenges with respect to interpretability: in particular it is not clear how much information about the underlying neural processes can be revealed by summary statistics generated by the method, such as the number of codewords and their length distribution.

The reviewer is correct that our analysis of the songbird data raises a number of important questions for future studies. Although these remain to be answered, we emphasize that before the biological interpretation of over/underrepresented neural patterns can be attempted, such patterns must first be identified. uBIA therefore represents a crucial advance in our ability to address these questions.

Most conclusions are reasonably supported by the data. The analysis of the irreducibility of the codewords has insufficient support based on the numerical simulations. Moreover, the generality of the tool and comparison to other methods are discussed in almost entirely theoretical terms, which makes the claim on immediate utility for other datasets less convincing, especially outside the neuroscience community.

We hope that the addition of the new comparison figure partially alleviates these concerns. Additionally, we point out that 3rd and 4th order words are long, as most others deal with just pairs, as illustrated in the new Figure 7. Indeed, it is not easy to fit an N = 20 Ising model with 4th order terms, because there are 20 ∗ 19 ∗ 18 ∗ 17/(4 ∗ 3 ∗ 2 ∗ 1) = 4845 terms in this model, which cannot be fit from just a few hundred samples, which is precisely why the Ganmor model fails in this case (Fig. 7).

Nonetheless, the idea is quite interesting and likely of broad interest for theorists interested in the development of unsupervised statistical tools for neural data analysis, with practical applicability for a range of modern systems neuroscience experiments that involve task specific ensembles as the building block of circuit computation.

I love this, because of course it had cultural relevancy in that it created a deep philosophy of education that gained international attention, but it's also just a good way of looking at the work that children are doing. Children are current citizens of a place and a culture and so the work they do inherently becomes a cultural project anyway that can then become visible.

Who is the author of the source? Are they an expert? I think this post did well at staying relatively neutral and keeping an unbiased deliverance of the facts. I think this "Arguments" section exemplifies that. It shows the opposition to the law and clearly labels the author's by their opinions. If you wanted a quote from an official that is part of the Sensible Voting Rights Policy side, you can use this. It has a verified reference with a link and shows that the quote wasn't distorted or rewritten to fit any type of biased language or persuasive outlook of the main article.

So very few modern sources describe annotation or note taking in these terms.

I find often in my annotations, the most recent one just above is such a one, where I start with a tiny kernel of an idea and then my brain begins warming up and I put down some additional thoughts. These can sometimes build and turn into multiple sentences or paragraphs, other times they sit and need further work. But either way, with some work they may turn into something altogether different than what the original author intended or discussed.

These are the things I want to keep, expand upon, and integrate into larger works or juxtapose with other broader ideas and themes in the things I am writing about.

Sadly, we're just not teaching students or writers these tidbits or habits anymore.

Sönke Ahrens mentions this idea in his book about Smart Notes. When one is asked to write an essay or a paper it is immensely difficult to have a perch on which to begin. But if one has been taking notes about their reading which is of direct interest to them and which can be highly personal, then it is incredibly easy to have a starting block against which to push to begin what can be either a short sprint or a terrific marathon.

This pattern can be seen by many bloggers who surf a bit of the web, read what others have written, and use those ideas and spaces as a place to write or create their own comments.

Certainly this can involve some work, but it's always nicer when the muses visit and the words begin to flow.

I've now written so much here in this annotation that this note here, is another example of this phenomenon.

With some hope, by moving this annotation into my commonplace book (or if you prefer the words notebook, blog, zettelkasten, digital garden, wiki, etc.) I will have it to reflect and expand upon later, but it'll also be a significant piece of text which I might move into a longer essay and edit a bit to make a piece of my own.

With luck, I may be able to remedy some of the modern note taking treatises and restore some of what we've lost from older traditions to reframe them in an more logical light for modern students.

I recall being lucky enough to work around teachers insisting I use note cards and references in my sixth grade classes, but it was never explained to me exactly what this exercise was meant to engender. It was as if they were providing the ingredients for a recipe, but had somehow managed to leave off the narrative about what to do with those ingredients, how things were supposed to be washed, handled, prepared, mixed, chopped, etc. I always felt that I was baking blind with no directions as to temperature or time. Fortunately my memory for reading on shorter time scales was better than my peers and it was only that which saved my dishes from ruin.

I've come to see note taking as beginning expanded conversations with the text on the page and the other texts in my notebooks. Annotations in the the margins slowly build to become something else of my own making.

We might compare this with the more recent movement of social annotation in the digital pedagogy space. This serves a related master, but seems a bit more tangent to it. The goal of social annotation seems to be to help engage students in their texts as a group. Reading for many of these students may be more foreign than it is to me and many other academics who make trade with it. Thus social annotation helps turn that reading into a conversation between peers and their text. By engaging with the text and each other, they get something more out of it than they might have if left to their own devices. The piece I feel is missing here is the modeling of the next several steps to the broader commonplacing tradition. Once a student has begun the path of allowing their ideas to have sex with the ideas they find on the page or with their colleagues, what do they do next? Are they being taught to revisit their notes and ideas? Sift them? Expand upon them. Place them in a storehouse of their best materials where they can later be used to write those longer essays, chapters, or books which may benefit them later?

How might we build these next pieces into these curricula of social annotation to continue building on these ideas and principles?

This my first time reading this, and I can by far, she is my favorite character. As all the characters in this play is going thought poverty, but Audrey is fighting for life as well. She comes up as this sweet character, generous, but deep down she is a complete mess. She mask herself to hide the scarves that she carries and that says a lot. she does not wish for money, nor does she wish for lavishness. All she wishes for his true happiness. To know what it feels to not hide behind that mask of her. And I truly love that about her

I love how Mushnik tries to please the costumer by saying they will give the costumer the FINEST roses but then when it's presented to the costumes it's just a bunch of dead roses. I was surprised that the costumer didn't return the roses and asked for a refund because the costume was paying double the price or they could have easily bought the Audrey two plant that caught their eye. What I'm still questioning is if the costumer found the Audrey two plant interesting and kept constantly asking about it why didn't they buy it?

I know Seymour and Audrey are both singing these lines kind of only because they're the leads and the love interests and they have to, but I also think it speaks to their individual characters specifically. They both declare that they would do anything they could to get out of Skid Row, and as we see, Seymour really means he would do ANYTHING. Even though they both want the same things (a better life somewhere nicer, to be with each other), had it been Audrey who discovered that Audrey II will only eat blood, I don't think she'd have gone nearly as far as Seymour does. Especially since she doesn't really seem like the kind of person who takes action to actually get the things she wants (or even needs). "Somewhere That's Green" is sung like it's a fantasy and an out-of-reach dream, but she herself says that the only thing that's stopping her from being with Seymour is herself, since she doesn't think she deserves him and won't break up with Orin even though she's miserable. I guess my point is that Seymour and Audrey both have similar roots and come from the same place (physically and emotionally), and I think ultimately what sculpts Seymour's character is his desperate need to please (to keep the shop open for Mushnik, to be a "botanical genius", to impress Audrey, etc.) and this drives what he DOES. Whereas Audrey has just as low self-esteem and needs the same validation, and this drives what she DOESN'T do.

I LOVE LITTLE SHOP OF HORRORS! However, as I've never read it on paper before and I'm already seeing that some of the character descriptions are a bit stereotypical and slightly problematic. I guess in a way it is okay in that the musical is supposed to be "tongue in cheek" and satirize many different things as stated in the author's note, but I think it also reinforces somewhat dangerous stereotypes. Then again, I think that is the point? I like that the descriptions are very specific and detailed, a lot of the time, especially in plays, it's just the age, gender, and maybe ethnicity.

Yes, it's not just learning about government. All power players in the political world are skilled in rhetoric--the art of arguing and convincing others of your viewpoint. A project like these letters can encourage students refine that skill. Obviously we have to understand the issues and learn about them in school and out of school, but technology can be a great way for students to practice their rhetorical skills and attempt to persuade one another as they prepare to enter the political landscape.

Explaining things is extremely hard, especially for some people. Therefore, you need to be ready to ask more specific questions

in grade school when teachers only taught "traditionally" the class felt like we weren't learning, it felt like the teacher was doing in to spite technology.

This is a very interesting take yet it's true. When we see a white man shoot up a school we don't assume this is something that christianity supports, yet when a terrorist blows up a building the media will be quick to assume that this was due to islam.

KEY IDEA: We shouldn't ask of culture "is it real or just in people's heads?" That doesn't matter. It's about figuring out how it makes sense to people.

This image really bothered me, to see that the search results for something as simple as "black girls" to turn up so much pornographic material is disgusting. It makes me curious what it is like today, if it's changed, or has just been reconfigured to be more politically correct. Though I am sure the algorithm has been modified, I still believe the Internet continues to support and drive up what serves the male gaze as a priority, mostly because they know that is what will keep them online longer. It's just really disappointing.

There are so many fast, in the moment comments on site like Twitter that I go elsewhere for thoughtful content. It's just too difficult to separate mixed content on the same site.

I'm wondering if there needs to be a platform which does everything.

Author arguments:

• There's a "bookish" quality to physical books that's not present for ebooks, which is why many people prefer the former. It's partly about tradition / nostalgia, partly missing features.
• Ebooks cater to specific users who read many books, and care less about the form. Annotations and bookmarks (which should be the prime feature of ebooks), are somewhat neglected.
• But this doesn't matter too much, because we can just continue reading physical books.

My thoughts:

• I like physical books primarily because they get me away from a screen, and because I can place them in bookshelves. That's in a way "bookishness".
• But I think digital annotation features would really add to the reading experience if done right. I'd be interested in indexing notes more easily, or what passage my friends thought especially good.

Learnings:

• There's some nostalgia about print books that people associate with worthwhile long form reads. Maybe this is a form of inner rebellion against the modern noisy world.
• Current (Kindle) Ebooks are made for popular best-selling genres. They are not made for heavy highlighting, note-taking, or bookmarking. That's an opportunity.

I can directly relate to the sentiment that I think upon first glance that excellence is a core value, but I've highlighted the bulk of this paragraph to remind myself that digging deeper over the course of a few days to find the connections between and roots of my values will hone in on the core few that matter most (and those will naturally bring along the secondary, perhaps more superficial values).... and it's also my first time using Outline, so I'm just type type typing to test the various features.🤓

Minor: I think it's good to discuss the issues you're interested in from your area of expertise with neuroscience. You even state outright that you'll be framing things from that perspective. However, the both the intro and that outright statement suggest the paper is written for a more general audience. That conflicts somewhat with the framing here, which seems to be geared towards neuroscientists familiar with the features of neurodata. Not a major concern though, this is just a question of whether you want to maintain a consistent audience throughout the paper. Arguably reconciled by substituting "We" with "neuroscientists". Same comment applies to other areas where this may pop up

Is there a way to parse out who “owns” such infrastructure now? I would guess mostly research institutions own their own if there’s a cluster and then, most of the time, it’s just the lab who owns some computers?

With behavior, this could easily be encouraged by having data repos, model zoos and implementations for a data layer across existing tools.

In general though, this is a crucial point -- it takes a lot of work to convert to HDF5/NWB. Is the goal just to be able to share it? It doesn't make it more compatible with other tools since there's not enough adoption on that end. It's often even less efficient than a bespoke format -- so who's gaining anything here?

From this simple description I really don't understand the concept of pinning or how that avoids the problem of loss of all peers. If it's just mirroring an entire server, that seems prohibitive, but I'm probably not understanding it. I don't think it's worth a complete description, but this paragraph needs to be clearer and also point to a source for a complete description.

This just shows how everything is very hypocritical in a way because theres certain things that we have to do like go to school, get a job but then its for nothing because it's not a fair system for everyone.

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

Learn more at Review Commons

Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)): **Summary:** The manuscript submitted by Djekidel et al entitled: "CovidExpress: an interactive portal for intuitive investigation on SARS-CoV-2 related transcriptomes" reports on a new web portal to search and analyze RNAseq data related to SARS-CoV-2 infections. The authors downloaded and reprocessed data of more than 40 different studies, which is available on the web portal along with all available meta data. The web portal allows to perform numerous differential expression and gene set enrichment analyses on the data and provides publication ready figures. Because of batch effects that could not be removed, the authors do not recommend to analyze data across studies at this point. The authors conclude that the web portal is unique and will allow scientists to rapidly analyze gene expression signatures related to SARS-CoV-2 infections with the potential to make new discoveries. **Major comments:** Based on the scientific literature, the web portal seems to be an unprecedented resource to search and analyze SARS-CoV-2-related RNAseq data and as such would certainly be a useful resource for the SARS-CoV-2 scientific community. The authors argue that new discoveries are possible by using their web portal in providing use cases. However, the section detailing the analyses the authors did to generate new hypotheses about genes potentially relevant in SARS-CoV-2 infections are very difficult to follow and without more guidance very difficult to reproduce with the web portal. It would require substantial expert knowledge in RNAseq data analysis without more information being provided. It also seems that key candidate genes identified by their analyses have all been studied or identified to be related to SARS-CoV-2 infections, so it is somewhat unclear whether new hypotheses can be generated by the reanalysis of RNAseq datasets, especially because combining the data from different studies is currently not recommended by the authors. The manuscript would benefit from providing fewer use cases but for each of them providing more information on how the portal and which studies were used to generate them and which findings were not described in the publication of the used studies. Some observations in the manuscript are not substantiated with significance calculations (see below). At times, the English writing (grammar) should be improved.

We thank the reviewer for the positive comments. We suppose the reviewer conclude it need substantial expert knowledge in RNAseq data analysis were due to lacking Video Tutorial. We have now put up several Video Tutorials and more tutorials would be added along later along with users’ feedbacks. We believed this would help ease reviewers’ concern.

In response to whether new hypothesis can be generated. Sorry if it’s not clear, for all the case studies and our “CovidExpress Reveals Insights and Potential Discoveries”, our portal has provided information not reported by their original publications, as listed below:

1. Case study #1: The original publication employed a multiomics approach to find the predictor genes between ICU and non-ICU patient. But it’s not obviously to know which genes were mainly due to expression level, which might be due to other data they included (e.g. mass spectrometry data). Our portal allow user to quickly check their expression level and find SESN2 does not have strong expression differences.
2. Case study #2: We replace this case study with bacterial-susceptibility genes to show such questions could be quickly asked and answered using our portal. Such investigation has not been reported before.
3. FURIN’s function have been well related to SARS-CoV-2. However, for all reports we could find, they focused on Furin cleavage sites of SARS-CoV-2 or whether FURIN were expressed in the SARS-CoV-2 sensitive tissues. SARS-CoV-2 infection could up-regulate FURIN expression have never been reported before. The study published the data didn’t mentioned FURIN at all. We have made this discovery simply by using CovidExpress portal to find the differential expressed genes and overlap with the literature-based gene list (Supplementary Table S2), we believe more discoveries could be made by users by selecting different data.
4. If we search OASL AND " SARS-CoV-2" on pubmed, only 5 results shown up indicated it’s under-studied. And none of them indicated OASL could be up-regulated both by SARS-CoV-2 infected lung and Rhinovirus-infected nasal in human. It is not clear to us if we might misunderstand reviewers’ suggestion as “fewer use cases”. Thus, we haven’t removed any use cases, instead we provided more details to help users understand what and how did we made those discoveries not reported by their original studies using CovidExpress.

At last, we have gone through substantial scientific editing to improve the grammar. **Minor comments:** Page 6 last sentence: The statement of this sentence is very much what one would expect. It remains unclear whether the authors mean this as a result to validate the processing of the RNAseq data or as a new discovery. Please, clarify.

We apologize for the confusion. We intended this statement to be a result confirming what we had expected. We have now amended the text to make this point clearer.

Figure 3A: The violin plots are so tiny that it is impossible to see any trends. It is also difficult to understand which categories one should compare with each other. If there is anything significant to observe, please, add a statistical test and better guide the reader.

We agree with the reviewer; therefore, we have removed this figure from the paper. The goal of this figure was to demonstrate how to use violin plots for exploratory analysis; however, in this case, the violin plot did not show a clear trend. By using more filtering and other plots (e.g., Figure 3B-C), we believe we now provide better insight.

Figure 3C: A legend for the color scale is missing. The signal (I guess expression amounts) for SESN2 seems very weak and the same between ICU and non-ICU samples. What is the significance for assigning this gene to the group of genes being upregulated in ICU samples? Also contrary to what the authors state on page 8, SESN2 does not seem to be highly expressed in ICU samples, however, without knowing what the colors represent (fold changes or absolute expression values?) this is somewhat speculative.

