i just think it's good to note that the author did a really good job making the connection between the message of the book and the message of the piece itself!

It's funny to see things like this because it's true that we have so many positive things towards the lgbtq community but for me personally I had no idea that we had such a huge drag group or a BDSM school because sex and sexuality is such a taboo topic for most utahns in the state. just reading this has opened my eyes to utahs drag community and now i'm interested in learning more about them and what they stand for. More people including me need to be educated on what is happening in the community under the taboo sheets of the states judgement due to the church.

This is an interesting quote but at the same time you could argue that the Author has always being dead. In reality The author or the painter or the artist in general has always being dead. Most of the time the creation that they have made has gone out of the real that they intended it to be seeing. For example books and music. Most stories even if they have their own agenda and message they want to get across. They tend to be taken out of context. Most of the time for the better. Fan theories and fan fiction is full of it. In a way we are all the author of any given work. Even if you create something you don't always own it. At least not fully. From what I have read so far it makes it seem like art as a science has slowly died. I could be interpreting it wrong. But I could argue that art did not loose it's meaning. We just couldn't ignore the pretentious nature of the animal that is art For example the Mona Lisa. In reality is the old time version of what we consider a selfie today. A lot of paintings that we study and learn about are basically glorified selfies of kings and such. So what's stopping us from going to Instagram and picking a selfie and put it under a magnifying glass and picking things that may have meaning and thus making it art.

So that leaves us with a couple of questions. Was the Author alive to begging with?

Who is the actual author? Is it the one taking and interpreting the work or is it the one creating it?

This line here helps illustrate the evolution of art. By illustrating it as a parent and child personification.. Because it illustrates how an art form can be born from another art form. Become it's unique entity while still being influence by the mother art form. To use one of this writing examples. Rock music was born from the passionate one night stand between Blues and Country music. And almost like a birth mark Rock was also knows as the devils music just like the blues were. Funny enough in the year 2020 I doubt either one is knows as that but when they were new and young. That was a title they both shared.

I've been thinking about how much I want to learn spanish. I would love to make life easier for someone, or just make them happy by being able to speak spanish and connect with them that way, especially in a school setting like the one I am entering where their native language skills are often not seen as a strength. It's been something I've wanted at every job I've ever worked. Wondering if I can take a spanish class as an elective for NYU classes and if that would be a good use of my credits or if I can take a class elsewhere.

It wouldn't let me highlight or annotate so I'm just going to quote it here:

"Good teachers make a difference in the academic growth of students. Bad teachers can slow down a student's academic progress."

It's good to keep in mind that we won't make or break our students education. There are tons and tons of outside influences that effect a child's development. Acknowledging this myth definitely helps with taking some of the pressure off and allow myself to do my best without any anxiety or stress of failing.

Ask questions that require complex/higher order thinking, not just questions requiring factual answers.

This is definitely true. Many people often think of themselves as annoying because they're feeling like they have talked too much or overshared. When meeting someone new or talking in a new setting, it's hard to understand where the lines are of what is oversharing and what is just sharing.

I think it's important to understand that not everyone is emotionally available to participate in every activity, especially now that we're all on Zoom for the majority of the day. For some people, just logging on and listening is enough for them.

Author Response

Reviewer #1:

This manuscript provides evidence that drug administration during a reconsolidation window does not necessarily prevent memory recall, as has been shown by many groups. The authors attempted to replicate several published experiments and despite demonstrating that the drugs had other effects on the animals' behavior and physiology (e.g. weight gain), no effects on memory were observed.

The paper is nicely prepared.

We sincerely thank the reviewer for these kind words and the support to publish our replication efforts.

Reviewer #2:

General assessment:

In this study, Luyten et al. aimed to replicate post-retrieval amnesia of auditory fear memories reported numerous times in the literature. They used a variety of behavioural approaches combined with systemic pharmacological treatments (propranolol, rapamycin, anisomycin, cycloheximide) after reactivation of fear memories. Interestingly, none of the treatments induced a significant decrease of freezing responses during subsequent retrieval tests. Authors strengthened their null results by using Bayesian statistics, confirming the absence of drug-induced amnesia.

Overall, the study is really interesting. Experiments and analyses are very well designed and bring some important findings to the debated topic of post-retrieval amnesia and its clinical relevance.

We are grateful that the reviewer appreciates our work and recognizes the general importance of our null findings. We genuinely thank them for the time that they took to evaluate our paper in detail and hope to provide some clarifications in our responses below.

I have nevertheless several comments for the authors to consider.

-Despite being very detailed, the authors should clarify and uniformize their Methods section and Supplemental information (e.g. number of CS, contexts used...) to improve the understanding of the different approaches. Similarly, methods for the reinstatement protocol (Exp 2) are missing.

We understand that the information in the main text is quite dense, but we explicitly chose to focus on the central message here, i.e., that we applied standard procedures that should have allowed us to detect amnestic effects in consideration of most of the published literature. In addition, the crucial overview of the number of training and test trials, as well as the context that was used for each session is depicted in Fig. 1-3, immediately above the results of the respective experiments.

In the Supplement, we provide a more extensive (and repetitive) report of the experimental procedures. The idea is that the reader can find the most important information in the main text, and all additional details in the Supplement (or in our preregistrations on the Open Science Framework: https://osf.io/j5dgx ). For example, in the main text, it is mentioned that reinstatement in Experiment 2 consisted of two US presentations in context A, one day before the final test (see p. 6 and Fig. 1C). The Supplement (p. 1) adds that the reinstatement session started with 300 s of acclimation, followed by the first US and 180 s later by the second US, and that the rat was removed from the context 120 s after last US onset. For all phases of Experiment 2, the US was a 0.7-mA, 1-s shock.

• In exp 5, tests 1 and 2 are supposed to have 12 CS each. However, only 8 dots are represented on the graph. Did the authors average some freezing values after the initial 4 first CS presentations?