We thank the reviewer for bringing this to our attention. We have now added a legend for the color scale in the revised figure. In Figures 3A-C, we are showcasing how an exploratory analysis can be performed using CovidExpress. As an example, we investigated the expression of the top 20 genes identified by the random forest classifier of Overmyer et al., 2021, as predictors of ICU and non-ICU cases. In the original Overmyer et al. paper, only the general performance metrics of the models are presented (Fig. 6c-g), but the authors do not show the expression patterns of the top predictors. Hence, we demonstrate how CovidExpress can be used to further investigate some questions not explored in the original paper. SESN2 was listed as a top predictor; however, its expression did not vary between ICU and non-ICU samples, as was also observed by the reviewer. We suspect SESN2 was a top predictor due to other data the Overmyer et al. paper included, such as mass spectrometry data. Our statement about SESN2 was not accurately reflected in the figure; therefore, we have rewritten this section to make it clearer.

Page 9 first sentence: Please, specify what you mean by "starting list". Furthermore, in this paragraph, how do your results compare to the results from the study that you re-analyze here?

We thank the reviewer for the question. By “starting list,” we meant the top genes from the Overmyer et al., 2021, article as predictors of ICU and non-ICU cases. We have now rewritten this section to make it clearer. We did not expect our results to differ from their data. Our goal was to ask which of their top predictors (by multi-omics data) show a difference in gene expression. When we downloaded their TPM values from their GEO records, the values were very similar overall (see below).

Figure 3F: Please add labels to your axes and is there a particular reason why in a correlation plot like this one, the y and x axis are not shown with the same range and why does the y axis not start at 0?

We thank the reviewer for this helpful comment. Our reasoning for presenting the figure in this way is that different genes can have very different expression levels but still be correlated. For example, if gene A expressed 1, 5, and 10 in samples 1,2, and 3, while gene B expressed 100, 500, and 1000 for samples 1, 2, and 3, then their range would be very different but still perfectly correlated (see panel A below). If we draw the x- and y-axes using the same range, this correlation will not be visually obvious (see panel B below).

This comparison is different from the correlation plots that compare the expression of one gene in different samples. We apologize for the confusion and to avoid misleading readers, we have enlarged the gene names in the Figure labels to ensure that readers notice their differences. We have also added an option to the correlation plot on our portal so that users can choose the optimal format (see below).

Page 9 second last sentence: It remains unclear which kind of analysis the authors intend to do here and what the starting question is. Please, try to rewrite with less technical terms (i.e. what do you mean by "precalculated contrasts"). In line with this, it remains unclear what Figure 3I is supposed to show. Please, provide some more information to readers who are not RNAseq analysis experts.

We thank the reviewer for this suggestion. To avoid any misleading claims, we followed Reviewer #2’s suggestion and replaced the coagulation gene list with a filtered gene list from the “Coronavirus disease - COVID-19” KEGG pathway (hsa05171) to showcase how to identify experiments in which this gene signature is enriched or depleted. We also replaced the related figures and text with new results and rewrote this section to avoid using technical terms.

Figure 3J is somewhat confusing. Why is the mean expression range indicated from 0 to 1 and why are all genes apparently having a mean expression of 1?

We thank the reviewer for this question. Because the levels of expression of different genes can vary greatly, in Figure 3J (new Figure 3A and 3I), we normalized the mean expression levels of the genes to their maximum values across groups to improve the visualization. We have now made this clearer in the figure, legend, and text.

Page 10 line 5-6. Are you referring to coagulation markers here or general expression patterns? In case of the latter, how does this statement fit to the paragraph about analyzing expression patterns of coagulation markers? Please, specify. And in line with this, are the highlighted genes in Figure 3K coagulation markers? If not, what is the relevance of these to make the point that one can use the portal to investigate the role of coagulation markers in SARS-CoV-2 infections?

As mentioned above, to avoid any misleading claims, we followed Reviewer #2’s suggestion and replaced the coagulation gene list with a filtered gene list from the “Coronavirus disease - COVID-19” KEGG pathway (hsa05171). This revision enables us to show how to identify experiments in which this gene signature is enriched or depleted. We have now replaced these figures and text with new results.

The appearance of describing batch effects and attempts to remove them from the studies was somewhat surprising on page 10 as I would expect this kind of results rather earlier in the results section before describing use cases of the data. You may consider changing the order of your results for a better flow.

We apologize for the confusion. However, we want to make it clear that the analysis before page 10 did not involve “batch effect”; all analyses were performed within each study. Thus, it is not necessary to change the order in which the results are presented. Also, based on Reviewer #2’s comments, we did not accurately use the term “batch effect,” because “batch effects are purely due to technical differences.” We have now revised the corresponding text to make this point clearer.

Page 11, second paragraph. Please, explain briefly what the silhouette score is supposed to reflect and thus how Figure S4G should be interpreted. The difference of both bars in Figure S4G is very marginal and thus, does not seem to support the statement of the authors that the ssGSEA scores-based projection is better unless you perform a significance test or I misunderstood. Please, clarify.

We thank the reviewer for this suggestion. We have now added an explanation of the silhouette score in the manuscript. Briefly, a silhouette score is a metric of the degree of separability of gene clusters from the nearest cluster. For a given sample, lets be the mean intra-cluster distance, and be the mean distance to the nearest cluster. The silhouette score (sil) will be calculated as follows

The silhouette score ranges between -1 and 1. A value near 1 means that the clusters are well separated, and a value near -1 means that the clusters are intermingled. Using a Wilcoxon rank test, we showed that using ssGSEA scores significantly improves the separability of global GTEx tissues (in Figure S4G; p=8.75e-26).

Page 11, third paragraph: Figure 4B, to the best of my understanding, does not support the claim that samples clustered less according to study cohorts using the ssGSEA approach. Please, quantify the effect and test for significance or better explain.

We apologize for the confusion. We quantified the separability between cohorts (GSE ids) by using the silhouette score. In Figure S4H (panel A below), we show that the TPM-based PCA leads to more separation by studies than does the Covid contrast ssGSEA scores in which the separation between studies is less prominent (p-value=0.0045, paired Wilcoxon test).

For the analyses described starting on page 12 it remains largely unclear whether they were conducted across studies or within studies and which studies were used. This section until the end of the results would especially benefit from providing more information on how the analyses were performed, either in the results or in the methods section.

We apologize for the confusion. The goal of the analysis on page 12 and the corresponding Figure 4G was to identify genes whose expression increased in both the SARS-CoV-2 infection lung and rhinovirus-infected nasal tissue. Hence, we did a log2(fold-change) vs log2(fold-change) comparison. The log2(fold-change) values were independently calculated for each study. Because we compared values by using the same ranking metric, the cross-samples comparison was possible, as shown in Figure 4G. We have now added more details to the Methods section to clarify this point.

Figures 4J and 4K miss axis labels and since we look at correlations, the figures could be redrawn using the same ranges on x and y axis.

We thank the reviewer for this suggestion. We have now added axes labels to the new figures. However, we have not used the same range on the x and y axes because they depict expression levels of different genes. For example, if gene A is expressed 1, 5, and 10 in samples 1, 2, and 3, while gene B is expressed 100, 500 and 1000 for samples 1, 2, and 3, their range would be very different but still perfectly correlated (panel A below). If we draw x and y axes using the same range, this correlation will not be visually obvious (panel B below).

This comparison is different from the correlation plots that compare the expression of one gene in different samples. We apologize for the confusion and to avoid misleading readers, we have enlarged the gene names in Figure labels to ensure that readers notice they are different genes. We have also added an option to the correlation plot on our portal so that users can choose the optimal format (see below).

Page 14 line 5: Is this the right figure reference here to Figure 4G? If yes, then it is unclear how Figure 4G supports the statement in this sentence. Please, clarify.

We apologize for the confusion. In Figure 4G, we labeled several important genes and used different colors to indicate whether the gene was regulated by SARS-CoV-2 only (purple), Rhinovirus only (black), or both(red). FURIN was the gene that is only significantly upregulated by SARS-CoV-2. The data in Figure 4G were from GSE160435(“SARS-CoV-2 infection of primary human lung epithelium for COVID-19 modeling and drug discovery”); that study used lung organoid alveolar type 2 (AT2) cells as the model. We think this confusion was caused by our failure to provide the details about the GSE160435 study. We have now amended the manuscript to include these details in the Methods section to avoid confusion. We also enlarged the gene labels in the figure to make them more visible. In the manuscript, we have changed from “our results found FURIN gene was also upregulated in SARS-CoV-2–infected lung organoid alveolar type 2 cells (Figure 4G, Supplementary Table S3).” to “We found that FURIN was upregulated in SARS-CoV-2-infected lung organoid alveolar type 2 cells (Figure 4G, Supplementary Table S4) (Mulay, Konda et al., 2021), it has reported that TGF-β signaling could also regulates FURIN (Blanchette, Rivard et al., 2001). Our gene enrichment analysis also found TGF-β signaling enriched only for up-regulated genes in SARS-CoV-2-infected lung cells (FDR correct p=7.58E-05, Supplementary Table S4), these observations implicated a positive feedback mechanism only for SARS-CoV-2-infected lung but not RV-infected nasal cells.”

Figure 2 is of too low resolution. Many details cannot be read. Please, provide a higher resolution figure.

We apologize for the inconvenience. However, we did not expect the reader to read the details on Figure 2, as it is just an overview of the CovidExpress portal. The aim is give the reader an impression about what functions CovidExpress could offer.

Reviewer #1 (Significance (Required)):

Providing a single platform for the analysis of SARS-CoV-2-related RNAseq data is certainly of high value to the scientific community. However, as the portal and manuscript are currently presented, for scientists that are not RNAseq analysis specialists, more guidance would be required to understand and use correctly the functionalities of the portal. Unfortunately, because batch effects could not be removed from the studies, the authors, correctly, do not recommend to combine data from different studies for analyses, however, this likely will also limit the potential of the resource to make new discoveries beyond what the original studies have already published. As indicated above, the authors could support their claim by comparing their findings with findings published from the studies they reanalyzed. The portal is only of use to scientists studying SARS-CoV-2. I am not an expert in RNAseq data analysis and thus cannot comment on the technicalities, especially the processing of the RNAseq datasets. We thank the reviewer for the positive comments. We apologize for the confusion and acknowledge that we should not describe our effort using the term “batch effect.” As described by Reviewer #2 (and we agree), batch effect should be used only to indicate a purely technical difference in the same biological system; for example, differences in experiments performed on different days or by different lab personnel. Thus, we cannot correct for “batch effect” by using CovidExpress. We hope that the reviewer realizes that what we did was correct for the effect caused by differences in software and parameters across the studies. For example, in our approach, the DEGs from GSE155518 and GSE160435 (both primary lung alveolar AT2 cells (both from Mulay et al., Cell Report, 2021) were significantly correlated (panel A below; p = 1.36e-24, F-test). However, when we downloaded the TPM values from their GEO records, GSE155518 appeared to have a genome-wide decrease in the expression of SARS-CoV-2–infected samples (panel B below). We suspect that this is because in their data processing, the expression of virus themselves were also considered. Thus, using the proceed data directly without careful reviewing the method might lead to false hypothesis.

At last, researchers can make new discoveries, such as our OASL and FURIN findings, by using many other features that CovidExpress provides.

Reviewer #2 (Evidence, reproducibility and clarity (Required)): Djekidel and colleagues describe a web portal to explore several SARS-CoV-2 related datasets. The authors applied a uniform reprocessing pipeline to the diverse RNA-seq datasets and integrated them into a cellxgene-based interface. The major strengths of the manuscript are the scale of the compiled data, with over one thousand samples included, and the data portal itself, which has useful visualization and analysis functions, including GSEA and DEG analysis. My primary concerns with the study are centered on the analysis examples that are presented and their interpretation, as well as the user interface for the data portal. **Major Comments:**

1. The literature analysis feels out of place and is not informative (Fig 1E), as the conclusions that can be drawn from literature mining are minimal. In evidence of this, the authors highlight that CRP is a top-studied "gene" and later voice their interest in how CRP is not a differentially expressed gene (pg6). This illustrates the problems with the literature-based analysis, since in the context of COVID-19, CRP is a common blood laboratory measurement that is used as a general marker of inflammation. Transcription of CRP is essentially exclusively in hepatocytes as an acute phase reactant (see GTEx portal for helpful reference), and would therefore not be expected to be found in the various datasets collected by the authors. The one exception might be liver RNA-seq samples from COVID-19 patients, but I do not think these are available in the current collection. I would therefore suggest to remove the literature analysis parts from the manuscript.

We thank the reviewer for sharing knowledge about CRP. As discussed in our manuscript, we agree that not all top genes from literature-based analysis were expected to be included in RNA-seq analysis. We apologize for the confusion, and we have amended our description to make this point clearer. However, we still believe that literature-based analyses are very useful in the following aspects:

1. This type of analysis bridges the gap between data-driven research and hypothesis-driven research. For example, we found many genes in our meta-analysis, but it is not feasible to describe the functions of all of them. Thus, in Figure 1F, we color-coded genes in red if they also appeared as top genes in the literature-based analysis and read related manuscripts to build confidence that the meta-analysis is useful. Then we expanded our review to more top genes and found more interesting evidence (Supplementary Table S2, “TopGenesbyDifferentialAnalysis” tab).
2. Literature-based analyses also reduce the time researchers spend prioritizing their investigations. For example, in our comparison of SARS-CoV-2–infected lung and Rhinovirus-infected nasal tissue, we found >2000 genes upregulated only in SARS-CoV-2–infected lung but not in Rhinovirus-infected nasal cells. It is not easy to derive a hypothesis from so many genes. When we overlapped the gene list with literature-based analysis, FURIN popped up as the most well-studied gene, and we did not find any report that mentioned that SARS-CoV-2 can regulate FURIN This raised our interest and led to a suggested mechanism in which SARS-CoV-2 could evolve to induce FURIN expression and gain superior infectivity. FURIN’s upregulation is significant but not among the top genes, in terms of fold change (>2-fold change, FDR p th by fold change). Thus, without the literature-based analysis, this observation could have easily been neglected.
3. Such analyses help researchers to prime their hypotheses for novel findings. For example, in our comparison between SARS-CoV-2–infected lung and Rhinovirus-infected nasal tissues (Figure 4G, Supplementary Figure 5D and E), we found many upregulated genes, but OASL was not in our literature-based analysis, which indicated that it is under-studied and worth highlighting. We hope the reviewer will agree that we should retain the literature-based analysis in our paper. These analyses were not meant to be conclusive but rather a way to prioritize investigations. Finally, we removed CRP from Fig 1E and the main text to avoid confusion.

1. The data portal, implemented through cellxgene, is accessible for non-programmers to use. However, it is very easy to end up with an "Unexpected HTTP response 400, BAD REQUEST" error, with essentially no description of the cause of the error or how to rectify it. When this occurs (and in my experience it occurs very frequently), this also forces the user to refresh the page entirely, losing any progress they may have made. I see that the authors describe this error in their FAQ page, but their answer is not very intuitive and I was unsure of what they meant: "This happens because the samples you selected doesn't contain all "Group by" you want compare for each "Split by" group. You could confirm using the "Diff. groups" buttons.".

We apologize for the confusion. This excellent point made by the reviewer required an improvement in the software engineering, which we have now completed. We have figured out how to avoid this error and have run thorough tests to ensure that it does not appear anymore. We also added a gitter chat channel to our landing page, so that users can report if they encounter this or other errors.

I would therefore ask that the authors provide more detailed tutorials (ideally step-by-step) on common analyses that users will want to perform, hopefully minimizing the amount of frustration that users will encounter.

We thank the reviewer for this suggestion. We have uploaded several video tutorials to our landing page and will gradually add more. We also added a gitter chat channel, so users can ask questions, report bugs, or suggest new studies to include in the portal.

1. Selection of samples is not very quick or intuitive. If I wanted to select only the samples from one specific GEO accession, I had to resort to individually checking the boxes of the sample IDs that I wanted. If I instead selected the GEO accession under the samples source ID, then used the "Subset to currently selected samples" button, I invariable got the HTTP error 400 message. Of course, this may simply reflect my lack of familiarity with cellxgene; I would nevertheless encourage the authors to improve the FAQ to include a step-by-step example for how to do common analyses/procedures.

We apologize for the confusion. To select an individual GEO accession, users can simply tick the box beside “Samples Source ID.”

Then all boxes would be clear for “Samples Source ID” that allow you to select only the one you want. We also have uploaded video tutorials to help users learn how to navigate the portal.

We apologize for the “HTTP error 400” messages. We figured out that users would encounter that message frequently after they encounter it once due to a back-end cache mechanism. We have now improved the portal from the software-engineering side. In our recent tests of the latest version, this error does not appear anymore. We also added a gitter chat channel on our landing page so that users can report encountering this or other errors.

1. The second case study, centered on coagulation genes, is misguided. Alteration of coagulation lab values in severe COVID-19 patients is reflecting the general inflammatory state of these patients, and would not be expected to manifest on the transcriptional level in infected cells/tissues. Coagulation labs are measuring the functional status of the coagulation cascade, which is far-removed from the direct transcription of the corresponding genes - proteolytic processing of clotting factors, etc. As with CRP (see above comment), most clotting factors are transcribed almost exclusively in the liver (check GTEx portal); I would not expect upregulation of coagulation factors in lung cell lines/organoids/cultures etc after infection with SARS-CoV-2. I would recommend the authors to pick a different gene ontology set for a case study, as the current one focusing on coagulation is confusing in a pathophysiologic sense.

We thank the reviewer for this suggestion. To avoid any misleading claims, we have replaced the coagulation gene list with a filtered gene list from the “Coronavirus disease - COVID-19” KEGG pathway (hsa05171) to showcase how to identify experiments in which this gene signature is enriched or depleted. We also replaced Figures 3G-J with new results.

1. The two large clusters of blood-derived samples vs other tissues is not surprising and the authors' interpretation is confusing. The authors write that "the COVID-19 signature was not able to overcome the tissue specificity and that immune cells might respond to SARS-CoV-2 differently." This should be immediately obvious given the pathophysiology of COVID-19 infection; the cell types that are directly infected by SARS-CoV-2 will of course have a distinct response compared to the circulating blood cells of COVID-19 patients, which are responding by mounting an immune response. There is no reason to expect a priori that the DEGs in the directly infected lung cells would be similar to that of immune cells that are mounting a response against the virus.

We thank the reviewer for these comments. We agree that it should be obvious that directly infected lung cells would differ from immune cells. However, this has never been shown in a large dataset. Also, it is not obviously whether all other different tissues would respond to SARS-CoV-2 differently. Thus, we believe it is important to present this overview. We have amended the description to deliver clearer message as “This confirmed immune cells respond to SARS-CoV-2 differently from other tissues also suggested the response of most other tissues might sharing similar features.”.

1. The authors devote considerable space in the manuscript to exploring "batch effects" and trying to minimize them (pg10-11 Fig 4A-D, Fig S4). However, given that the compiled datasets are from entirely different experimental and biological systems (e.g. in vitro infection vs patient infection, different cell lines, timepoints after virus exposure, diverse tissues, varying disease severity), it is inappropriate to simply refer to all of these differences as "batch effects" alone. Usually, the term "batch effect" would refer to the same biological experiment/system (i.e. A549 cells infected with CoV vs control), but performed on different days or by different lab personnel - in other words, batch effects are purely due to technical differences. This term clearly does not apply when comparing samples from entirely different cell lines, or tissues, etc, and the authors should not keep describing these differences as batch effects that should be "corrected" out.

We thank the reviewer for the insight. We apologize for the confusion caused by using the phrase “batch effect correction” to describe our approach. We agree that the difference between studies should not be referred to as a “batch effect correction” and have now amended the descriptions to avoid confusion.

Indeed, the authors themselves state that the main point of their "batch effect correction" efforts is only for PCA visualization. I therefore feel this section contributes very little to the overall manuscript, especially given the authors' own recommendation that all analyses should be performed on individual datasets (which I certainly agree with). I assume that the authors were required to provide some sort of dimensional reduction projection for the cellxgene browser, but this is more a quirk in their choice of platform for the web portal. Thus, this section of the manuscript should be deemphasized.

We thank the reviewer for these comments and again apologize for the confusion caused by our use of the term “batch effect correction” to describe our approach. However, we believe these parts of the paper should be retained for the following reasons:

• In practice, sample mislabeling can happen. PCA or simple clustering approaches are very useful for helping raise researchers’ attention, so they could further check the possibility of sample mislabeling.
• Even within a study, one sample can be an outlier due to low or unequal sample quality. Removing outliers would help boost the significance of real findings. Without our approach, it would be harder for users to notice and remove outliers from their investigations.
• Finally, these efforts are useful for generating hypotheses. For example, although we collected a lot of data, it is not feasible for us to read all the details in all the manuscripts published. We observed a similarity between SARS-CoV-2–infected lung samples and Rhinovirus–infected nasal samples by exploring our portal’s capabilities (Figure 3E-F). Then we read the manuscripts in which those data were published and found that our discovery was consistent with the original studies’ results. We believe these efforts are essential to help researchers generate or refine their hypotheses. As we update the database with more samples, this approach will become increasingly powerful.

  1. Given the limitations of any combined multi-dataset analyses, one very useful feature would be to conduct "meta-analyses" across multiple datasets. For instance, it would be informative to find which genes are commonly DEGs in user-selected comparisons, calculated separately for each dataset and then cross-referenced across the relevant/user-selected datasets.

We thank the reviewer for this comment. Indeed, we agree that “meta-analyses” are useful and have now compiled Supplementary Table S2 and Figure 1F to demonstrate the commonly regulated genes. To enable user-selected comparisons across studies on our portal, we need to design a thoughtful user interface. Otherwise, the results from our portal could easily cause fatal misinterpretation. For example, GSE154613 includes samples like DMSO, Drug, SARS-CoV-2, and DMSO+SARS-CoV-2. If a user simply selected to compare SARS-CoV-2 versus Control, the results would be SARS-CoV-2 and DMSO+SARS-CoV-2 versus DMSO and Drug. Such functions need time to design and implement; therefore, we will consider this suggestion for further development of our portal.

**Minor comments:**

1. Fig S1G, color legend should be added (I understand that these colors are the same from S1H).

We thank the reviewer for the comment. We have now added information about the colors in the figure legend.

1. Mouseover text for trackPlot on the data portal is incorrect (it says the heatmap text instead).

We thank the reviewer for this comment. We have now corrected this bug.

1. Abstract should be revised to describe only the 1093 final remaining RNA-seq samples after filtering/QC steps.

We thank the reviewer for this comment. We have now amended the Abstract to include this information.

1. Text in many figures is too small to be legible. I would suggest pt 6 font minimum for all figure text, including the various statistics in the figure panels.

We thank the reviewer for this comment. We have now amended the font sizes and will provide high-resolution figures in revision.

1. Are the DE analyses in Fig 1F specifically limited to control vs SARS-CoV-2/COVID-19 comparisons? Many of the samples included in this study are from other respiratory infections (labeled "other" in Fig 1B).

We thank the reviewer for the question. Figure 1F was not originally limited to control vs SARS-CoV-2/COVID-19 comparisons, because we thought control vs virus, drug vs mock, or difference between time points would also be interesting. If we narrow the analysis to contrasts only between control vs SARS-CoV-2/COVID-19, Figure 1F would be still look similar (as below) because the genes in that comparison comprise the largest share of genes included in the original graphic.

In the end, we replaced Figure 1F to avoid confusion and added more details in the Methods.

1. The word cloud format is not conducive for understanding or interpretation. It would be much more informative to simply have a barplot or similar to clearly indicate the relative "abnudance" of a given gene among all 315 DE analyses.

We thank the reviewer for this comment but respectfully disagree with this point. Visualization of the relative “abundance” of genes with word clouds is a relatively novel concept in computational biology. However, we believe, that in this case, it has certain advantages over visualization using traditional bar plots for example. The word cloud format allows us to highlight genes relative to their importance, with the word “importance” being used here in the sense of combined metrics from DEGs, as shown in Figure 1F, or the frequency with which genes are mentioned/discussed in various literature sources, as shown in Figure 1E. For this purpose, the exact values will most likely not be important for most users/readers. Be presenting a word cloud visualization, readers can easily discern the top genes and use them in the exploration of their own data or the CovidExpress portal. However, if users want to analyze raw values, we provide in Supplementary Table S3 a full list of all genes and gene sets that can be download from our landing page (section “CovidExpress Expression Data Download”) in GMT format. Also, when we visualized the ranks of genes by using bar plots as the reviewer suggested, the results were much harder to read (as shown in the bar graph below) than simply looking at the raw data in supplementary tables.

1. Claims of increased/decreased dataset separability should have statistical analysis on the silhouette score boxplots (Fig S4G-I).

We thank the reviewer for the reminder. We have added statistical tests to referred silhouette score boxplots (Wilcoxon rank test)

1. Regarding Fig 4E-F - what are the key genes that contribute to PC1, and how do they relate to the DEGs in Fig 4G?

We thank the reviewer for this question and apologize for the confusion. In Figure 4E-F, the PCA were based on ssGSEA score, as each gene set would have a score for a sample, not individual genes. Thus, the top contributed to PC1 were gene sets upregulated or down-regulated in certain contrasts. We provided on the portal’s landing page detailed results for top gene sets (for the ssGSEA approach) and genes (for the TPM approach) that contributed to various PCs (“Clustering Results for Reviewing and Download” section). This allows users to download and further explore these data.

1. Statistics describing the relation between OASL And TNF/PPARGC1A should be included to justify the author's statements. This could be correlation, mutual information, regression, etc.

We thank the reviewer for this suggestion, and we have updated Figures 4J-K to show the correlation values and corresponding F-statistics. The Pearson correlation between OASL and TNF was significant (Pearson Correlation=0.75 and p-value = 6.85e-72), but the correlation between OASL and PPARGC1A had a negative slope and showed a moderately significant p-value (Pearson Correlation=-0.08 and p-value=0.12), confirming to a certain degree our statement. We have now updated the corresponding text in the manuscript.

1. There are several studies now that have performed scRNA-seq on the lung resident and peripheral immune cells of COVID-19 patients. To more definitively tie in their analyses in Fig 4J-K/Fig S5D-E (to affirm "its important role in the innate immune response in lungs"), the authors should assess whether OASL is upregulated in the lung macrophages of COVID-19 patients vs controls.

We thank the reviewer for this suggestion. Indeed, Liao, et al. recently reported “BALFs of patients with severe/critical COVID-19 infection contained higher proportions of macrophages and neutrophils and lower proportions of mDCs, pDCs, and T cells than those with moderate infection.” (Nature Medicine, 2020, https://doi.org/10.1038/s41591-020-0901-9). They further refined macrophage data into subclusters and reported top enriched GO terms as “response to virus” (group 1), “type I interferon signaling pathway” (group 2), “neutrophile degranulation” (group 3), and “cytoplasmic translational initiation” (group 4). When we investigated their data, we found that group1 and group2 both identified OASL as a marker gene, indicated OASL might response to virus and help type I interferon signaling. Furthermore, another data set (from Ren et al., Cell, 2021, https://dx.doi.org/10.1016%2Fj.cell.2021.01.053) showed several clusters in patients with severe COVID-19 (left panel below) that were enriched for OASL expression(right panel below).

We have now added these observations to strengthen our hypothesis about the role of OASL.

1. The visualization and analysis functions in the data portal appear to work reasonably well out of the box. However, the download buttons for plots did not work in my hands. I realized that a workaround is to right click -> "Save image as" (which then downloads a .svg file), but this is not ideal and should be fixed to improve usability. I had tested the data portal on both Firefox and Edge browsers, using a Windows 10 PC.

We agree with the reviewer. Due to some technical issues with the figure javascript plugin, the download feature does not work unless the figure is saved as a file on the server side. To avoid any security issues, we tried to minimize new file generations, hence, for the moment we have disabled this feature. Users can still download high-resolution .svg figures by using the right-click -> “save image as.” This information is now included in the FAQ section on the portal’s landing page.

Reviewer #2 (Significance (Required)): The data portal appears to have useful analysis and visualization features, and the data collection appears to be quite comprehensive. I would strongly encourage the authors to continue collecting datasets as they become available and further improving the usability of the portal. As noted in the above comments, I think there is potential for their cellxgene-based browser to be useful to non-computational biologists, but at present, the data portal is not as simple to use as it should be. With further efforts to developing step-by-step tutorials for common analysis/visualization tasks, more informative case studies, and the other revisions suggested above, this study could be a valuable resource for the community. Of note, this review is written from the perspective of a primary wet-lab biologist with extensive bioinformatics experience but limited web development expertise.

We thank the reviewer for the positive comments. We understand the importance of data updating. Our plan is to complete quarterly updates once this manuscript has been accepted or when 10 new studies have been either collected by us or suggested by users. This information is also now included in the FAQs of the portal’s landing page. We have also uploaded several tutorials videos to the landing page and will gradually add more. We also added a gitter chat channel, so users can ask questions, report bugs, or suggest new studies to add to the database.

**Referee Cross-commenting** I agree with the comments of the other reviewers. Reviewer #3 (Evidence, reproducibility and clarity (Required)): **Summary:** The ongoing COVID-19 pandemic is a big threat to human health. The researchers have conducted studies to explore the gene expression regulations of human cells responding to COVID-19 infection. A website that integrating those datasets and providing user-friendly tools for gene expression analysis is a valuable resource for the COVID-19 study community. The authors collected published RNASeq datasets and developed a database and an interactive portal for users to investigate the gene expression of SARS-CoV-2 related samples. This website would be of great value for the SARS-CoV-2 research community if the batch normalization problems are solved. **Major comments:** 1) The major concern of CovidExpress is the batch effects from different studies. As the authors have shown and mentioned in their discussion that "For the current release, we strongly suggest investigators to perform gene expression comparison within individual study." This limits the usage of CovidExpress as integrating analysis from multiple datasets of different studies is the key value and purpose of CovidExpress.

We thank the reviewer for the comment. Reviewer #2 reminded us, and we agree, that differences between studies should not be considered “batch effects.” We apologize for the confusion. The GSEA function provided in the portal does not suffer from batch effect, because all the pre-ranked lists of genes are based on contrasts from the same studies. Although we cannot correct for the differences between studies, we did correct for effect caused by differences in software and parameters used. For example, in our approach, the DEGs from GSE155518 and GSE160435 (both studies of primary lung alveolar AT2 cells from Mulay et al., Cell Report, 2021) were significantly correlated (below panel A, p-value = 1.36e-24, F-test). However, if we simply download the TPM values from their GEO records, GSE155518 appears to show a genome-wide decrease in expression in SARS-CoV-2–infected samples (below panel B). These errors might lead to false hypotheses.

2) The authors should include experimental protocols as one key parameter in the description and further integrating analysis of different datasets. As the authors showed that QuantSeq is a 3' sequencing protocol of RNA sequencing. However, it is not convincing to me that simply excluding QuantSeq samples is the ideal solution for downstream integrating analysis as QuantSeq has been shown that it has pretty good correlations with normal RNASeq methods in gene quantifications. It is interesting that there are 21.2% of samples were biased toward intronic reads. What protocol differences or experimental variations would explain the biases?

We thank the reviewer for the comment and apologized for not being clearer. One of our main goals re-processing all samples is to correct for pipeline processing–related batch effects. We tried to reduce those effects introduced by using different software or parameters. QuantSeq or similar protocols are heavily bias to 3’ UTR; thus, the software and parameters used for RNA-seq data will not be suitable. In contrast, we agree that the downstream results from QuantSeq have good correlation to RNA-seq (we observed a correlation of ~0.75, when compared to the log2 fold-change from Quant-Seq to RNA-seq). However, we could not reconcile QuantSeq always correlated well with RNA-seq, in terms of individual quantification. For example, Jarvis et al. recently reported only ~0.35 correlation between QuantSeq and RNA-seq (https://doi.org/10.3389/fgene.2020.562445). Theoretically, the correlation would be weaker for genes with a small 3’ UTR. Thus, we will not include QuantSeq data in this portal. However, if we collect enough studies in the future, we will consider uploading a separate portal just for QuantSeq using a pipeline optimized for protocol bias to 3’ UTR.

For the 21.2% samples that were biased towards intronic reads, we believe they reflect differences in the kits used. For example, of the 162 samples “BASE_INTRON (%)” >30% (Supplementary Table S1) that passed QC, 76 samples were total RNA obtained using the SMARTer kit and 36 were total RNA obtained using the Trio kit. Given that we have 105 samples of total RNA derived using the SMARTer kit and 38 samples of total RNA derived using the Trio kit, we conclude that the Trio kit was more biased toward introns, and the SMARTer kit was also strongly biased. This finding is consistent with those of others who have reported the bias of the SMARTer kit (Song et al., https://doi.org/10.1186/s12864-018-5066-2). Users can find these results in our Supplementary Table S1. We have also uploaded the protocol information to our portal.

3) How do the authors plan to update and maintain CovidExpress?

We thank the reviewer for this question. We understand the importance of data updating. Our plan is to update the database quarterly once this manuscript has been accepted or when 10 new studies have been collected by us or suggested by users. We have added this information to the FAQs on the portal’s landing page. We also understand the importance of maintaining the service for a feasible amount of time for research. Therefore, we will keep the server activated for at least 2 years after the WHO announces that COVID-19 is no longer a global pandemic. We will also ensure that, even after we take down the server , scientists with programming skills will be able to create local servers based on the data provided on CovidExpress.