Thank you for noticing this. We did not average freezing values, but just did not measure freezing on all trials, as we were not specifically interested in the concrete freezing levels on each trial, but rather in the overall extinction curve. As mentioned in the legend of Fig. 2, freezing during CS5-7-9-11 was not measured (and hence also not shown). In other words, the 8 dots on the graph represent CS1-2-3-4-6-8-10-12.

-There is an obvious difference in baseline freezing response before the test in Exp 7 (Figure 5A-B). Discussion of these differences is an important point and was thoroughly discussed by the authors in the Supplement.

Thank you for pointing this out.

-Ln 384-387: "... additional Bayesian analyses were carried out that collectively suggested substantial evidence for the absence of an amnestic effect". Despite the "substantial effect" given by the meta-analysis, I am a bit confused by the meaning of an "anecdotal evidence against drug < control" reported in half of the experiments. How do the authors interpret these results?

In short, Bayesian analyses provide evidence that is categorized starting from ‘no evidence’, to ‘anecdotal’, ‘substantial’, ‘strong’, etc. depending on the obtained Bayes factor. Grouping studies with anecdotal and substantial evidence in a meta-analysis can result in overall substantial evidence, which is what we observed here.

Addressing this remark in more detail, we want to point out that the use of frequentist analyses (ANOVAs and t-tests) allowed us to conclude that we could not replicate the amnestic effects of previously published studies – we did not obtain a statistically significant amnestic effect although we had sufficient power to detect the effect sizes that had been previously reported. However, those analyses do not permit us to make inferences about the evidence against an amnestic effect. Bayesian analyses, on the other hand, do allow us to quantify the obtained evidence against an amnestic effect (i.e., the null hypothesis) for each single experiment or by combining the results of several studies. When a single study suggests only anecdotal evidence against an amnestic effect, this implies that we cannot conclude based on that study alone that we have proper evidence for the absence of an effect. Rather, we can only conclude that we have no evidence for the presence of an amnestic effect and weak (‘anecdotal’) evidence for its absence. However, a collective analysis of our studies does lead to the conclusion of substantial evidence for the absence of an amnestic effect overall.

-The effect of cycloheximide on memory consolidation is indeed unexpected. Even if beyond the scope of the current study, what is the authors' hypothesis to explain that cycloheximide in their conditions induced a pro-mnesic effects on the consolidation of fear memories but altered the consolidation of extinction?

As indicated by the reviewer, this is beyond the scope of the current study. We have no additional data on this effect and can only guess at its meaning. Also note that the effect was rather small and disappeared quickly during the test under extinction.

One purely speculative hypothesis is that the injection with cycloheximide was more arousing than the vehicle injection, either due to sensations caused by the substance during injection or due to the rapidly emerging malaise it induced (or a combination of both), which we have documented in the Supplement (p. 5).

In line with work by McGaugh, Roozendaal and colleagues, such arousal around the time of training could, in theory, enhance consolidation of a fearful memory, and thus explain greater fear memory during test (see e.g., Roozendaal & McGaugh (2011), https://doi.org/10.1037/a0026187 ). Then again, a similar argument could be made for improved consolidation of the extinction memory (de Quervain et al. (2019), https://doi.org/10.1007/s00213-018-5116-0 ), which we did not observe. One could suggest that – assuming that we have observed ‘true’ effects here – the arousal component had the upper hand during the consolidation of the fear memory, while the protein synthesis inhibition overruled such effects during consolidation of the extinction memory. As this is all highly speculative, we prefer to not add this to the Discussion.

-Cycloheximide seemed to induced post reconsolidation amnesia of fear memory after extinction training (Exp 8, Fig 3G) but not after single CS reactivation. Can the authors please develop this point? Is it possible that several presentations of the CS is required to destabilise the initial memory trace?

First of all, it is important to emphasize that cycloheximide-treated rats in Experiment 8 (Fig. 3G) froze more during the CSs of Test 2 than control animals, arguing against a drug-induced reconsolidation blockade of the initial fear memory. Furthermore, the obvious within-session extinction during Test 1 in Experiment 8 suggests that it did not function as a typical reactivation-without-extinction session (Merlo et al. (2014), https://doi.org/10.1523/JNEUROSCI.4001-13.2014 ).

In light of the current literature, reactivation with a single CS is by far the most common way to destabilize a memory trace that was formed with one or three CS-US pairings. As mentioned in our paper, this should provide an appropriate degree of prediction error for the memory to become malleable (p. 12).

Theoretically, it is indeed possible that more than one (e.g., two) CS presentations could allow for destabilization of the memory trace, although others who have used reactivation sessions with more than one CS presentation did not find the amnestic effects that they did observe with a single CS (Merlo et al. (2014); Sevenster et al. (2014), https://doi.org/10.1101/lm.035493.114 ).

Reviewer #3:

Luyten et al's study examines the phenomenon of drug-induced post-retrieval amnesia for auditory fear memories in rats, and report that after several experiments using Propranolol, Rapamycin, Anisomycin or Cycloheximide that they essentially observe no disruption of reconsolidation, (i.e., no amnesia). This is a well-executed, written and meticulous study examining an important phenomenon. The author's lack of observing amnesia using these "reconsolidation blockers" highlights an important fact that systemic administration of these drugs at the time of memory retrieval may not robustly influence reconsolidation processes despite what the existing literature may collectively indicate. The author's data clearly indicate this point and it is important the scientific community be made aware of these difficulties in blocking reconsolidation using systemic administration of these drugs.

We are thankful for these generous comments and value the reviewer’s thorough and thoughtful assessment of our work. We also appreciate the reviewer’s position that it is important to get this message across to the scientific community.

This group has previously published similar studies disputing similar phenomena. First highlighting a lack of amnesia following the reconsolidation-extinction paradigm and then more recently demonstrating a lack of amnesia attempting to block the reconsolidation of context fear memories. This is now their third installment focusing on Cued fear memories. Certainly, these findings are important, but arguably the novelty of such findings may be diminished a bit.

We appreciate that the reviewer is well aware of some of our other work in this domain that supports a more general and widespread reproducibility crisis in this field.