**Minor comments:** 1) Some texts in figures are not readable. For example, Fig2B, 2C, 2D, 2E.

We thank the reviewer for this comment. We have now increased the font sizes and provided high-resolution figures in revision.

2) The authors could use Videos to demonstrate how to use CovidExpress on the website as they have shown in Fig3.

We thank the reviewer for this suggestion. We have uploaded several video tutorials to the landing page and will gradually add more. We also added a gitter chat channel so that users can ask questions, report bugs, or suggest new studies to include in the database.

Reviewer #3 (Significance (Required)): The ongoing COVID-19 pandemic is a big threat to human health. Many molecular and cellular questions related to COVID-19 pathophysiology remain unclear and many researchers have conducted studies to explore the gene expression regulations of human cells responding to COVID-19 infection. However, there is no database/website that integrating all RNASeq data to provide user-friendly tools for gene expression analysis for COVID-19 researchers. The authors collected the published RNASeq datasets and developed a database and an interactive portal, named CovidExpress, to allow users to investigate the gene expressions response to COVID-19 infection. CovidExpress is a valuable resource for the COVID-19 study community once the batch normalization problems are solved. The users who came up with ideas about the regulation of COVID-19 response could use the system to test their hypothesis, without experience in bioinformatics and RNASeq data analysis. This will be more important when more RNASeq data from samples with different tissues, cell lines, and conditions are integrated into the database.

We thank the reviewer for the positive comments. We apologize for the confusion and acknowledge that we should not describe our effort using the term “batch effect.” As described by Reviewer #2 (and we agree), batch effect should be used only to indicate a purely technical difference in the same biological system; for example, differences in experiments performed on different days or by different lab personnel. Thus, we cannot correct for “batch effect” by using CovidExpress. We hope that the reviewer realizes that what we did was correct for the effect caused by differences in software and parameters across the studies. For example, in our approach, the DEGs from GSE155518 and GSE160435 (both primary lung alveolar AT2 cells (both from Mulay et al., Cell Report, 2021) were significantly correlated (panel A below; p = 1.36e-24, F-test). However, when we downloaded the TPM values from their GEO records, GSE155518 appeared to have a genome-wide decrease in the expression of SARS-CoV-2–infected samples (panel B below).

Thus, using the proceed data directly without careful reviewing the method might lead to false hypothesis. At last, researchers can make new discoveries, such as our OASL and FURIN findings, by using many other features that CovidExpress provides.

This is the kind of thing I tell myself while I practice.

I just which I had logged the hours I’ve already put towards my 10 000, there is no way of knowing. What I can know if I assume it’s 0, the longest it will take me the get to mastery is 10 years, if I do my 3 hours daily. And faster if I spend more time. 3h a day is not much dude, I'm doing way more than than in making permanent notes and I guess it will be a great idea to keep a tab on my progress, how many hours are left in reaching that 10000 hour mark, Any idea Wasiq?

1. Assess how deep are they in. 1.1. if they're a true believer and they're really into this stuff the best we can do is nudge them here and there and not expect much at all. 1.2. If the person is moderately interested in conspiracy theories this is where we can have the most positive influence. You can push a bit, you can joke a bit, but you should still use a light touch. Pressure doesn't work. Nobody likes getting lectures. Don't call people out, don't ridicule them.
2. Just ask questions. When a claim someone makes seems far-fetched just ask questions about how it works. You don't have to know if the theory is real or not. Maybe it's a real conspiracy. Who knows? Just ask questions. This can help people think something through out loud and maybe arrive at a conclusion on their own.
3. Don't repeat conspiracy theories. Be careful about repeating conspiracy theories because we know that people use familiarity as a shortcut for truth. When we hear something a lot we tend to believe it's true or somewhat true. So very simple if you mention conspiracy theories in the wild frame them as being untrue and avoid repeating their actual claims.
4. We all have cringy beliefs. We all hold some weak ass theories about politics, or health, or relationships, or whatever. And this awareness can help us approach these interactions with a spirit of sympathy.
5. We need to endure. (All predictions about the future of society should be taken with much more than a grain of salt but) my guess is this conspiracy theory thing isn't going away anytime soon and it might get worse. It's better to be prepared to endure for the long run than expecting to vanquish this foe shortly and then getting cynical and pessimistic when things get worse. Ultimately this stuff will destroy itself because it doesn't work. It fails because it's indifferent to reality and at some point reality will get its way. But this can take a very long time and right now we need to slow conspiracy theories spread as best we can.

This distinction is interesting. I guess what they're saying is that it's not just about what sparks happiness in you but also about what you actually need. Tons of records might spark joy in you, but are those things essential? I think that also ties back to the article about consider the environment when you purchase or get rid of something. Perhaps also we living in a culture that is more about sparking joy than about focusing on the essential? Is that why I end up with a dog Halloween costume from Target? It certainly makes me happy and makes me laugh, but do we really need it? And what's the effect on the environment with fast fashion?

So if we go with the interpretation of Woyzeck being the horse, this line asks a question, what is the cause of Woyzeck's metamorphosis? If an animal is just a human without the ability or expression of the ability of reason, and Woyzeck finds himself in a that kind of state by the "end" of the play, what was the catalyst for this transformation?

It was being the horse. Woyzeck, for all his lack of propriety and crassness and paranoia is still a person who is worthy of care and respect. But characters like the Captain and the Doctor, and the conditions of living in poverty, having to give up your dignity and happiness for the sake of survival striped Woyzeck of his personhood, which in turn cost him his family. He was not a human, he was a utility. And that is what drove him mad.

This entire section fascinates me. I definetly understand and can see everyone else's interpretations of the horse's point, what Büchner is trying to say through the Showman's missive about the horse and it's humanity, but I think that like the monkey, the horse is Woyzeck. The monkey represents Woyzeck in his subservience to those in power, a dance monkey that has to do whatever they ask of him because his livelihood depends upon them and their continued satisfaction with him. But I think the horse does as well due to a horse means.

Horses are working animals, and in 1837 they were the backbone of European society. On land, horses are what kept the world moving before the invention of trains and automobiles, they were necessary for transportation, for farming, for trade, and for war all the way up until practically the First World War. So horses on the whole would have been indespensible, but individually? Well, it's just a horse. If you need another you can find another. Expendable in the same way that a poor, untitled solider is expendable. It might be sad for the Captain, if they ever went into battle and Woyzeck didn't come back alive, but there will be other low-rung soldiers willing to do his laundry or chop his wood and shave his face. The same goes for the Doctor, neither care for Woyzeck as a person, only what utility he serves to them. Woyzeck is called almost animalistic by the Doctor many times, and by the end you could say that he's right considering his succumbing to the basest of instincts; jealous, violence, paranoia.

An overview of how Ton Zijlstra uses Obsidian as part of his workflow

Use of tools has ‘gone back’ to what he used in the 90s, which is to say, he’s moved away from things like Evernote (silo'd) towards using mostly text with Obsidian (note-taking) and Tinderbox (on the Mac, for writing).

Didn’t try to bring all of his Evernote notes into Obsidian, because it was a massive dump of content - an unorganised mess - and would have just created a lot of noise. Instead, he mined his blog posts for the most useful information. Still has the Evernote notes as markdown files, so still available when necessary. So he started using Obsidian with a blank slate.

Uses Obsidian as a viewer of his text/markdown files. Plain text files make it easy to maintain your notes across different platforms and systems, while allowing creativity and innovation to happen as a layer on top of the files.

Tools change. Every tool is temporary, so avoid having tools that drift too far away from the core feature set. If you're going to install plugins that provide additional, more powerful functionality, that functionality isn't going to be present when you look at your notes in plaintext. Avoid plugins that are going to make it difficult to view the content of your notes in other tools.

You need to be able to move on to other tools with low/no friction.

Uses folders to organise information, mostly related to projects, with an ‘overview’ page that links to other parts of the project.

Finding a new tool provides a certain level of fascination. But, we need to ask if the tool is genuinely useful with respect to the work we do, or is it a shiny new hammer to hit nails with?

He has replaced several tools with Obsidian (notes, to do, outliner). He says that Obsidian has replaced about 2/3s of the steps in his system. This is my problem at the moment…I’m happy with the number of steps in my system, but not that each step needs it’s own tool.

Don't write about the tool. Write about how you use the tool to do real work. You see this in the YouTube channels of people who spend hours talking about the tools they but they don’t show themselves doing real work with the tool.

Still not happy with the relatively high friction of turning notes notes into output. Works in Obsidian a lot to generate notes but still struggles to push out artifacts. I also want to figure out how to move my notes and reflections into output, with less friction.

There’s a tension between writing for yourself and writing for an audience. Do you want to publish your daily notes? Would this lead to self-censorship? Would you be more self-conscious?

Use blogs as part of an obligation to explain. If you need to search for information to do something, maybe you should blog that process to try and make it easier for others to do things. I like the idea that we have an ‘obligation to explain’ because we have all benefited from others who have explained things for us.

Tools will come and go but your process should be more stable over time.

It’s what I already said earlier just being able to reduce text on the screen, put more photos and things that makes sense to voice over so that they can actually hear what I’m saying versus putting a text and not understanding the context of my voice.

For level one, I’ll plan on implementing this by just changing my tone of voice when it’s needed and make sure that I use my voice an appropriate manner and using it at the right time. Not only that by using hand gestures and other things in that nature

It’s what I already said earlier just being able to reduce text on the screen, put more photos and things that makes sense to voice over so that they can actually hear what I’m saying versus putting a text and not understanding the context of my voice.

Just being able to differentiate the important material whether it's the color of the text or the way I underline it. Honestly I’m pretty organized with these kind of stuff.

Real creative humans don't tend to "create" "content". They write essays, sing, do sketch comedy, dance, take photographs, etc. This is a long-running peeve of mine but I'm bringing it up because the corporate language obscures the nature of this complaint.

Imagine: "I've read some really good plays, but it's not always obvious what's going on. Playwrights should write out prose between the pieces of dialogue so it's as easy for readers to understand as possible."

Anyone can see this would be silly, since the form--drama rather than prose--is part of the intended experience.

If you are consuming Twitter threads equivalently to long-form content, you are missing out on part of their value, since each tweet is open to its own replies. This means that people can chime in with their own stories or anecdotes or objections non-linearly.

Cory's thread doesn't do this because it's so long it breaks the Twitter UI's conventions of loading first the thread, then people's replies. I would cede that makes Cory's Twitter thread relatively ineffective.

However, it can still be the case that someone can excerpt via RT a particularly interesting detail they want to highlight without viewing an entire thread as worth the time.

So we get back to intent: some writers would hate that for sentences out of their blog posts. Some writers like to make that form of discourse part of what they're doing. (It's pretty common for a thread's author to further engage with replies to particular Tweets) If you don't engage with that, then yeah, of course it looks preferable to just copy-paste the text into a blog post to read. But that doesn't mean that people are mindlessly "forcing" each other onto a "bandwagon" because they "get a tonne of followers."

A reprehensibly techie comparison: If you don't use stuff like transclusion and non-linear structure, outliners seem like they just introduce noise into what could just be paragraphs of text. But... the whole point is the part you're not using.

Why is this intuitive? It's not intuitive to me. It seems to say that bettors who bet on Horse A to win are trying to actually make Horse A to win, when they really are just predicting that Horse A will win with a sufficient probability.

If Nate Silver really wanted Obama to win, he wouldn't bet on it, but would have donated to Obama's campaign.

If bettors really wanted Horse A to win, they wouldn't have betted on it, but would have fed it steroids or something.

When life insurances first came out, pretty much the exact argument was used against it, that it's "gambling on death". Eventually culture absorbed it, and even though life insurance is still gambling on death, it no longer feels like it.

Similarly, shorting stocks of a medical company that's developing a breakthrough cancer treatment, is, in an indirect sense, rooting for the cancer treatment to fail, and thus more people to die.

Betting on people dying is not a dealbreaker, even if human psychology never evolves. Just add enough cultural excuses and decorations around it, and it will no longer irritate the mind. Imagine a clam covering up an irritating sand until it becomes a smooth pearl.

Hi copy editor! Many thanks!

I’ve done very minimal editing on the Word files, with the exception of bibliographies, which I cleaned up significantly.

I think it would make the most sense for us to focus on readability. Feel free just to highlight sentences and deem them “awkward”—I’m happy to make the changes myself—or of course feel free to go further; I’ll happily make whatever substantive edits you propose. Consider breaking up paragraphs, since paragraph breaks are one of the things that easily get lost in transfer from Word. And of course whatever else you see….the only thing I’ll say is that in my opinion it’s not worth our eyesight to spend much time looking at those Works Cited lists; a couple are, well, REALLY long.

Hi copy editor! Many thanks!

I’ve done very minimal editing on the Word files, with the exception of bibliographies, which I cleaned up significantly.

I think it would make the most sense for us to focus on readability. Feel free just to highlight sentences and deem them “awkward”—I’m happy to make the changes myself—or of course feel free to go further; I’ll happily make whatever substantive edits you propose. Consider breaking up paragraphs, since paragraph breaks are one of the things that easily get lost in transfer from Word. And of course whatever else you see….the only thing I’ll say is that in my opinion it’s not worth our eyesight to spend much time looking at those Works Cited lists; a couple are, well, REALLY long.

Hi copy editor! Many thanks!

I’ve done very minimal editing on the Word files, with the exception of bibliographies, which I cleaned up significantly.

I think it would make the most sense for us to focus on readability. Feel free just to highlight sentences and deem them “awkward”—I’m happy to make the changes myself—or of course feel free to go further; I’ll happily make whatever substantive edits you propose. Consider breaking up paragraphs, since paragraph breaks are one of the things that easily get lost in transfer from Word. And of course whatever else you see….the only thing I’ll say is that in my opinion it’s not worth our eyesight to spend much time looking at those Works Cited lists; a couple are, well, REALLY long.

Hi copy editor! Many thanks!

I’ve done very minimal editing on the Word files, with the exception of bibliographies, which I cleaned up significantly.

I think it would make the most sense for us to focus on readability. Feel free just to highlight sentences and deem them “awkward”—I’m happy to make the changes myself—or of course feel free to go further; I’ll happily make whatever substantive edits you propose. Consider breaking up paragraphs, since paragraph breaks are one of the things that easily get lost in transfer from Word. And of course whatever else you see….the only thing I’ll say is that in my opinion it’s not worth our eyesight to spend much time looking at those Works Cited lists; a couple are, well, REALLY long.

Hi copy editor! Many thanks!

I’ve done very minimal editing on the Word files, with the exception of bibliographies, which I cleaned up significantly.

I think it would make the most sense for us to focus on readability. Feel free just to highlight sentences and deem them “awkward”—I’m happy to make the changes myself—or of course feel free to go further; I’ll happily make whatever substantive edits you propose. Consider breaking up paragraphs, since paragraph breaks are one of the things that easily get lost in transfer from Word. And of course whatever else you see….the only thing I’ll say is that in my opinion it’s not worth our eyesight to spend much time looking at those Works Cited lists; a couple are, well, REALLY long.

Hi copy editor! Many thanks!

I’ve done very minimal editing on the Word files, with the exception of bibliographies, which I cleaned up significantly.

I think it would make the most sense for us to focus on readability. Feel free just to highlight sentences and deem them “awkward”—I’m happy to make the changes myself—or of course feel free to go further; I’ll happily make whatever substantive edits you propose. Consider breaking up paragraphs, since paragraph breaks are one of the things that easily get lost in transfer from Word. And of course whatever else you see….the only thing I’ll say is that in my opinion it’s not worth our eyesight to spend much time looking at those Works Cited lists; a couple are, well, REALLY long.

Wow, this is fascinating. The difference between written and photographed representations

isolated, just like school work. a problem is give that you already know how to solve. What happens when you don't know how to solve the problem because it's outside the lines that school taught?

we are told we need high education to succeed in life and if we don't succeed in school then we are good for nothing. But maybe it's just a belief?

This sounds 3000% correct.

This seems like it's talking about something really important, but "Adtech is the technology that decides how worth is distributed" is something other than the boiled-down version of that thing.

Cultivating dependency, not just food crops!

This statement is right on. It has now become a question about the RATE of system change we can achieve to avoid a degraded future. The faster we act, the less degraded it will become.

in my summary I added, "according to the author". Because it's just a more solid sentence. I find that it adds more intensity and seriousness to the situation.

Such a powerful statement! The ways in which media has worked to destroy any positive image of black culture and black people has been used throughout history in the variety forms of media expression. It's sad to hear the expression that it's been used as a weapon against black people. It reaffirms the intention and creation of systemic racism and oppression.

I find this a very important thing. it happens to me a lot, as I find it the easiest way to let a conversation flow. Now, thinking about it I've realised it's something really annoying when being interviewed and, from the interviewer side does not add anything new to the information gathered!

ResearchQuestion: Does AI help to combat racism, sexism, etc., in healthcare? I Googled this question and found opposite sides of it.

1. This article talks about racial bias in the software that makes decisions on healthcare. https://www.nature.com/articles/d41586-019-03228-6
2. This article argues the other side of it. https://www.forbes.com/sites/robertpearl/2021/02/16/how-ai-can-remedy-racial-disparities-in-healthcare/?sh=6a03df1830f6 And that was just what I found with less than five minutes of searching. I would need to print these and dig into them more, and find other stuff, to really get into these questions, but, it's a start!

Who is she speaking to; who is you? I feel like there are two options. One, she is speaking to a lover who she cannot be with, forbidden love if you will, or an affair gone depressive and toxic and just terrible. Two, I see this as being the human that Kane longs to be. The person that she is "supposed" to be. This persona does not long to be in Kane's body though, and this is where the conflict arrises. Also, a few lines later, Kane asks if it's possible for a person to be born in the wrong body. I am wondering if Kane would have transitioned or been non-binary in another life. Such beautiful words to describe something aching so deeply inside of her.

Something that Sarah Kane does really is utilizing her structure of the script to make certain things stand put. I remember talking about this in my play writing class a few semesters ago but everything from font, word placement, size, format, etc, goes into the delivery and tone of the script. Kane plays around with the structure a lot to make specific things stand out in this dark play.

I like to read a lot, it is one of my favorite hobbies. I have realized that I would rather read a play than watch it. When you are reading something, your mind comes up with how the story would play out visually. How the characters would look, dress, etc. I like how in this play there are no character names , which leaves much more room for imaginative creation. In my head, there are questions forming like, why isn't there a bigger distinction between the doctors and the main character? Maybe this person is actually talking to themselves and there really are no doctors? or is it a memory of a past conversation between them and the doctors?

This is 20% lower than what was shown in the survey that just went out. It's in the news because we have all been locked in the house for so long and everyone (at least that I know about) have been going a little crazy.

Lost in Translation

In the film, Lost in Translation, Bob and Charlotte begin their conversation learning what each of them is doing in Tokyo.

Bob: What do you do?

Charlotte: I’m not sure yet, actually. I just graduated last spring.”

Bob: What did you study?

Charlotte: Philosophy.

Bob: Yeah, there’s a good buck in that racket.

Charlotte: (Laughs.) Yeah. Well, so far it’s pro bono.

(33:45)

Edge Effects

In ecology, edge effects are changes in population or community structures that occur at the boundary of two or more habitats. Areas with small habitat fragments exhibit especially pronounced edge effects that may extend throughout the range. As the edge effects increase, the boundary habitat allows for greater biodiversity.

Wikipedia: Edge effects

“The truth is that all of the gold that was mined out of the Klondike was under Indigenous land. There was no treaty with any of the Indigenous peoples in the Yukon.”

“That land was stolen by the Canadian state and that gold was whisked away by private interests. The Federal Government only signed land claims with Indigenous peoples in the Yukon in the 1990s, but by that point, almost all the gold had been mined out of the ground.”

“The Klondike gold rush was a rolling disaster that captured tens of thousands of people. When the first European explorers came to the Americas, they came here looking for gold. In the 1890s, that lust for precious metals eventually led men to the farthest reaches of this continent.”

“Today, instead of 100,000 people descending on a small patch of land, you have large corporations digging treasures out of the ground. But the legacies these mining operations leave behind are just like what happened in the Klondike: workers with broken bodies, environmental destruction, the dispossession of Indigenous land, sexual violence. The gold rushes never stopped. They just morphed into something different.”

Canada is Fake

“Canada is not an accident or a work in progress or a thought experiment. I mean that Canada is a scam — a pyramid scheme, a ruse, a heist. Canada is a front. And it’s a front for a massive network of resource extraction companies, oil barons, and mining magnates.”

THis is not about regulation, this is about learning to co-exist. And Schmidt is avoiding the question whether this scale is too big, too inhuman - focuses on how to make it work

The thing to understand about NFT

hyperknowledge indeed

plugin ecosystem

not just end up on medium

feed contolled by algorithm for engagement

It's easy for us in a lot of ways, because this is a PD course, but this is how we've approached the evaluation structure for this course: it's more about your narrative of what you've learned than it is about ours.

Though I'm just now learning about doxxing, I can already tell that it is a very negative term. It's wrong to post such confidential things about someone on the internet for everyone to see just to see them hurt. That's just another way of saying cyberbullying.

It's interesting here that the "wet" motif includes not just water, but oil and tar. With the personification of the river sweating, it can be viewed as the river laboring and its products are pollution that are harming most likely harming everything around it.

Bad! That's really bad! It's also bad that "families" are sectioned off from both.

The discussion of what is lawful versus what is right, what is just, is such an interesting part of Antigone that I've always loved, but I wonder if this conflict works within this context. Maybe it's just me, but it seems to me that the army and government that launched and fought the dirty war are unquestionably in the wrong here. I think Coryphaeus taking on the image of Creon might be a bit misleading to me, since I cast him in the role of the authority, the Argentinian government. But I think he represents more the general people who worry about and uphold the laws and morals of the society they live in just because they do not harm them personally and hence they feel no urge to challenge them. He takes on and off the airs of Creon, of the authority, eventually becoming simply Coryphaeus who stands by and allows the demise of Antigona to happen.

Going into this play with the understanding of the plot of "Antigone" I already had, I was waiting for a moment like this where the alignment of these new characters I don't recognize would be revealed. Even though it seems that Antinous relents just a few lines later, it's clear that the two are not quite of the same mind when it comes to Antigone's actions, or at least her motives anyway.

Reviewer #2 (Public Review): 

The authors investigated sex differences in explore-exploit tradeoff using a drifting binary bandit task in rodents. The authors tried to claim that males and females use different means to achieve similar levels of accuracy in making explore-exploit decisions. In particular, they argue that females explore less but learn more quickly during exploration. The topic is very interesting, but I am not yet convinced on the conclusions. 

Here are my major points: 

1) This paper showed that males explore more than females, and through computational modeling, they showed that females have a higher learning rate compared to males. The fact that males explore more and have lower learning rates compare to females, can be an interesting finding as the paper tried to claim, but it can also be that female rats simply learn the task better than male rats in the task used. 

(a) First, from Figure 1B, it looks like p(reward, chance) are similar between sex, but visually the female rats' performances, p(reward, obtained), look slight better than males. It would be nice if the authors could show a bar plot comparison like in Figure 1C and 1E. A non-significant test here only fails to show sex differences in performance, but it cannot be concluded that there are no sex differences in performance here. Further evidence needs to be reported here to help readers see whether there are qualitative differences in performances at all. 

(b) The exploration and exploitation states are defined by fitting a hidden Markov model. In the exploration phase, the agent chooses left and right randomly. From Figure 1E and 1F, it looks like for male rats, they choose completely randomly 70% of the times (around 50% for females). The exploration state here is confounded with the state of pure guessing (poor performance). 

(c) Figure 2 basically says that you can choose randomly for two reasons, to be more "noisy" in your decisions (have a higher temperature term), or to ignore the values more (by having a learning rate of 0, you are just guessing). It would be nice to show a simulation of p(reward, obtained) by learning rate x inverse temperature (like in Figure 2C). From Figure 2B, it looks like higher learning rates means better value learning in this task. It seems to me that it's more likely the male rats simply learn the task more poorly and behave more randomly which show up as more exploration in the HMM model. 

(d) From figure 3E, it looks like female rats learn better across days but male rats do not, but I am not sure. If you plot p(reward, obtained) vs times(days), do you see an improvement in female rats as opposed to males? Figure 4 also showed that females show more win-stay-lose-shift behavior and use past information more, both are indicators of better learning in this task. 

Taken the above together, I am not convinced about the strategic sex differences in exploration, it looks more like that the female rats simply learn better in this task. 

2) I do like how the authors define exploration states vs exploitation states via HMM using choices alone. It would be interesting to see how the sex differences in reaction time are modulated by exploration vs exploitation state. As the authors showed, RT in exploration state is longer. Hence, it would make a conceptual difference whether the sex difference in reaction times is due to different proportions of time spent on exploration vs exploitation across sex.

Самое важное - софт-скиллы и интересы каждого

PTSD is very common for sexual assault victims and it is rarely recognized as something to be treated by the VA.

Do you mean in comparison to the other piece, or just that this is its weakest feature? It's a little unclear from the phrasing here.

Okay - you're starting to get into some argument territory here, just needs a little more synthesis throughout so it's clearer that you want all this points to connect to a larger claim.

And an aristocratic upbringing! Cf. the anger of the downwardly-class-mobile, who received it without its "deserved" reward.

A lot of mothers, particularly, driving themselves mad over this.

The narrative also doesn't allow considering fallback. What kind of world should my child have if he's just "not bright enough" to win the class mobility lottery? You can't ask it if you've internalized the good/bad meritocracy moral coloring. How would I have to work differently to help him have a good life? What do the winners and losers of that lottery owe each other?

Those are then the questions that can drive progress, even in the absence of sparks of resentment.

Here Flamineo, having “riseth,” chastises the cunning and ingratious wives, adding to the negative portrayal of women but also establishing a moral compass that sets an important backdrop for the aftermath of not just Flamineo’s story but truly Eliot’s. Following the reference to Jerusalem and Jesus’ resurrection, it’s not implausible to view a “moment’s surrender” as a sin which “an age of prudence,” or repentance , “can never retract,” or absolve one from responsibility. From here, Eliot paints a grim outlook where one’s legacy cannot be ensured (the spider not spinning one’s tale), possibly referencing Arachne and the way her legacy was truncated by her hubris. As we near the end of the poem, this hinting at legacy makes me wonder if these characters just suspended in a perpetual purgatory, defined by this one “moment’s surrender” or have some sort of afterlife/sequel Eliot envisioned (404).

For service and product advertisements, the internet has become an important medium now. Every company that can afford radio, print, and TV advertisements have also ventured on the Internet into placing ads.

But, how can you make sure that your ad can be seen by your target customers with millions of websites out there being visited by millions of computer users, and that your leads will be followed by these users? In this article app promotion company, prepared for you a few tips are here.

1. Place ads on your target customers' websites frequented. You need to define who will be your target customers before doing this. There will gain attention by placing an ad on sites that they usually visit will increase the possibility of your ad and your target customers will be followed.

Consider also the website’s traffic, search engine placement, external linking, and the other ads placed on it. If there are other sites that link to this site, external linking will determine it. This will mean that even if they are browsing a different site, people can get to the site where your ad is placed. Just check if any of your competitors have ads on this site.

1. On some sites placing ads cost some money. There are also sites that can place a link to your product, service or site, in exchange for having their link being displayed on your site. About the newsletters sent by site owners to its mailing list members regularly, it is also true.

Having sites link to your pages and having helpful links placed on your site also help in boosting search engine ranking. To make a statement that your site’s content is important, this is similar that other sites have links to your web pages.

The first suggestion for those who have the product and want to market products through the internet:

• Learn first, how much the ‘market demand’ for the goods you want to sell.

• Type the name of your product or your ‘target market’ that is approximately related to the product.- Do research, how much the desire to market your goods.

• If the result has many, this means good news! you just develop strategies, but before all of these strategies, of course, you must create a website and used good a website script.

• Conversely, if the product does not have an interested buyer, you can still do something!

One thing that makes the internet amazing to me, even though sometimes we want a selling product that does not exist or maybe more appropriate if I say “not yet” there, then we can “create” a new target market through our creativity!

For instance, I have a traditional puppet factory. Then I would like to sell my “traditional puppet” through the internet. After I do market research, it appears that very few people who find puppet on the internet, because so many foreigners who do not know about puppets. So what, I will give up and then close my traditional puppet factory? Of course not! If so, it’s not a business!

Our team mobile app promotion services  advise you always thinking creatively. And to be creative, are required to always practice. A person can be creative at the time he does start to put aside a little time to think about what he must do to complete a problem.

The more our brain is trained to continue to think creatively, then our creativity will be more tested. And who knows with the elapsing time, also with the experience, training, trial, and error, you may become a ‘creative genius.

And this is my main problem with method acting. I have no issues with it as long as it doesn't make others lives' miserable. And Carrey's commitment to the character no doubt created issues on set, and like Bax, I feel that the movie takes a one sided approach to this problem

This manuscript is in revision at eLife

The decision letter after peer review, sent to the authors on June 30 2020, follows:

Summary:

This paper addresses a critical issue in neuroscience: what's the question, and can we answer it? The questions the author proposes are ones that have been considered, in one form or another, reasonably often by experimentalists. And the author shows rigorously that there's a reasonable chance that they are simply not answerable.

Essential Revisions:

We believe that this is an extremely important issue, and the approach the paper takes is a reasonable one for addressing it. Our main worry, though, is that mainstream neuroscientists will ignore it, for two reasons. One is that it's not a message they want to hear. Second, the example circuits are sufficiently abstract that they can be dismissed as yet another musing by your typical uninformed theorists. (That is not, we should emphasize, our view, but it's not an uncommon one in the field.) Our goal, therefore, is to fix these potential problems, so that people will have to pay attention.

The premise of the paper is that if you understand a neural circuit, there are certain questions about it that you should be able to answer. The author proposes six such questions, and then shows that in the worst case they are exponentially (in the number of neurons) hard to answer.

The success of this program hinges on two things: a sensible set of questions, and a demonstration that answering those questions is hard. We're not ecstatic about the questions, but we believe that's not an insurmountable issue (more on that below). More problematic is the result that the questions are hard to answer. What's really shown is that there is at least one circuit for which, in the worst case, answering the questions is exponentially hard. While this is certainly correct, it doesn't make a convincing case that answering these questions will be hard in the brain. First, the worst case may not be the typical case. The 3 SAT problem, for instance, is NP complete, but is hard to solve only for a narrow range of parameters. Second, answering the questions for actual circuits found in the brain may not even be exponentially hard in the worst case.

This brings us to two critical comments. First, it needs to be crystal clear that this paper does not demonstrate that answering the proposed questions is guaranteed to be exponentially hard, only that it might be. This was stated in the manuscript, but not emphasized. For instance, on lines 138-140, it says

"Using techniques from Computational Complexity Theory, we ask what is the smallest number of experiments necessary, in general, in order to answer these questions, in typical experimental settings."

Here "in general" means worst-case. For neuroscientists, though, "in general" means "most of the time". It should be clear that you mean worst case, and that the typical case may be very different.

In fact, this needs an expanded discussion. Whether or not it will take an exponential number of experiments to answer the questions depends on the circuit. We might get lucky, and only a small number of experiments are needed. Or we might get unlucky, and a large number are needed. This analysis can't tell us that, and this should be clear in the paper.

Second, what's really needed is the analysis of a more realistic circuit, ideally with both positive examples (for which it is possible to answer the questions) and negative examples (for which it isn't). This is hard, but we have a few suggestions, some of which can probably be done without a huge amount of work.

a. Linear network, y=Ax+noise. For this (and possibly in all realistic) situations, "perform the task" needs to be replaced by "achieve a certain level of performance". For instance, if there's a true mapping y=f(x), then "perform the task" would be replaced with "<(y-y*)^2> below some threshold". The questions should be answerable in polynomial time for this network; otherwise, one should worry.

b. In 2000, Hopfield and Brody came up with a simple circuit which we think of as "understandable" (Hopfield and Brody, PNAS 97:25, 13919-13924, 2000). It would be nice to determine whether the questions can be answered in polynomial time for this circuit. Again, if they can't, one should worry.

c. Deep networks. Again, "perform the task" would have to be replaced with "performance is above some particular threshold". Here we suspect that the questions are not answerable; if that could be shown, it would be a huge step forward.

d. A made-up model of a deep network. Assume that in a deep network, whenever you delete a neuron, performance drops. That's probably not so far from the truth -- and also not so far from what we think would happen in the brain. (With some exceptions; occasionally I hear talks where performance is better when two areas are ablated rather than just one, but let's ignore that.) How much performance drops depends, of course, on which neurons are deleted, so there's not a simple mapping between performance and which neurons are present in the circuit. Can the questions be answered in this case? This sounds like a problem computer scientists have considered, so possibly rigorous analysis could be done.

We believe it's critical to consider a case that is not far from what one might find in the brain. Otherwise, it will be too easy to dismiss this work as irrelevant to real neuroscience. The above are only possibilities, and a and d may be pretty easy, but the author is welcome to come up with his own examples. Note that rigor is not absolutely necessary here, since there's already one rigorous example. Plausible arguments would be fine.