Regarding the novelty, one key point to stress here, which is also articulated in the paper (p. 3, 13), is that the current rodent findings (which we could not replicate) are the ones that provide the most direct basis for the clinical translations that have been proposed (e.g., by giving patients a propranolol pill after retrieval of a traumatic or phobic memory, see e.g., https://kindtclinics.com/en/ or Kindt & van Emmerik (2016), https://doi.org/10.1177/2045125316644541 ), and are therefore critical in their own right, not only because of their fundamental scientific relevance, but certainly also in light of their clinical reach.

In one of the "control" experiments where the experimenters administer anisomycin immediately post training, they observe a paradoxical result - they observe memory strengthening instead of the expected blockade of consolidation and amnesia. This result highlights a number of things to consider when we interpret these overall results. For one protein synthesis inhibitors(PSIs) are toxic and when administered systemically usually result in inducing the animals to have diarrhea and generally just makes them sick. This of course will make the animals stressed and agitated and result in increasing their stress and likely amygdala activity. All of this could likely be the reason why the animals exhibited memory strengthening or no impairment in consolidation even with a PSI on board. See PMCID: PMC7147976. Figure 6. In this study, they could rescue the impairment of PSI on consolidation by increasing BLA principal neuron firing. Thus an important take away is something like this could easily be happening in the reconsolidation experiments - that there is no blockade because the animals are stressed either due to PSI on board or because some issues with experimenter/animal interactions, etc lead to higher BLA neural activity and rescue of the reconsolidation process.

We agree that (systemic) protein synthesis inhibitors can induce signs of sickness in the animals (particularly in the first hours after injection) and have provided a detailed description of our relevant observations in the Supplement (p. 4-5). The reviewer is completely correct in stating that this may cause some amygdala activation which could interfere with the amnestic effects that we expected to see, as described in the paper by Shrestha, Ayata et al. (2020), and in line with our reply to Reviewer #2’s first comment regarding our cycloheximide experiment. Yet, effective induction of amnesia with these drugs has repeatedly been reported in the literature.

Nevertheless, although relevant, the current remark has relatively little implications for our findings. In the large majority of our experiments, we did not use these toxic protein synthesis inhibitors (PSIs) (such as cycloheximide and anisomycin), but drugs that have generally been administered systemically throughout the literature (with successful amnestic effects). Furthermore, in the experiments where we did administer systemic cycloheximide or anisomycin, we observed no differences compared to vehicle-treated rats in contextual freezing (e.g., 9% on average in Experiment 7) immediately prior to the crucial test tones (Test 1, 24h after injection) – which argues against high levels of stress or agitation. Moreover, a blinded experimenter could not tell the difference between PSI-treated versus vehicle-treated animals while handling the animals for the test session, and observed no behavioral abnormalities, nor signs of pain or distress, as mentioned in the Supplement. We acknowledge that these experimenter observations may not entirely reflect what is happening in the animals’ amygdala, but they at least go against the notion that PSI-treated animals would be too sick to be tested properly.

I don't think the authors go far enough articulating the important differences between systemic and intra-cranial administration of these drugs. Time is a potential factor. Immediate administration of the drug at high concentration in the target brain region (BLA) versus many minutes until the drug gets to the target region with uncertain concentration levels that may not mirror levels reached with intracranial administration. It's unfortunate the authors were not able to include intra-BLA administration of these drugs in this study. I do not necessarily expect them to do such experiments, since they have already done so much and it is not clear the laboratory has the appropriate expertise to conduct such experiments, but this comparison would be helpful.

We fully agree that our results do not provide any information about the replicability of intracranial administration of drugs to induce post-retrieval amnesia of cued fear memories. We had already clearly acknowledged this in the first version of the paper (p. 11), but have now added an extra section to the Discussion (p. 13) to highlight this point in the new version posted on BioRxiv (Version 2). Notwithstanding the expertise of our laboratory to carry out intracranial infusions, we agree with the reviewer that such experiments are beyond the scope of this article.

It is, however, noteworthy that the drugs that we used in 6 experiments did not necessarily rely on intracranial administration in prior successful studies. Rapamycin, for example, has generally been used systemically (not intracranially). Propranolol has been used either systemically or intracranially in rodents and always systemically in human subjects (healthy and patients). Bearing in mind the timing issue that was raised by the reviewer, we moreover included an experiment with pre-reactivation administration of propranolol (Experiment 4), where the drug was injected 5-8 minutes before the rats heard the reactivation tone.

I think it is important that the authors make some statement of training conditions on cannulated versus cannulated rats. For example, every animal in Nader's 2000 study was bilaterally cannulated targeting the BLA. In contrast every animal in this study underwent no such surgery. I think this is relevant. In my experience non cannulated animals are a bit smarter than cannulated animals and the training conditions across these two differing groups may not equate to the same level of learning. And of course, differences in learning levels can lead to differences in the ability of the retrieved memory to destabilize.

Thank you for pointing this out. We are aware that there may be differences between operated and non-operated animals and already briefly discussed this matter in the Supplement (p. 4). We have now also added this issue to the Discussion in the new section (p. 13) where we emphasize the differences between systemic and intracranial drug administration in relation to the previous comment.

That being said, the comment regarding (non-)cannulated rats only really applies to Experiment 7 where we tested the effects of systemic anisomycin or cycloheximide. Prior cued fear conditioning studies indeed used intracranial administration of these drugs. The argument does not hold for Experiments 1-6, as systemic propranolol and rapamycin have repeatedly been reported to have amnestic effects in non-operated rats, with procedures identical to or closely resembling ours.

The authors mention possibly examining markers of memory destabilization. GluR1 phosphorylation, Glur2 surface levels, protein degradation/ubiquitination have all been used to assess if destabilization has occurred. I do not fully agree with their reasons for not performing such experiments. They could examine some or one of these phenomena across differing training conditions between retrieval, no-retrieval animals. This likely could be informative. However, the authors may not possess the necessary expertise to conduct such experiments, so I'm not stating these experiments need to be completed, but certainly the study could be strengthened with such data.

We agree that including yet more control experiments, using different experimental approaches could further strengthen the study. Nevertheless, the main conclusion of our paper – i.e., reconsolidation blockade using systemic administration of several drugs is considerably more difficult to reproduce than what the literature collectively indicates – is strongly and sufficiently supported by the data that we already report here. Overall, we believe that our conclusion does not require such additional controls. Moreover, even though the comparisons suggested by the reviewer could indeed be scientifically interesting, it is still unclear whether such experiments would provide sufficiently clear cut-offs as to which experimental condition would then allow for adequate memory destabilization and interference.

Experiment 3E - Propranolol without reactivation. I don't see any data for this on the graphs. Am I missing something?

Our apologies for the confusion. The legend shown next to Fig. 1F applies to all panels of Fig. 1, but only Experiment 1 (shown in Fig. 1A-B) contained a no-reactivation group as an additional control. Experiment 3 (shown in Fig. 1E-F) did not. We have moved the legend to the bottom of Fig. 1 to clarify this.

The authors should probably cite this paper too, PMID: 21688892. The authors in this study find no evidence that propranolol inhibits cued fear memory reconsolidation.

Thank you for bringing this to our attention. We were aware of this paper, but it had slipped through the cracks. We have cited it in the new version of the paper (p. 11).

Reviewer #3:

Luyten et al's study examines the phenomenon of drug-induced post-retrieval amnesia for auditory fear memories in rats, and report that after several experiments using Propranolol, Rapamycin, Anisomycin or Cycloheximide that they essentially observe no disruption of reconsolidation, (i.e., no amnesia). This is a well-executed, written and meticulous study examining an important phenomenon. The author's lack of observing amnesia using these "reconsolidation blockers" highlights an important fact that systemic administration of these drugs at the time of memory retrieval may not robustly influence reconsolidation processes despite what the existing literature may collectively indicate. The author's data clearly indicate this point and it is important the scientific community be made aware of these difficulties in blocking reconsolidation using systemic administration of these drugs.

This group has previously published similar studies disputing similar phenomena. First highlighting a lack of amnesia following the reconsolidation-extinction paradigm and then more recently demonstrating a lack of amnesia attempting to block the reconsolidation of context fear memories. This is now their third installment focusing on Cued fear memories. Certainly, these findings are important, but arguably the novelty of such findings may be diminished a bit. In one of the "control" experiments where the experimenters administer anisomycin immediately post training, they observe a paradoxical result - they observe memory strengthening instead of the expected blockade of consolidation and amnesia. This result highlights a number of things to consider when we interpret these overall results. For one protein synthesis inhibitors(PSIs) are toxic and when administered systemically usually result in inducing the animals to have diarrhea and generally just makes them sick. This of course will make the animals stressed and agitated and result in increasing their stress and likely amygdala activity. All of this could likely be the reason why the animals exhibited memory strengthening or no impairment in consolidation even with a PSI on board. See PMCID: PMC7147976. Figure 6. In this study, they could rescue the impairment of PSI on consolidation by increasing BLA principal neuron firing. Thus an important take away is something like this could easily be happening in the reconsolidation experiments - that there is no blockade because the animals are stressed either due to PSI on board or because some issues with experimenter/animal interactions, etc lead to higher BLA neural activity and rescue of the reconsolidation process.

I don't think the authors go far enough articulating the important differences between systemic and intra-cranial administration of these drugs. Time is a potential factor. Immediate administration of the drug at high concentration in the target brain region (BLA) versus many minutes until the drug gets to the target region with uncertain concentration levels that may not mirror levels reached with intracranial administration. It's unfortunate the authors were not able to include intra-BLA administration of these drugs in this study. I do not necessarily expect them to do such experiments, since they have already done so much and it is not clear the laboratory has the appropriate expertise to conduct such experiments, but this comparison would be helpful.

I think it is important that the authors make some statement of training conditions on cannulated versus cannulated rats. For example, every animal in Nader's 2000 study was bilaterally cannulated targeting the BLA. In contrast every animal in this study underwent no such surgery. I think this is relevant. In my experience non cannulated animals are a bit smarter than cannulated animals and the training conditions across these two differing groups may not equate to the same level of learning. And of course, differences in learning levels can lead to differences in the ability of the retrieved memory to destabilize. The authors mention possibly examining markers of memory destabilization. GluR1 phosphorylation, Glur2 surface levels, protein degradation/ubiquitination have all been used to assess if destabilization has occurred. I do not fully agree with their reasons for not performing such experiments. They could examine some or one of these phenomena across differing training conditions between retrieval, no-retrieval animals. This likely could be informative. However, the authors may not possess the necessary expertise to conduct such experiments, so I'm not stating these experiments need to be completed, but certainly the study could be strengthened with such data.

Experiment 3E - Propranolol without reactivation. I don't see any data for this on the graphs. Am I missing something?

The authors should probably cite this paper too, PMID: 21688892. The authors in this study find no evidence that propranolol inhibits cued fear memory reconsolidation.

I love these little moments of meaning-making in the play. These "characters" are clearly interested in the author's intentions, and the consequences of the play, but the characters who have these moments are typically women (Sabina), and it's pretty poignant that this excellent little analysis comes from Ivy, a young woman of color who isn't really even in the play, when you might expect it to come from Mr. Fitzpatrick, or Mrs. and Mr. Antrobus

Sabina takes the role of God by refusing to act the next scene and thus diverting the flow of the play-within-a-play. As a result, we recognize that the ground she stands on is merely a setting for theatrics, the "world as a stage," and those theatrics can be adjusted according to actor preference. In addition, Sabina's call out to the audience draws attention to her identity as an actor. She expresses concern for having to act in a manner that would arouse negative emotions for a personal guest in the audience. We are conscious of her feelings as an actor and not her feelings as a character; we confront the fact that Sabina is a conscious actor on the stage.

Consider the thematic contrast created in this description: stern civil war soldier and friendly woodworking show host. You'll note that this is carried through the fundamental thesis of Saunders' work below that it balances a "cool rigor" with "enormous compassion."

I like the concept of the piece. But, I just feel as though the end was presented in a condescending tone and the last paragraph lacked the intensity that it could have had.