Finally, "understand" needs further discussion. That's partly because the approach here is a little non-standard. Most people try to directly define "understanding". Instead, the statement is "if you understand a circuit, you should be able to answer these questions". This has to be made crystal clear -- especially since people aren't expecting it. In addition, a discussion of the more standard approach, a direct definition, should be included. The usual definition is something like "A short description of what is being computed, along with a description of an algorithm for computing it." It should be clear how this, more standard, definition compares to the one in the paper. For instance, under the standard definition it may be possible to understand a circuit without being able to answer any of the questions. For instance, I believe we can "understand" (by the more standard definition) the synfire chain circuit. This doesn't mean that one definition is better than the other, but their differences should be acknowledged.

MVT

I was fortunate enough to experience a “nurturing” response (from a writing professor) that not only impacted my risk-taking abilities, but also brought out the best in myself as a writer. So, in my first year at college, I remember visiting my writing professor—let’s call him J—to talk about a final writing assignment (it was something to do with evaluating Wikipedia as a source of knowledge). I don’t recall why I disliked that assignment, but I remember I found some courage to share my honest thoughts and feelings. I wasn’t sure how J would respond…but guess what happened? J paused, looked at me thoughtfully and then warmly said, “Write what you want to write…just make sure it’s your best writing.” I was shocked but truly delighted!

Today, I wonder why J had listened and responded with kindness, respect and trust. I believe he was one of those beautiful souls who probably applied the Namaste concept or its equivalent in his classroom. His powerful words truly nurtured my thinking and writing, and I ended up writing my very “first” fiction piece in his class. It’s phenomenal how J’s nurturing response supported my growth, transformation and risk-taking abilities as a student writer. Yes, J had chosen kindness, in that he made sure his students’ writing experiences were made as rewarding and fulfilling as possible. It was indeed J’s kindness that he chose to bend rules and make exceptions to support my writing needs. If J had not demonstrated kindness, I would have certainly completed that Wikipedia assignment, but I wouldn’t have experienced what it feels to be heard, understood and nurtured in an academic setting. I am truly grateful to J for not only demonstrating respect and kindness toward me, but also shaping my path to do the same for my students today. Thank you, J, for everything you did. You have my highest respect and deepest regards.

1:23 I think this part of the video really stands out following me reading the other assignment. That assignment put into perspective that our time is limited no matter what, we just don't know when it's up. And yet, when we are given a diagnosis of a shortened yet determined fatality date, we do everything we would want. However, we don't do that while we live life with no guaranteed time (yet still determined) date. Truly makes one wonder why we don't prioritize having all the experiences we want to have over a life of struggle resulting in flourishing, so I suppose that might make me at least slightly hedonistic in thinking this.

This is really frustrating and disgusting. Though it may not be appropriate, it's one thing to make an innuendo to your boyfriend or group of friends, it's another to make assumptions about strangers and put them on blast. Just another reminder to think before you speak.

Paraphrase: Mettelus attacked a village that wasn't ready for battle whatsoever. Interpretation: He seems to be paranoid and power hungry. He is attacking everywhere and wouldn't accept any bribes. It's also interesting that the village wasn't ready for war at all. It could show the complexity that they knew they would lose, so they just didn't prepare for anything.

maps deduplicates cross references

This is actually one of the cases that should definitely be exempted from anything really same as with rape. I don't think it's right for something to be taken away from someone just because they don't have the access to it or had something serious happen in their lives.

Having just been around my first exposure of language deprivation and seeing the effects that will probably be undoable is heartbreaking. The child tries so hard in school but the lack of exposure during the critical years catches up and behavior outbursts sometimes happen. With me being new to the school and the environment, it's a huge shock for me to see how the behavior is handled and I want to help and be so patient with them but it then hinders the other children.

This is a powerful example that class consciousness and class fears have driven the building of America since its inception.

It's been built into our DNA and thus will be difficult to ever stamp out fully so that people will enjoy greater equality, equity, and freedom.

There is nothing that gets in the way of success more than avoidance. We avoid hard conversations. We avoid certain people. We avoid hard decisions. We avoid evidence that contradicts what we think. We avoid starting a project until we're certain of the outcome. ... Sometimes we muster half the courage. And here’s the interesting thing. Half-efforts tend to make things worse, not better. When things don’t get better, it only reinforces that we shouldn’t have said anything in the first place. Avoiding isn’t better, it’s just easier. Everything becomes harder until we stop avoiding what's getting in the way. The longer you wait the higher the cost.

As we discussed yesterday, this is something I feel is lacking when observing social dynamics. We had school pictures just the other day and the photographer was trying to make my student laugh using a "baby voice" and puppet...I remember the way he was looking at her with eyes that seemed to say I'm a human being why are you speaking to me this way? This is just one example but it's the first that comes to mind. I feel that behavior like this is almost derogatory in a way as you would never speak to an adult peer this way!

I cannot agree more. I think pedagogy should teach the way of thinking, not just the knowledge which can be easily standardized and tested. I think it's absurd to train students from K-12 to be good standardized test takers but then expect them to be innovative employees when they enter the workforce.

It's devastating that all this happens around us and we're so blind to it, and even when we do hear about it, we turn a blind eye to it, just because it's not happening to us.

On top of that, the topic is on civiliation CENTURIES ago. It's a little hard to put out an assumption that they lacked protein if they were from a long time ago

That is the worst part of the education system. Societal expectations is to get your degree and finish school. Why would school be promoted if it couldn't have it's truly entitled faults being taught to students or even brought to mind by students. The entire message and concept of student debt and loans are just pushed to the side and these effect students lives way too much to just be tossed to the side.

Programming is equally or more about communications between humans as much it is about humans communicating with computers

I think this is the biggest problem that crypto space has at the moment. People treat like a stock market, which it is not, or at least it's not what the intention was.

It is very natural for people to see complete bozos earning hundreds of thousands of dollars on Bitcoin trading in the early days and just imitate it in many different ways. These are very much like good old Ponzi Schemes, where the intention is to inflate the value, cash out and then who care what happens.

At some point such coins will very likely collapse as in the case of Dogecoin and many, many other similar tokens. Many people will get hurt, many people will get rich.

However, that doesn't mean that the whole industry will go down. The event described above will remove all the people who are in it just for the quick buck and only those who believe in the blockchain and relating technologies will remain.

This sentence really interests me because it almost reads as a callout to other countries with bad histories, including the US. It's almost as if Nieman here is telling the others to step it up. While I don't agree with an atonement monument being built just to stop backlash or to keep up with other countries, I feel like nations need to acknowledge that some of their past actions really did have negative consequences that hurt a lot of people.

An underreported sad state of Thunder Bay norm. When I was living in Montreal I had a Kenyan roommate. A model actually. There would be times when I'd happen to be walking benind her on a city sidewalk....and some of the glares and comments were unimaginably cruel. "...fucking parasite." "Invader!" "Can't see her til she smiles eh? haha so dark". People from Kenya are commonly very dark-skinned, tall, and slim. Completely beautiful in my opinion. It was through the glares and under-the-breath comments in passing, or louder ones if only spoken to me directly. There were times I felt such a sense of disgust and embarrasssment that I wasnt sure if I should pity the person making the comments or hate them. Usually it was complete embarrasment. The stunning Evangeli Anteros:

I think this relates back to the term psychic fantasy because of the myth created that black youth play basketball instead of being educated (whether or not the myth created is the truth, it is still the image one had in mind). I think it's also interesting that black youth are expected to reach "higher goals" than just playing basketball, and it shows that the expectations for black people tend to be much higher than white people in order for them to prove their knowledge. A white man vs a black man playing basketball are judged by society very differently. If black kids do not live up to and exceed societies definition of success, they are looked down upon and often called lazy or stupid, when it is even more difficult for black people to reach this definition of success because of discrimination and lack of resources compared to white folks.

Wow, that's incredible to see, but also very predictable. This has been a result of the culture throughout our world's history where women have been perceived to be "servers" to men and that they would stay in the kitchen and take care of the house while the men went to work. Fortunately, that culture has begun to shift but this example shows that there is still work to be done to transition people's minds into understanding that there are other roles for women. Also, it emphasizes that women are capable of being able to be leaders in sports if they're just given a fair opportunity. Now that doesn't mean to hire someone =just because they're a women or a person of color, which is one potential drawback that could occur. However, it's all about giving those who have generally been marginalized throughout history a fair opportunity so succeed.

This review reflects comments and contributions by Ricardo Carvalho, Joachim Goedhart, Sónia Gomes Pereira, Pratima Gurung, Samuel Lord, Claudia Molina, Arthur Molines, Gregory Redpath, Mugdha Sathe, Sagar Varankar. Review synthesized by Ewa Sitarska.

This preprint introduces a recombinant profilin that has a flexible linker to a genetically encoded fluorescent tag (either mApple or Halo). Fluorescent protein tagging is a popular and accepted method to study the properties of a protein of interest in solution and in cells. A careful analysis of the tagged protein relative to the untagged, native protein is crucial to understand whether the tagged protein faithfully reflects the behavior of the native protein. Therefore, studies like these are very valuable and the current manuscript is a good example of how such a study should be performed. The flexible linker presented here overcomes challenges observed in previous papers that found that linking a fluorescent protein to profilin disrupted some of its actin-related functions in cells and in vitro.

This study is carefully conducted and nicely describes the properties of a fluorescent protein tagged profilin in a detailed manner. In particular, the authors use various in vitro assays as well as rescue experiments to demonstrate that their tagged version of profilin appears to behave similarly to wildtype profilin. The manuscript is written in a clear manner and was an enjoyable read. The comments below cover a couple of experiments, clarifications and questions for consideration to further add to the work. Regardless, this work is likely to contribute to the field, as anyone studying profilin is likely to try this construct in their future experiments.

General comments:

• Why was mApple chosen as a tag (as opposed to the popular and best known fluorescent protein mEGFP)?
• The mApple is prone to photochromicity/photoswitching (https://doi.org/10.1038/nmeth.1209, https://doi.org/10.1038/nmeth.4074). This should be mentioned to warn future users of this fusion protein.
• It would be advisable to be consistent with the naming of ‘untagged profilin’ throughout the manuscript. Currently unlabeled, untagged, wild-type or rescue are used interchangeably.
• In Figures 4B, 5A there appears to be differences between untagged and tagged profilin in the images. Maybe a more representative image would be beneficial, where applicable.
• Depositing the plasmids from this paper at addgene.org would be beneficial for the public (the plasmids can be deposited under condition that these will be released only after publication of this work in a peer-reviewed journal).

Title

‘Functional fluorescently-tagged human profilin’– suggest clarifying in the title and throughout the text that the fluorescent tags are genetically encoded.

Abstract

‘high cellular concentrations (121 µM)’– This is a very precise number for such a general statement. It seems that the number is derived from a specific cell line, so it would be beneficial to present it as a number from this cell line or change it to an approximation (~100 µM).

Introduction

'Some profilin outcompetes actin bound'– suggest some rewording to clarify the fragment, for example, it could be mentioned whether it refers to F-actin or G-actin.

Results- Design of tagged profilin

‘Profilin is considerably smaller than the smallest fluorescent tags’ – clarifying that it is a genetically encoded fluorescent tag would be of advantage. There are no smaller fluorescent proteins (FP) yet, but genetically encoded FP of a similar size exist, for example miRFP670nano is 17kDa. https://doi.org/10.1038/s41467-018-08050-8

‘Traditional direct labeling approaches are cytotoxic and disrupt actin-based functions’ – is there data showing the new fluorescent profilin side-by-side with one without a flexible linker (or other version used previously) to show that the latter disrupts profilin's functions? It’s not essential but it would strengthen this point and confirm the improvement over prior work.

‘Estimates of cellular profilin concentration are very high depending on the cell type’ – would be nice to provide a rough estimate at this point, similarly to the introduction part.

‘with an mApple fluorescent probe or as Halo-tagged single molecules’ - What is the meaning of 'single' here?

‘We cloned mApple- or Halo-tags fused to a ten amino acid flexible linker on the N-terminus of human profilin-1.’ – As in the introduction, it is stated that “Positioning a GFP-derived fluorescent tag on the C- or N-terminus disrupts PLP- and PIP-binding interactions, effectively rendering the fluorescent version flawed for critical measurements in cells”, it would be beneficial to state the rationale for tagging profillin at the N-terminus? Also, how was the linker composition and length determined and how is it related to other linkers used in compromised fusions of profilin?

Figure 1

• In panel A, it would be helpful to indicate the N-terminus, as this is the side where the fluorescent protein is attached.
• In the legend, PFN1 is introduced for the first time and thus, it could be replaced with ‘tagged-profilin (PFN1)’.

Results - mApple-profilin binds phosphoinositide lipids with similar affinity as untagged profilin

‘PIP’ - PIP is usually an abbreviation for Phosphatidyl Inositol Phosphate (which is a lipid). Phosphoinositide is the same thing, but is not abbreviated as PIP. Recommend using Phosphatidyl Inositol Phosphate = PIP. These types of lipids can be indicated as PIPs (without the addition of lipids).

‘Profilin also binds PI(3,5)P2 which regulates critical signal transduction events through intracellular vesicles to the early endosome’ - The prevailing consensus now is that PI(3,5)P2 is involved in late endosomal trafficking to the lysosome. This could be different for profilin specific purposes, but this statement could be updated with recent references to PI(3,5)P2.

‘Thus, mApple-tagged profilin retains functional interactions with two important PIP lipids.’ – Testing other phospholipids, including PIP3 (Lu et al., 1996 showed an interaction with profilin) and some negative controls would be beneficial. Covering most phosphoinositide species by using the phospholipid-binding dot blots would make this figure stronger.

Figure 2

• In panel B, it would be easier to compare profilin and mApple-profilin binding affinity to each of the PIP lipids. For that, profilin and mApple-profilin samples could be run side by side in the same blot. The suggestion being that panel B includes profilin and mApple-profilin incubated with PI(3,5)P2, and panel C profilin and mApple-profilin incubated with PI(4,5)P2. For clarity, can it be specified in the figure legend or in the figure that the profilin-1 lane is the negative control pellet lacking the liposomes as well as that S and P stands for supernatant.
• Also, in panel B, what are the loading controls? The quantification and western-blots are unclear. For example, it is indicated that 1µM PFN1was used in this experiment, but the pellet band for mAp-PFN1 is not comparable to PFN1 pellet band. Despite this, quantification in 2D shows that they are similar. Please clarify and add the corresponding loading controls.
• Panel D: Please mention what is quantified here (supernatant, pellet or overall level),
• Panel D and E: the shades to pink may be tricky to distinguish, more contrasting colors and/or shapes might be useful.
• It is highly appreciated that the antibodies and dilutions are mentioned in the figure legend.

Results - Direct visualization of fluorescent-profilin with polymerizing actin filaments

‘We used fluorescence anisotropy to measure the binding affinity between profilin and Oregon-Green (OG)-labeled monomeric actin (Figure S2).‘ - This appears confusing with the 3D results. If a change in fluorescence anisotropy with OG-actin is not detected, then is it okay to use OG-actin for bulk polymerisation assay. Maybe the OG-tag interferes only during fluorescence anisotropy, but not during fluorescence microscopy.

‘Several studies demonstrate that thymosin b4 (Tb4) competes with profilin to bind actin monomers‘ – It is worth mentioning that this refers to untagged/unlabeled actin monomers.

Figure 3

• Please consider if it is relevant to compare the competitive and non-competitive data on the same graph.
• In panel A description, ‘10 nM GFP-thymosin b4 (GFP-Tb4) mixed with increasing concentrations of unlabeled actin.’ – Would ‘10nM unlabelled actin monomers in presence of increasing concentration of Τβ4’ be more appropriate?
• In panel D, the curves would be better visible when the y-axis runs from 0-30.
• In panel E, the mApple-Profilin samples show longer filaments, maybe quantification of the filament length could be performed?
• In panel G, are the errors bars based on the means of the technical replicates or on all aggregated data? The first option is preferred, as this plotting strategy was also used in 3F.
• In the panel description, ‘Data were quantified from four separate reactions (FOV) each.’ – could clarify whether the data derive from 4 totally independent reactions, or from the analysis of 4 different fields of view (FOV) from the same experimental procedure?
• In the panel description, ‘ns, not significantly different from 1 µM actin alone control; a, compared with control (p <0.05)‘ – such labeling may be confusing for the reader, it would be beneficial to state that the first ‘ns/a’ are related to the actin alone, and the second ‘ns’ are comparing labelled and unlabeled profilin or omit showing the statistical test in the plot, and show it in a table instead.

Results - Fluorescent profilin stimulates formin-based actin filament assembly

General: Based on the competition and interaction experiments it seems important to generate a dose-dependent inducible construct for profilin to govern the stoichiometry of interactions and study their relevance in the cells.