Serious "Religion for the Nonreligious" vibes (Tim Urban.) Gotta have clear perception -- addendum here is not just of bad, but of good too.

people try to force everyone to do anything what ever one person wants to do if they are with an other or others they will try to get them to do what they want to do

Victor doesn't associate body with soul. Her body doesn't make up who she is/was. It's interesting that he thought he could bring a body back to working order and not think about the soul that would come back with it, when he created the Creature. Also it's interesting that later in the text when Victor is on his death bed he seems to have a dualist perspective. He mentions getting to see his loved ones again (including Elizabeth), so perhaps he seems to have less attachment to the actual body. He kinda just leaves it there... right?

In many of my high school history textbooks the discussion on slavery was "it happened, it was bad, it's over," but the fact is that it isn't over, it's just produced in a different way now. No country wants to look like the bad guy in a given situation, so often times they think a short handed apology will solve the problems and they can move on. Acknowledge it happened, and that's good enough is how they see it. However, they fail to really reach deeper and make connections to today's society and how the past affects the present and the future. I understand the skepticism on if the promised land can even exist because America still hasn't taken full responsibility for the history of the nation, the history the nation is built on. There is no yearning to explore deeper meaning and deep emotion surrounding slavery, and thus there is no desire to teach about how it impacted the society we live in and how it is still present in society today.

This reminds me of a talk I attended at Playfair Library by Alistair Hudson, director of Middlesbrough Institute of Modern Art, who was promoting his idea of the 'useful museum' as 'art school for everyone'. I was impressed by his enthusiastic outlook towards full inclusivity and enjoy this quote from the following article: https://civicroleartsinquiry.gulbenkian.org.uk/resources/mima-middlesbrough-institute-of-modern-art

“all people learn together this role that art has in society of transformation. That includes cooking, gardening, politics, housing, healthcare, everything. Once you start to get that message embedded into people’s minds, that’s when you stop them saying we don’t need an art gallery any more, or we don’t need art, or we don’t need culture. Because actually you don’t choose whether you have culture or not, it’s just a question of what kind of culture you have.”

Yes! Just the justification for my gap quarter that I was looking for ;)

I'm not sure how ethnic studies can be standardized. It's almost as if you're saying, " yeah, we can just teach them the bare minimum of what they need to know about other people's cultures and generalize the critical analysis of the disparities that exist".

A lot of misogyny is radiating from these lines. Victor is implying that his female creation might be so ugly that even his male creation will be offended by her existence one he sees her. But on the other hand, what if his creation isn't her type and just abandon's him? It's interesting to see how much thought Victor puts in when it comes to making a female creation...I thought he was trying to create a new species?

it affects the reader's minds and planting the idea of this parenting into their heads, so no not everyone has profited. At the end, it's the kids that suffer

Some Egyptian parents can be like this, for example, they might force their children to enter a major that they don't like just because they believe it's the better choice. but, what about the children's preferences. Why are they disregarded? children tend to become more encouraged to work if they're studying what they love

without the safety precautions we are suggested to follow then the US cases will just continue to increase

I'm still confused. Does mobile-first indexing just mean that it gets indexed before the desktop version? Or does it mean that it indexes mobile first despite the mobile interface? This didn't really clear up what "mobile-frist indexing" means

Yes!! It's been a mission to continue to challenge those who say "a child's brain is like a sponge just soaking up everything around them" - they are SO much more than that! They are not passively experiencing or observing the world around them. They are active in their exploration and have opinions about their experiences!

Incredible! If we just give them a safe place to talk and discuss. This would be one of those "scary" topics that in the past we would have avoided.

Absolutely love this! It really shows that children are smarter than believing when a teacher says, "just a few minutes left" or in a minute. They wish for concrete knowledge of time. They wish for honesty and specific clarification.

According to the secondary source for Unit 2, one of the reasons Zeus was so successful was because he swallowed Metis and she lived inside him, giving him her wisdom. It's definitely problematic of him to devour his wife, but it's nice to know the Ancient Greeks recognized the capability of women. Just goes to show that you can't paint all pre-modern civilizations with the same misogynistic brush!

Well said

Good answer

She keeps making assumptions that make Qanon look like rationalists.

Just because her and her peers require outside noise to make their ego stay afloat, doesn't mean it's a lead!

Its a dude that wants to be left alone. Not harassed

This made me laugh because I feel like Collins is just making fun of self-righteous of Christianity with such a ridiculous statement like this. If someone is concerned with the whole of their stockings being “spotless”, they clearly care a LOT about their appearance (which shows them as somewhat vain and maybe hypocritical in the case of the comparison to Christianity because the stockings are an article of clothing that no one really pays attention to). Also, Miss Clack’s comment about stockings “ready to put on a moment’s notice” suggests that it’s both expected and normal to take off your faith, so faith is just something that you put on to maybe look nice or be presentable in front of others?

This is pretty bold coming from a man who just pinned Sergeant Cuff to the wall in a moment of anger a few pages ago. I think there are a lot of behaviors that Betteredge identifies as feminine that he himself exhibits throughout the novel (this being one example). At several points in the novel he comments on the nosiness of the women servants, but he himself is snooping around and gossiping for his entire narrative. Additionally, in the power dynamics of the house, he is in a subservient position to "his Lady" and Rachel who, in the absence of a man in the family, are the masters of the house.

Here we can clearly observe Betteredge's double standard. Just a few lines above, he ascribes a contradictory behavior to women, and now he exhibits it gloriously himself. It's very apparent throughout the story that Betteredge cares a great deal about whether someone is "a Dustman or a Duke." He never fails to talk highly about people of rank, and scarcely about people of no particular hereditary rank. It even manifests itself in the way he values himself (albeit with some insecurity), as opposed to his servants. But here, he so blatantly declares himself free of any prejudicial behavior... This is one of various instances in which his hypocrisy is showing face.

When I'm searching, I much prefer to land on EXACTLY what I was looking for. If Google prefers one "comprehensive page" won't that encourage "bouncing" if I don't want to scroll through an entire page just to see if it's what I'm looking for? This feels like undercutting the user experience, even if this seems like a "white hat" method that's mostly harmless.

In my experience, the defense of most people who argue for the continuation of "he" and "she" pronouns is convention, like stated here. But, I have found that they really don't care about convention as much as they care about forcing people into boxes, and then care when people don't fit these boxes. A big problem with the whole "I identify as..." is that people hear the term "identify" and feel that these people are choosing to identify as something other than a "she" or a "he", when, really, that's just who they are. Language is a way for us to communicate parts of ourselves to others, to be known, and now is a better time than ever to explore the ways in which language fails us in terms of gender identity, especially when that gender identity falls outside our scope of language. I am a huge supporter of whatever the hell people want to call themselves. One, I am so happy that they feel comfortable enough to defy societal norms, and two it's really nobody's business except theirs.

Most people use some form of the gender neutral "they" in their typical speech, as I just did. When you don't know the specific gender of the person you are discussing, you use a gender neutral pronoun, like "you" or "they." It's not consciously agender, but it's so ingrained in our language that it's natural without thinking about it. The author makes a great point, that although many people consider the use of those pronouns as wrong when they are actively thinking about it, it actually isn’t noticeable in regular use. There is no real rule against it. Even if there were, rules of language change all the time. Additionally, gender neutral terms have been in use for centuries, really since the beginning of English. Some of them have fallen out of use because they were too obtrusive in text, but some, like “they,” have stuck around because they fit so effortlessly into our natural dialogue.

Pronouns, like other grammar tools, are very important. We've been using them for so long that they have become a serious part in our conversations about people other than ourselves. I wouldn't necessarily say that they've just suddenly become cool. I would say our interest has recently increased towards the subject when it comes to using the correct pronoun, as it shows our understanding of the identity of the person we are speaking about. Because someone's identity is important, their pronoun should be just as important. I think looking at pronouns as simply just a "part of speech" as Baron says is only looking at the basic layer of what a pronoun means. Messing up someone's pronoun can spark some conflicts, depending on the individual mentioned.

But when you look at "they" (singular) versus they (plural), their doesn't seem to be an event where conflict would arise. I may have used they in reference to a singular being before, but I don't think there has been a time where it's crossed my mind to contemplate if what i'm saying is weird. Then again, I can't really think of many clear examples where I've used they as singular. I typically use he or she, since I tend to know the correct pronoun for the identity of the person i'm referring too. I guess myself, like others, may not see the distinction between the two as such an important topic, so we overlook it. But, since we've discussed how important semicolons and oxford commas are, maybe I'll start paying attention a little more.

She is signaling acceptance of sorts, but why didn't she say anything to him?

So, If Gurdon is able to get the epithelium nuclei into an egg and it grows, it will show that it is not the DNA that changes, just how it's expressed

I think it's bigger than just unempowerment of women in employment. if I'm talking about Egypt for instance in specific, sometimes there are certain job criteria that require specific category of women which might cause discrimination of others.

I agree, and it's not just due to time constraints but also due to trial and error. Often when children try something on their own in their quest for autonomy, they do it in a way that is different than how we would do. Teachers have a tendency to want to correct the children and do it for them... this also strips their autonomy.

So this is one of my favorite speeches the king makes. I mean it's just to the audience so I guess it's not really a speech? anyway, I love this because henry is pouring his heart out to the audience. He is telling us everything that is on his mind and how conflicted he is which shows how human he is! This entire time everyone has been putting him on such a high pedestal and now all the sudden he brings himself down! We can see his pain in this speech and really the only conclusion we come to is that the king will never sleep as soundly as a peasant.

So I always thought it was interesting he said "guest" I look at Henry as a good guy and a hero, but when he said all that stuff about letting the real horrors of war show it's not under his control because then his soldiers will just be running wild and that's hard to keep them under control. But I really think that was all talk. Henry was trying to win the french over with words since they were loosing with soldiers. LETS NOT FORGET HIS MEN STILL HAVE BLOODY FLUX AND THAT WAS HONESTLY HELL. And historically, this was a 5 day truce, if the Dauphin didn't send help with in the 5 days, the French would surrender. this was not a victory. Henry lost about 1500 to flux alone and I think around 2000 were sent home??? Henry is a good guy and he'd doing anything he can to win, and taking a neutral stance you can notice how anyone might say something like that during that time. It's not right but I get it. so saying guest makes it less mean? you can almost tell henry is tired and wants his men to rest.

I like to think the Canterbury and Ely are not just listing what they love about the King, and putting him on such a high pedestal right off the bat, but listing what could be an obstacle to get the king on their side. Also we don't have much Idea what they're talking about, only some clues so it's fun to think that they need something from Henry.

It's kind of ironic that the current king of France holds his throne due to female succession, but the french are denying Henry's legitimacy due to only being related through a female. This seems to be very common for the time due to conflicts that have ensued. The Wars on Roses is a good example of how English royalty was constantly in conflict and just about anybody wanted to claim the crown. This conflict seems to have been brought to France during the 100 years war.

I really don't like the author apologizing to me. It sounds like a mopy ex who is trying to talk to you about their sob story. But with this in mind, it sounds like the Ribbon is a metaphorical thing that people wear. People who have shame specifically, like people who were taken advantage of at a young age sexually or through other malicious means. Not being able to cope or talk to someone about it, which she was clearly afraid of. Led to the ribbon staying on late in life. It made her rather sexualized and at the same time shameless in a sense. Sexuality liberated her but as from what she knew growing up, this further sent her into a spiral of shame until she waited until decades after being with this man to reveal her ultimate secret and sin. She herself is the antagonist and the protagonist as the only person in the end beginning of the conflict and the end is her and her fear of being accepted or being rejected. To her living in a world who truly embodies what she thinks is okay is somewhat scary and unsettling, and by hiding in conformity finds safety. It was honestly a frustrating read but this and how it dodged action by throwing in odd wives tales really solidified this point too. That our main character is probably alive and doing the context of living in the story between 1970 and 1980 I'd say. Because there is no mention of men and war, but also because it references parents marrying even young for their generation. So when I think of the green ribbon I think of like an old Victorian style ribbon that is old and almost out of date, not something slick and shinny but something that is fashionably strange and in many ways objectified like a gift or a present. In this context it's the opposite. You are the present that's opened decades later and frankly, emotionally it's the same thing. You wait too long to deal with your own emotional turmoil and it just doesn't feel the same when you waited so long to find your solution.