‘Similar to experiments in assessing only profilin-actin interactions (Figure 3F), we counted significantly fewer actin filaments in reactions containing actin and either profilin (Figure 4D)’ - In Figure 3F the plot shows around 45 actin filaments per FOV with 1 uM actin (20% oregon green actin). In Figure 4D the plot shows more than 100 actin filaments per FOV with 1 uM actin (10% alexa 647 actin). Surprisingly, the elongation rate are similar in Fig 3F and Fig 4D. Is the Oregon-Green actin known to be less efficient at nucleating filaments while retaining the same polymerization ability? If it is the case, it would be worth making a mention.

‘Thus, fluorescent profilin stimulates formin-mediated actin filament nucleation similar to the untagged version.’ - The data seem to suggest that the presence of profilin inhibits actin filament nucleation and polymerization, a clarification would be appreciated.

‘The ac-celerated rate of actin filament’ – Please change to ‘accelerated’.

Results - Profilin directly binds tubulin dimers and enhances the growth rate of microtubules in vitro

‘microtubule stability index‘ – The values indicated on the y-axis for the plot in Fig 5E are confusing (0 / 25/ 50 / 75 / 1). Is the index expressed as a percentage and the max value supposed to be 100? Or is the index supposed to be the number of rescues per catastrophe?

’This suggests a mechanism where profilin accelerates microtubule polymerization by directly binding to tubulin dimers to promote microtubule assembly and then diffusing along the sides of the microtubule lattice to further stabilize microtubule growth.‘ - Microtubules are more stable the faster they grow (catastrophe frequency scales inversely with polymerization rate). In this condition where profilin increases polymerization rate by around 5x, it is unclear how much of the increased stability is due to the lattice binding. The fragment could be softened regarding the role of the transient lattice binding in microtubule stabilization.

Figure 5

• In panel B, intensities are quite different, would it be possible to comment on this?
• In panels H an I, a black/magenta merge is tricky to see. Although it breaks consistency, a green/magenta or cyan/magenta merge may be more informative visually.

Results - Profilin regulates the morphology of N2a cells through actin and microtubule crosstalk

‘We used quantitative western blots to determine the level of endogenous profilin as well as levels of profilin in CRISPR knockout cells following transfection with plasmids containing untagged profilin, mApple-profilin, or Halo-profilin.’ - The levels of profilin are quantified from a blot, which is a bulk measurement. The transfection of profilin will show substantial cell-to-cell variation (some cells may have much higher or lower levels than the measured average). Mentioning it and discussing its implications would be advisable.

‘We chose this parameter because N2a cells have unique actin filament and microtubule cytoskeletal features but do not efficiently perform other classic cell processes that require intact cytoskeletal crosstalk (i.e., migration or division).‘ – While looking at cell shape is one strategy, an additional experiment looking into a migration phenotype may strengthen this point. Another interesting experiment to strengthen this point and providing a direct measure of profilin function could be performing a pulse chase experiment using drugs to depolymerise actin/microtubules. In such an experiment, a distinct change in depolymerisation should be noted between WT, KO, and the profilin rescue cells. This could show that the mApple-profilin can substitute for WT profilin.

‘super resolution confocal microscopy to image fixed cells.‘ – how is super-resolution achieved here? Or is ‘super’ unnecessary here?

‘the ratio of endogenous cell area to other cell conditions’ - This metric is a ratio of areas and it is only valid as an assessment for shape if the cells from each condition cover similar areas. If it is not the case, then the two parameters (shape and area) are convoluted and the ratio measures both the difference in shape and in area covered. It would be good to provide the average area of the cells in each condition for clarification.

‘We also stained these cells for actin filaments (Figures 6H and 6I) and micro-tubules (Figure 6J and 6K) and used a similar morphology parameter to detect broad differences in cytoskeletal architecture.’ – Please clarify the reasons for using the cell area ratio metric for quantification of cell morphology. How is the quantification metric (area) used for sub-cellular network like actin and microtubule? F-actin stained with phalloidin looks different in endogenous PFN1 vs mApple-PFN1, but by using area metric there is no morphological difference. Microtubule, on the other hand, appear similar in all the cells.

Figure 6

• In panel A, it does not seem like mApple-PFN1 and Halo-PFN1 reach endogenous levels. Maybe a quantification would be beneficial.
• In panel B, please report the number of independent replicates. Also, may be worth commenting on why tagged PFN1 rescue cells were not included?
• For panel E, please provide the error bars and the legend that contains the information from where the data derived or from how many independent experiments.
• In panel F, it may be advantageous to use red green and blue as colors for the overlay. This will generate unique colors for the different combinations of the three images.
• For the context of panel F, unprocessed images of the tagged profilin in living cells could be presented somewhere in the main text. They could be larger than the small panels here, and not be segmented into binary images. The point of the fluorescent profilin is that it can be used for live-cell imaging without substantially disrupting the typical profilin interactions. This should be confirmed by presenting live-cell images of the profilin construct. To avoid problems with high cytoplasmic concentrations of profilin drowning out any localization signal, maybe the fluorescent version could be expressed at a very low level or the Halo version could be used with a low concentration of fluorophore.
• In panel G, it is clear that the dots are from different cover slips, how many cells were analyzed per coverslip? Data could be shown from individual cells (not just their average). Also, please clarify if this quantification was made 24 hours after transfection as well?
• For panel I, actin morphology was calculated from actin filament signals similar to the cell morphology index. These calculations could be explained further in the methods. Does this mean that the actin morphology index is the ratio of actin area between the two conditions? Is the actin image somehow thresholded before taking the ratio?
• Panel L, is really appreciated and helpful to understand the "competition" between actin and microtubules for profilin. It would be also nice to represent the plasma membrane and the lipid-binding activity of profilin as well as binding to nucleation promoting factors (the proline-rich motifs of VASP and WASP), as this is mentioned throughout the paper.

Discussion

The fact that mApple and HaloTag both are entirely different and non-disturbing gives confidence that profilin can be fused with other tags, without losing functionality. Mentioning this in the discussion could give new insights for the readers.

‘Our genetic analyses in mammalian cells indicate that mApple-profilin and Halo-profilin are fully inter-changeable with the endogenous version.‘ - Authors have given the field an excelled tool which will be quite useful to study cellular functions of PFN1, its interaction with its several binding partners. Currently, cell shape is the metric used to determine if the tagged versions are fully inter-changeable with the endogenous version. Whether the tagged PFN1 can replace untagged PFN1 for other cellular functions will require further exploration. Also, high concentration of PFN1 will remain an issue even with the mApple-PFN1 developed here. Do the authors suggest mild over-expression as a strategy to go around the high concentration issue?

‘Based on localization experiments using the pan-formin inhibitor, SMIFH2, some interactions between profilin and the sides of micro-tubules are thought to be indirect.’ – suggest clarifying how SMIFH2 treatment leads to conclude that interactions between PFN1 and microtubules is indirect.

Methods

Statistical significance tests do not demonstrate that conditions are identical. That is, when two conditions are not statistically different, it is not possible to say that these are equal (https://doi.org/10.1053/j.seminhematol.2008.04.003). Suggest avoiding the use of "n.s." in graphs to indicate that the data are similar. It is clear from the data that (in many cases) the tagged and untagged profilin show similar properties. If equality needs to be demonstrated, recommend carrying out an equivalence test.

‘Different shades of data points show technical replicates.’ – Please rephrase to clarify, for example “Different colors represent biological replicates. Similar colored dots reflect technical replicates“. Does "technical replicate," mean repeated measurements within each independent experimental run? Or different experimental runs? If the shading is supposed to denote paired experiments (e.g. darkest shading in different conditions are from the same experimental run), that can be stated in the caption.

How do the speaker's remarks reflect the impacts of globalization on the Arab Spring?

It's truly saddening to see how this emphasis on marriage caused such harm to many women's mental health. The opposition to marriage lead Wells, and no doubt many other women to believe that there was something somehow wrong with them, when in reality, they just didn't want something like that. Unfortunately, this is a problem that many LGBTQA+ people still face today.

I believe that that's where learning happens. Sometimes when children are having snack and they find themselves discussing different ideas and somehow get stuck in rigidity of thinking. It's worth spending more time to understand different perspectives and see that most of the things are not only black and white but there's a a range of different colors that make our lives so colorful.

This part of the play illustrates how money is valued over the individual. Uriah says, "you can replace a man anytime, but a paybook is sacred if anything is." I feel like this quote shows us that these characters believe that human life is easily replaced. However, if we must keep living what is the point if we can't get paid. Life is hard enough so if you're going to fight for anything it shouldn't be to survive but to get the paycheck. yikes.

I actually agree with this. I feel like it's useful if I really don't feel like putting much effort into an assignment. It just feels boring and the same as every other essay I've had to write. I would always think about if that's how my future college professors expected my work to sound like, but I'm just so used to this structure already.

Cho vay: y hệt như ngân hàng làm trung gian gửi tiết kiệm và cho vay, tuy nhiên là không có ngân hàng

This situation is just so scary and after a bit of thinking "that's such a horrible thing to do," I realized it's actually super realistic as it is literally what happens in real life whenever there is a natural disaster or well, a pandemic. A great example is the one we are still in, at the height of the lockdown when everyone was scrambling to buy toilet paper and food and other essentials, people began gouging prices. And even after everything, things got more expensive and still have stayed that way. I can't imagine going through it all the time like these people are. This seems like a connection to the real world and what the government and businesses do to those less fortunate during times of crisis.

These lines immediately make me a bit skeptical that I'm about to read a religious play where every action the characters take is somehow rooted in "God's will,". But it also is a little contradicting seeing as they're not exactly being positive about God or any of this. They treat it more like a passing thing that just happens and you have to deal with it. If God says it has to be a certain way, it will be that way and you just have to deal. It does come off a bit confusing but definitely interesting to see if this is a bigger message or maybe it's just a passing line of dialogue.

I can't see to shakes off the feeling that when he said this, it had something to do with the story he just told. honestly, I got so lost reading this. there was a part about the customers complaining about hum not telling happy stories, but yet they are still coming back the next day. Maybe that's why he said it depends on you. I remember him saying that when it's time for him to tell the story that they so long for, he will tell it, but in the mean time, he will tell other stories. they said they come to this café to enjoy his time not to become depressed and overwhelmed, but again yet they still come and why is that? simply because of curiosity. if they stop coming they will never hear the story they want, so they will keep coming and hoping that they will hear it

The author is making an emotional appeal by telling the reader how the app can have "long-term effects" and that how it is violating human rights and privacy protections, it can be very dangerous just image someone having all the information about you, even if it is the government it is never really 100% safe. Even though the app was mainly put into action to stop the Coronavirus from spreading it is still not safe, even making it a televised game, but someone's privacy protections is not a game it can lead to someone being hurt.

The style/form in this poem is quite interesting it's almost conversational, as if the narrator is talking to someone. With that there are no punctuation's allowing the poem to flow cohesively within the stanzas. The use of enjambment is noticeable.

It’s pretty amazing that these areas all speak French but they all speak different types of French. This just shows that it’s not just English that you could speak a different kind of English in every language you can speak differently.

It's interesting that the connection between the genocide in German Southwest Africa and the Holocaust was slowly made. Considering the genocide in Southwest Africa has just recently been considered a genocide, it's easy to assume that the similarities between both events in history seemed to go unnoticed in Germany.

Author Response:

Reviewer #1 (Public Review):

Summary of what the authors were trying to achieve

Background: Myopia (short- or near-sightedness) is an ocular disorder of increasing concern to human individuals and health-care systems; these days one speaks of a "myopia epidemic" in developed countries. Usually it is due to excessive elongation of the optic axis of the eye during the ages of most rapid growth (ca. 5-16 years in humans), causing images of distant objects to be blurred at the retinal photoreceptors. The optical error can be corrected with lenses or corneal surgeries, but this does not reduce the risk of continued progression and vision loss. Despite extensive epidemiological and animal studies in the past several decades, the underlying causal mechanisms remain poorly known, and therapeutic options are limited. Therefore, further discovery of new candidate mechanisms, drug targets and drugs for inhibiting the onset and progression of myopia is urgently needed.

Rationale: The axial length of the eye is regulated mainly by qualities of the visual environment, including light intensity, spectrum, and spatiotemporal characteristics of images on the retina. Thus the retina encodes and integrates visual information over time, and ultimately sends regulatory "grow" or "stop" signals via the choroid - a vascular plexus behind the retina - to the sclera, the fibrous outer coat of the eye. Changes in size (area) of the sclera are responsible for changes in axial length, and thereby, refraction. The choroid is in a critical position, not only to relay "stop" or "go" messages to the sclera, but also potentially to critically modify those signals (or generate signals of its own) and further modulate ocular elongation and refraction. Importantly, very little is known about how the choroid fulfills either of these roles.

Aims of the Study: The authors' purpose was to test, in juvenile chicken models, whether the 'pro-inflammatory' cytokine, interleukin-6 (IL-6) - synthesized and released in the choroid - might play a key role in the developmental regulation of axial elongation and refraction of the eye.

Major strengths and weaknesses of the methods and results

Strengths:

1) The studies are focused on the choroid, which must be important in regulating ocular growth and refraction, but whose role is still not well understood

2) Expert use of front-line tools for quantifying mRNA and protein (microarray, RT-PCR, ELISA)

3) Immunohistochemistry: Good choice of antibody (raised to chicken IL-6), appropriate specificity control (preabsorption with chicken antigen)

4) IL-6 mRNA in choroid was impressively increased during recovery from form-deprivation myopia (FDM) (preliminary results, Fig. 2) - i.e., during strong positive (myopic) defocus - a defocus-dependent effect confirmed by a similar effect of lens-induced myopic defocus (Fig. 5).

5) Good data for the time-course of IL-6 mRNA content in choroid, with some confirmation of protein levels (though at only 2 treatment intervals) (Fig. 3)

6) Choroidal IL-6 mRNA also shown convincingly to increase, going from darkness to light (Fig. 4).

7) It's clever to compare the growth- and myopia-inhibiting effects of positive defocus, with those of other treatments known to do the same - in this case, atropine and nitric oxide (NO). The evidence shows that the effects of these agents on choroidal IL-6 mRNA are similar to the effect of positive defocus, with an NO-donor increasing the amounts of IL-6 mRNA and protein in isolated choroid (Fig. 7), and a NOS-inhibitor decreasing the mRNA levels at an intravitreal dose that inhibits scleral growth (Fig. 6).

8) If my calculations are correct, 0.1% atropine sulfate solution has a molarity of something like

1.3 mM. Since alpha-2A adrenoreceptors are present in the choroid, of mammals at least (e.g., Wikberg-Matsson et al., 1996, Exp Eye Res, 63(1):57-66), it might be interesting to explore the possibility that atropine is stimulating IL-6 production in the choroid by acting as agonist via these receptors (cf. Carr et al., 2018, IOVS, 59m2778-2791). The isolated choroid, with IL-6 mRNA and protein synthesis as read-outs, should be an exceptional (and novel) model for testing this and other possible signalling pathways in the choroid.

Weaknesses:

1) Immunolocalization of IL-6: The images (Fig. 1) are not good enough to identify cellular localization of immunoreactive structures; identification of RPE is questionable (no DAPI+ nuclei in labeled 'RPE'); nucleated erythrocytes should be visible in vessel lumina.

We have increased the magnification and resolution of images in Figure 1 to better distinguish immunoreactive cells. Additionally, we have included as Figure 1 - figure supplement 1, both H&E stained and immunolabelled images from adjacent serial sections (both longitudinal and cross sections) of control choroids in order to compare immunopositive cells with the histoarchitecture of the choroid. From these images, one can see that nucleated erythrocytes are located in some of the vessel lumina. The nuclei of the RPE label weakly as compared with those of choroidal fibroblasts or nucleated blood cells. In order to visualize the RPE nuclei, we had to increase the intensity of the DAPI channel (blue, 405 nm) to a level that is not optimal for viewing IL-6 immunolabelling (green). Therefore, we included an additional supplemental figure (Figure 1 – figure supplement 2) in which RPE nuclei are readily visible.

2) Many important details of methods have been left out. Spectral peaks of LED light-sources need to be given, lines 409-412; that's just one of many examples.

We have included graphical and tabular data describing the frequency spectra for each of the three LED light sources used in the present study (Figure 4 - figure supplement 1), and in Sheet 3 of the source data file for Supplementary figures. Additionally, we have added the method of obtaining the frequency spectra in the methods section (lines 499 – 501).

We have also included details of the antigen retrieval procedure performed during histological processing of ocular tissues and details on the methodology and analysis of microarray data (lines 544-547).

3) Intensity (illuminance) of "red" and "blue" lights seems unnecessarily low (58 and 111 lux, respectively, far below the "medium" and "high" intensities of white lights that were used; Fig. 4).