This writing is honestly getting a little frustrating. "They do not have to make a cut..." and then they make the cut. It's just frustrating because it's written in past tense as if she is trying to tell us what happened but as if it's happening at the same time and the flow of the story just isn't right - right now for this to sound good, and this is a trend of the overall story that I can't get past.

Do not just be nice. Give constructive criticism.

This is an example of intertextuality, a connection/reference to the ancient story of Adam and Eve. Although "Skywoman Falling" is a contemporary text and does not influence the story of "Adam and Eve," this quote affects the way the reader may view the other story now. From an Indigenous perspective, Eve's exile is directly related to breaking a relationship to the land rather than to God (as many others would see). Another perspective is developed for the reader through this use of intertextuality.

I feel like this is a reoccurring theme in all the texts and vides we are viewing. It's sad that some people excited to see their own culture portrayed in film and books just because it's not like the "normal" books they were exposed to.

It's interesting that the child seems passive and non-reactive to what's happening to her, but Margret knows what's wrong, and feels angry for her. But it seems like a child should know when they are being treated wrong--like being spat at. Was the child just used to it? Or forced to stay quiet?

This line and the next one were particularly stood out to me as Harper's house is where the play begins and ends. I think it's interesting to think about how this space changes throughout the course of the play especially in regards to mood and time. How many years have passed since the audience was last in this house and how has it changed, is the mood different now that the audience has obtained more information about this world? Was it ever really a place of safety? One might say that in the beginning of the play, when the world seemed 'normal,' it might have appeared to be a place of safety, but once the reader (and Joan) witnessed more of the world, the concept of safety and the protection that one might feel from being surrounded by these four walls, completely dissipates. It seems as if this particular space provides the characters with a false sense of safety because they feel as though, the space is purely just a house, unaware that anyone might be able to see in and know more. Lastly, another thing regarding time, obviously years have past, but we also are seeing this house in the daytime now rather than at night like in the beginning and for me, that begs more questions about this world. Does daytime bring more of a sense of safety, how does it change the mood of the world? In other worlds, mood changes around nighttime, darkness brings other things to light. With time changes (whether that be years, or simply the contrast between day and night- both of which we witness) come mood changes and I think that is true and intentional for this world.

Imagine looking at this moment without context, it could be just a reflective thought said in a solemn tone; yet following that prisoner marching scene, it is such a blood-curdling line that it completely disrupts the audience’s expectation. The time should be staccato, as if a silent shriek instantaneouly strikes in a vacuum. I imagine the climate to be paralysing cold, in a way that parallels to Todd’s indifference to the burning of bodies in this dystopian realm. The mood is neither restrained or violent, but restrainedly violent, and sepulchral, as if someone suddenly bursts out laughing at a funeral in the most unsuitable manner, followed by awkward uneasiness.

It's interesting how in the midst of analyzing the two statue in depth, the authors just allow the civic memorial to stand on its own without much analysis.Only when they had everything set up did they introduce the memorial. That ending of "Today Vinegar Hill is just a memory" has more emotionally impact when the readers know what had happened rather than not understanding the full meaning from the start.

This has happened to me various of times, and it's so embarrassing

Carson points out how even in Ancient Times, the language barrier was a gigantic issue in public affairs. Even though Joan could have been innocent in what she was saying, the translators for Latin bended her words in translation to make it seem like she was guilty which evidently led to her death. Not only does it show an injustice in the system, it shows how it alters the facts and what was actually said which can throw historians off in discovering the actual trial facts. It's honestly quite amazing how different regions of the world can come up with their own languages and come together to figure out how to translate each and what word is associated with which in the other. With the fact that there is deliberate falsifications with translation, will this make historians question the credibility of many primary sources?

I see what Carson is getting at here, but honestly the example confuses me a bit. The word cliche is used in its original French form because it's easier to understand the meaning that way, perhaps, but it's not like the word "cliche" is completely untranslatable. The word itself has no direct translation, but if you look up synonyms for the word, you find things like "commonplace" or "overworked phrase". You can still describe what "cliche" is in English or define the word. So I feel that this example is a bit different than the other that she uses for metaphysical silence. MOLY is literally untranslatable because we are not given a translation. This feels like true metaphysical silence, because there is no meaning attached to the word whatsoever. We can read the sentence that it's in a million times and still come no closer to knowing what MOLY exactly is. Maybe this was Carson's point - or maybe I'm just not one hundred percent understanding what metaphysical silence is. Can you think of any other words in the English language that are "untranslatable" or that we use the original foreign term for?

This is just not true because if freedom means you can do anything then you will be able to do things that you are not supposed to do because it is breaking the law depending on what you are doing. There are laws that must be follow in order to use that freedom in a civil manner otherwise it's chaos and anarchy.

I’ve seen some version of this conversation happen more times than I can remember. And someone will always say ‘it’s because you’re too used to thinking in the old way, you just need to start thinking in hooks’.

But after seeing a lot of really bad hooks code, I’m starting to think it’s not that simple — that there’s something deeper going on.

It’s confusing enough, in fact, that the React team don’t trust you to write correct hooks code without using the special lint rules they devised.

But even that introduces new problems. The only difference between the app on the left and the one on the right is that on the right, I’m using a custom useLocalStorage hook instead of useState. It behaves equivalently. So why do I have this lint warning all of a sudden?

It turns out that certain built-in hooks have special treatment hard-coded in. So when you’re told that hooks are just functions, and you can do all the things you normally do with functions, it’s true… but there are some interesting caveats around that statement.