We agree with this comment. The intensities of the red and blue LED lights were limited by the red LED lights. We set them at their maximum intensity setting which registered at 58 lux. To compare their effect with that of the blue light, we felt we should set the blue LED lights at a similar setting, which required us to use its lowest setting, which registered at 111 lux. We realize that these intensities are low, compared with the medium and high settings of the white LED lights. Perhaps at higher intensities we might have observed a differential effect on IL-6 mRNA with red light compared to blue light. We have added this explanation and possibility in the Discussion section (lines 333 – 353).

4) Also, given that red and blue lights have been found to have opposite effects on FDM in chicks (e.g., Wang et al., 2018, IOVS, 59(11):4413-4424), the similarity of IL-6 responses to red and blue in the present study strikes me as a point against a role for IL-6 in regulation of eye growth.

We respectfully disagree with this statement. The Wang et al., paper reported that continuous exposure to red LED lights for five days had no significant effect on refraction or axial length in either control eyes or form deprived (myopic) eyes. In contrast, continuous exposure to blue LED light caused a significant hyperopic shift in refraction in both control and form deprived eyes, but had no significant effect on axial length in either control or form deprived eyes (although a trend toward a decrease in axial length was observed after five days in both control and form deprived eyes). Since refraction was significantly affected (in blue light-reared chicks), but axial length was only minimally affected, we suspect that continual exposure to blue light may have affected other ocular parameters (such as corneal curvature) that would have a significant impact on refraction. We predict that choroidal IL-6 expression is involved in the choroidal and scleral remodeling processes at the posterior pole of the eye that result in changes in vitreous chamber elongation, as opposed to having effects on the anterior segment of the eye. Our data shows short term (6 hr) exposure to red or blue LED light had no effect on IL-6 gene expression. If IL-6 gene expression is involved in the regulation of eye growth, we would expect that exposure to red or blue LED light would have no effect on scleral remodeling, vitreous chamber depth, or axial length, which in fact, is consistent with the results of the Wang et al., paper. We have added this interpretation in the Discussion section of the paper (lines 333 – 353).

5) I admire the thoroughness of confirming that some of the treatments did in fact have the predicted effects on ocular enlargement, by performing assays for scleral proteoglycan synthesis. This might not be essential to this work, although it is well done, and the scleral data won't detract from the value of the paper if retained. But the induction of opposite effects on eye (scleral) growth by such manipulations is well established, and much simpler (cheaper and faster) refraction and/or caliper measurements would have served the same purpose.

We elected to use scleral proteoglycan synthesis as a “read out” for axial elongation, since we can detect significant changes in scleral proteoglycan synthesis much earlier (within 6 hrs) than we and others can detect changes in axial length or vitreous chamber depth in chick eyes (≥2 days). Since our studies involved very short term exposure to myopic defocus (6 hrs), we felt measurements of scleral proteoglycan synthesis would be more likely to establish causal relationships between IL-6, nitric oxide and scleral remodeling.

6) I don't buy the argument that the source of NO is not in the choroid (lines 337-340), based on the failure of L-Arg to change significantly the amount of choroidal IL-6 mRNA (Fig. S1). Several thoughts come to mind here: (a) It is solidly established that the choroid is richly innervated with NO-synthesizing nerve fibres, and that its content of NOS is very high [e.g.: "NOS activity is widely distributed in the eye, (choroid > retina > CP > TM) …"; Geyer et al., 1997, Graefes Arch, 235(12):786-93; also (among others): Wu et al., 2007, Brain Res., 1186m155-63; Hashitani et al., 1998, J Physiol, 510(1):209-223; Fischer & Stell, 1999, cited in the present MS.]. So, there clearly are sources of NO within the choroid, in chicks as in mammals. (b) It's extremely unlikely that "NO, released from the retina … diffuses to the choroid to stimulate IL-6 synthesis", because NO is highly reactive and has a short half-life, restricting its diffusion. But yes, NO generated by iNOS in the RPE certainly could reach choroidal targets; is there anything in the literature to indicate that iNOS mRNA and protein are increased in the RPE, under conditions or treatments that inhibit axial elongation? (c) The critical experiment to test this idea - treating the isolated choroid with a NOS-inhibitor, to block synthesis of NO by cells in the choroid - was not performed here.

That would be a complicated and difficult exercise, however, requiring the invention of a way to stimulate NOS activity to a new base-level, and then being able to detect effects due to the inhibition of NO-synthesis. It would be good to discuss the issues raised in this point, but acceptable to suggest this as another of the questions that would be suitable to address by further experimentation beyond the scope of this paper.

Based on these comments (and the comments below) by Reviewer #1, we carried out additional experiments on isolated choroids using 50 mM KCl to depolarize the plasma membranes of choroidal cells in the presence of L-arginine (0.05 mM – 5 mM). We found that in the presence of KCl, treatment of choroids with L-arginine caused a significant increase in IL-6 mRNA. In contrast, L-arginine in the absence of KCl had no significant effect on IL-6 mRNA (as we found in our original experiments). We interpret these new results to indicate that choroidal IL-6 can be upregulated by endogenous sources of nitric oxide. We have included this data in new Figure 8 of the revised manuscript. We thank the reviewer for providing this insight!

7) Since it's overwhelmingly likely that NO is synthesized and released locally in the choroid, alternative explanations must be considered for why the NO-donor, PAPA-NONOate, caused increases in IL-6, while L-Arg didn't. Might it have been the case, for example, that the NOS- containing choroidal cells already were fully loaded with L-Arg, under these particular experimental conditions? or that the administered concentration of L-Arg was sub-optimal? or that the proportions of cellular mass to fluid volume in the choroidal samples were highly variable, causing high variance of the individual values? or that the parent compound PAPA- NONOate, however attractive 'his' name, had destinations in mind (molecular targets, actions) in addition to or other than sGC? Any one of these hypotheses might account for the fact that L-Arg reduced the mean level of IL-6 mRNA by almost 50%, but with p=0.14 despite the sample size n=16.

Please see explanation under item 6 above.

8) The results of the bulk assays - of whole choroids - are a good beginning, starting to build a map of largely uncharted territory; but they will never be completely satisfactory for constructing signalling pathways or networks for visual regulation of scleral expansion, and will leave one struggling to make sense of it all (cf. lines 345-347). Better immunolabeling, with better image definition and resolution, the addition of single-label images and bright-field images (to locate the RPE securely), and possibly FISH would be helpful for this. If you're rich, have great local resources, and/or are well connected with others who do, scRNA-seq of dissociated choroidal tissue (with RPE and sclera as controls) would have great potential here. If the tissue has been perfused intravascularly or well washed and drained, to get rid of blood cells, there shouldn't be very many cell types to characterize (but, my, wouldn't it be exciting and illuminating if there were!)

As stated under point 1 above, we have increased the magnification and resolution of images in Figure 1 to better distinguish immunoreactive cells and we have included both H&E stained and immunolabelled images from adjacent serial sections (both longitudinal and cross sections) of choroids in order to compare immunopositive cells with the histoarchitecture of the choroid in supplemental Figure 1 - figure supplement 1. We agree that scRNA-seq would yield valuable insights into gene expression changes amount individual cell populations within the choroid. We feel those studies are beyond the scope of this paper, but are ones that we are currently undertaking.

9) The relationships between the studies and outcomes reported in this manuscript, and the possible role of choroidal IL-6 and other inflammation-signalling molecules in myopia, is hardly touched upon at all - just a short, very general statement near the end of the Conclusions (lines 368-374).

We have added additional discussion regarding the possible role of inflammation in ocular growth regulation (lines 358-365, 370 – 372)

Reviewer #1 (Public Review):

Summary of what the authors were trying to achieve

Background: Myopia (short- or near-sightedness) is an ocular disorder of increasing concern to human individuals and health-care systems; these days one speaks of a "myopia epidemic" in developed countries. Usually it is due to excessive elongation of the optic axis of the eye during the ages of most rapid growth (ca. 5-16 years in humans), causing images of distant objects to be blurred at the retinal photoreceptors. The optical error can be corrected with lenses or corneal surgeries, but this does not reduce the risk of continued progression and vision loss. Despite extensive epidemiological and animal studies in the past several decades, the underlying causal mechanisms remain poorly known, and therapeutic options are limited. Therefore, further discovery of new candidate mechanisms, drug targets and drugs for inhibiting the onset and progression of myopia is urgently needed.

Rationale: The axial length of the eye is regulated mainly by qualities of the visual environment, including light intensity, spectrum, and spatiotemporal characteristics of images on the retina. Thus the retina encodes and integrates visual information over time, and ultimately sends regulatory "grow" or "stop" signals via the choroid - a vascular plexus behind the retina - to the sclera, the fibrous outer coat of the eye. Changes in size (area) of the sclera are responsible for changes in axial length, and thereby, refraction. The choroid is in a critical position, not only to relay "stop" or "go" messages to the sclera, but also potentially to critically modify those signals (or generate signals of its own) and further modulate ocular elongation and refraction. Importantly, very little is known about how the choroid fulfills either of these roles.

Aims of the Study: The authors' purpose was to test, in juvenile chicken models, whether the 'pro-inflammatory' cytokine, interleukin-6 (IL-6) - synthesized and released in the choroid - might play a key role in the developmental regulation of axial elongation and refraction of the eye.

Major strengths and weaknesses of the methods and results

Strengths:

1. The studies are focused on the choroid, which must be important in regulating ocular growth and refraction, but whose role is still not well understood

2. Expert use of front-line tools for quantifying mRNA and protein (microarray, RT-PCR, ELISA)

3. Immunohistochemistry: Good choice of antibody (raised to chicken IL-6), appropriate specificity control (preabsorption with chicken antigen)

4. IL-6 mRNA in choroid was impressively increased during recovery from form-deprivation myopia (FDM) (preliminary results, Fig. 2) - i.e., during strong positive (myopic) defocus - a defocus-dependent effect confirmed by a similar effect of lens-induced myopic defocus (Fig. 5).

5. Good data for the time-course of IL-6 mRNA content in choroid, with some confirmation of protein levels (though at only 2 treatment intervals) (Fig. 3)

6. Choroidal IL-6 mRNA also shown convincingly to increase, going from darkness to light (Fig. 4).

7. It's clever to compare the growth- and myopia-inhibiting effects of positive defocus, with those of other treatments known to do the same - in this case, atropine and nitric oxide (NO). The evidence shows that the effects of these agents on choroidal IL-6 mRNA are similar to the effect of positive defocus, with an NO-donor increasing the amounts of IL-6 mRNA and protein in isolated choroid (Fig. 7), and a NOS-inhibitor decreasing the mRNA levels at an intravitreal dose that inhibits scleral growth (Fig. 6).

8. If my calculations are correct, 0.1% atropine sulfate solution has a molarity of something like

1.3 mM. Since alpha-2A adrenoreceptors are present in the choroid, of mammals at least (e.g., Wikberg-Matsson et al., 1996, Exp Eye Res, 63(1):57-66), it might be interesting to explore the possibility that atropine is stimulating IL-6 production in the choroid by acting as agonist via these receptors (cf. Carr et al., 2018, IOVS, 59m2778-2791). The isolated choroid, with IL-6 mRNA and protein synthesis as read-outs, should be an exceptional (and novel) model for testing this and other possible signalling pathways in the choroid.

Weaknesses:

1. Immunolocalization of IL-6: The images (Fig. 1) are not good enough to identify cellular localization of immunoreactive structures; identification of RPE is questionable (no DAPI+ nuclei in labeled 'RPE'); nucleated erythrocytes should be visible in vessel lumina.

2. Many important details of methods have been left out. Spectral peaks of LED light-sources need to be given, lines 409-412; that's just one of many examples.

3. Intensity (illuminance) of "red" and "blue" lights seems unnecessarily low (58 and 111 lux, respectively, far below the "medium" and "high" intensities of white lights that were used; Fig. 4).

4. Also, given that red and blue lights have been found to have opposite effects on FDM in chicks (e.g., Wang et al., 2018, IOVS, 59(11):4413-4424), the similarity of IL-6 responses to red and blue in the present study strikes me as a point against a role for IL-6 in regulation of eye growth.

5. I admire the thoroughness of confirming that some of the treatments did in fact have the predicted effects on ocular enlargement, by performing assays for scleral proteoglycan synthesis. This might not be essential to this work, although it is well done, and the scleral data won't detract from the value of the paper if retained. But the induction of opposite effects on eye (scleral) growth by such manipulations is well established, and much simpler (cheaper and faster) refraction and/or caliper measurements would have served the same purpose.

6. I don't buy the argument that the source of NO is not in the choroid (lines 337-340), based on the failure of L-Arg to change significantly the amount of choroidal IL-6 mRNA (Fig. S1). Several thoughts come to mind here: (a) It is solidly established that the choroid is richly innervated with NO-synthesizing nerve fibres, and that its content of NOS is very high [e.g.: "NOS activity is widely distributed in the eye, (choroid > retina > CP > TM) ..."; Geyer et al., 1997, Graefes Arch, 235(12):786-93; also (among others): Wu et al., 2007, Brain Res., 1186m155-63; Hashitani et al., 1998, J Physiol, 510(1):209-223; Fischer & Stell, 1999, cited in the present MS.]. So, there clearly are sources of NO within the choroid, in chicks as in mammals. (b) It's extremely unlikely that "NO, released from the retina ... diffuses to the choroid to stimulate IL-6 synthesis", because NO is highly reactive and has a short half-life, restricting its diffusion. But yes, NO generated by iNOS in the RPE certainly could reach choroidal targets; is there anything in the literature to indicate that iNOS mRNA and protein are increased in the RPE, under conditions or treatments that inhibit axial elongation? (c) The critical experiment to test this idea - treating the isolated choroid with a NOS-inhibitor, to block synthesis of NO by cells in the choroid - was not performed here.

That would be a complicated and difficult exercise, however, requiring the invention of a way to stimulate NOS activity to a new base-level, and then being able to detect effects due to the inhibition of NO-synthesis. It would be good to discuss the issues raised in this point, but acceptable to suggest this as another of the questions that would be suitable to address by further experimentation beyond the scope of this paper.

7. Since it's overwhelmingly likely that NO is synthesized and released locally in the choroid, alternative explanations must be considered for why the NO-donor, PAPA-NONOate, caused increases in IL-6, while L-Arg didn't. Might it have been the case, for example, that the NOS- containing choroidal cells already were fully loaded with L-Arg, under these particular experimental conditions? or that the administered concentration of L-Arg was sub-optimal? or that the proportions of cellular mass to fluid volume in the choroidal samples were highly variable, causing high variance of the individual values? or that the parent compound PAPA- NONOate, however attractive 'his' name, had destinations in mind (molecular targets, actions) in addition to or other than sGC? Any one of these hypotheses might account for the fact that

L-Arg reduced the mean level of IL-6 mRNA by almost 50%, but with p=0.14 despite the sample size n=16.

8. The results of the bulk assays - of whole choroids - are a good beginning, starting to build a map of largely uncharted territory; but they will never be completely satisfactory for constructing signalling pathways or networks for visual regulation of scleral expansion, and will leave one struggling to make sense of it all (cf. lines 345-347). Better immunolabeling, with better image definition and resolution, the addition of single-label images and bright-field images (to locate the RPE securely), and possibly FISH would be helpful for this. If you're rich, have great local resources, and/or are well connected with others who do, scRNA-seq of dissociated choroidal tissue (with RPE and sclera as controls) would have great potential here. If the tissue has been perfused intravascularly or well washed and drained, to get rid of blood cells, there shouldn't be very many cell types to characterize (but, my, wouldn't it be exciting and illuminating if there were!)

9. The relationships between the studies and outcomes reported in this manuscript, and the possible role of choroidal IL-6 and other inflammation-signalling molecules in myopia, is hardly touched upon at all - just a short, very general statement near the end of the Conclusions (lines 368-374).

Whether the authors achieved their aims, and whether the results support their conclusions:

The authors have made a very convincing case that myopic defocus stimulates the synthesis of IL-6 mRNA and protein in the chick choroid. The effects of atropine and NO-related drugs further support the association between this action and the inhibition of excessive axial elongation and myopia.

Likely impact of the work on the field, and the utility of the methods and data to the community: This work - and in particular the use of assays for IL-6 in choroidal explants to assess the actions of candidate signalling molecules in the choroid - should be seen as an important step forward. The methods are up-to-date, but well established and straightforward, and could be easily duplicated by most workers in the field. The chick models for myopia induction and recovery have been used and refined for decades, so they are easy and almost foolproof (used successfully by many undergraduates in my lab). Once the foundations have been laid by studies in chicks, they can be translated rather easily for making similar studies in mammalian models such as guinea pigs and NHPs