Functions have lots of interesting and useful properties. You can isolate them. You can compose them. You can memoize them. In other words, functional UI feels correct, and powerful, in a way that wasn’t true of whatever came before it. I think this is why people get quasi-religious about React. It’s not that it’s Just JavaScript. It’s that it’s Just Functions. It’s profound.

We should always be learning from the people around us. It relates to the concept of positioning yourself around people who are better than you so that you can improve yourself. Continuous learning is important especially lately!

There has been too much building up over many years of neglect. No rage, no voices being heard, just silence. At some point if things just keep building up with no change or anything, there will come a point where it just goes to hell.

The way that quote is set up seems a little awkward to me. The first thought from beck's article is set up with "she talks about" and then the quote is set up with "Beck states." It seems a little repetitive

THIS. It's hard to think these men are fighting for actual liberty when they are simultaneously fighting to keep others from it. Is it actually even liberty, or is it just a new form of patriarchal imprisonment?

jurassic world idea...to enthralled with the idea that we can do it that we didnt think about if we should

Could the "ego" in this sense be the "limited image" of a person?

• This is like the 'GRAVEN IMAGE' that was forbidden to describe Yahweh in [because it's too limited]

1. Like trying to experience a video through one frame!

It's remarkable to me that this sentence still applies how many hundreds of years later. It reminds me of how the law is oftentimes unjust, particularly in the case of the increased incarceration and conviction rates of minorities. Law doesn't reflect morality, it reflects the will of the elite.

1. I find it interesting that it's noted "that there is no gender-neutral term to convey the idea of a person who achieves gloire." (p.181) Great man is the highest level of success, so even people who don't identify with the male sex or gender want to reach that level but aren't able to. There should be a new word/s for a person reaching a high level of success created by the marginalized people! Then there will be great people other than just the cis-male.

2. I see that there's this double standard again for female artists. A soft touch to painting and an artificial/decorative style was seen as feminine. Where as a strong touch of and a natural/truthful style of painting was seen as masculine. But if you painted too soft your skills were completely dismissed and on the other hand if you had a "masculine" touch to painting it was praised. Labille-Guiard was labeled a man because of her painting style. Was this a way for artists and critics to say Labille-Guiard's skill is of a man's but she isn't a man so she'll never be a great artist/man?

This is my favorite thing about the study of history, because it isn't just about the past it's also largely about what that means for the present and future. Its hard to understand the world around us without first understanding how it got this way

Author Response

Reviewer #1:

Major comments:

1) The title and the conclusion that SON and SRRM2 form nuclear speckles are not supported by the data. The data show that SON and SRRM2 are necessary for nuclear speckle formation. They do not rule out that another factor is necessary, such as SRRM1, which interacts with SRRM2 and itself harbors an intrinsically-disordered domain. That is, the authors have not shown that SON and SRRM2 are also sufficient for nuclear speckle formation. Such a test is necessary to draw the strong conclusion the authors make, and precedence for such a test has been established in the study of Cajal bodies. Specifically, central factors to Cajal body formation were shown to nucleate Cajal body formation at a specific site in chromatin when such central factors were localized to that site. The authors either need to perform such a sufficiency experiment or moderate their conclusions (and title).

2) In principle, in the immunofluorescence studies, the disappearance of mAb SC35 signal on depletion of SRRM2 does not alone prove that SRRM2 is what is visualized by the mAb SC35 in such assays. Given that this paper seeks to establish rigorously that mAb SC35 marks nuclear speckles by recognition of SRRM2, given that SRSF7 is recognized by the antibody on blots, and given that SRSF2 has been traditionally presumed the target of mAb SC35 in nuclear speckles, the rigor of this study demands that SRFS7 and SRSF2 be visualized in cells in the presence of an SRRM2 truncation to rule out that either SRSF7 or SRSF2 phenocopy SRRM2 in this assay.

This is a valid concern and we have thought of the same principal that is if any strongly speckle-associated intrinsically disordered domain containing protein, such as SRRM1 or RBM25, two proteins that are also frequently used as NS markes, would have a similar impact on NS formation as SRRM2 has. To this end, we performed a co-depletion of SON and SRRM1 (shown in Supplementary Figure 10) in a cell line that has a TagGFP2 inserted into SRRM2 gene locus. As it can be seen from the imaging presented in this figure for 4 individual cells (but also more generally on 10 independent field imaged, (data not shown)) we did not score a reduction in the GFP intensity, or dissolution of the spherical bodies as is the case in SON-SRRM2 co-depleted cells. We observed the nuclear speckles have the round-up morphology, that is seen upon SON-KD, but are not dissolved shown with PNN staining and SRRM2-TagGFP signals. Moreover, we performed a co-depletion of RBM25 (another strongly NS-associated protein also used as a NS-marker) and SON which did not result in the dissolution of nuclear speckles (Supplementary Figure 10). Therefore, we have reached to the conclusion that SON and SRRM2 form nuclear speckles with the contribution of SON being more important for the formation and titled our study accordingly.

Traditionally, because of the Fu & Maniatis 1992 paper, as pointed out by the reviewer, it is assumed that SC-35 recognizes SRSF2 in immunofluorescence experiments and potentially multiple SR-proteins in immunoblots. The former point, to the best of our knowledge, has never really been proven in any type of rigorous experiment. Fu lab. has generated SRSF2 K/O mice, but never provided an immunofluorescence image that shows that SC-35 signal disappears in K/O cells.

Just to summarize our line of reasoning here:

1) We do an unbiased IP-MS experiment, which shows that SRRM2 is the top candidate protein, at least an order of magnitude away from any other protein in the dataset by any measure. This strongly suggest that SRRM2 is the primary target of this antibody, although doesn’t prove it due to technical reasons i.e. no input normalization, some proteins produce more ‘mass-specable’ peptides than others, and larger proteins tend to produce more peptides.

2) We carry out a biased screen of 12 SR-proteins and find that SRSF7 is strongly recognized by mAb SC-35

3) We do IP-western blotting experiments, which correct for input and are not affected by relative ‘mass-specable’ peptide issues or protein sizes, which reveal a strong enrichment of SRRM2 (>10% of input), some enrichment for SRSF7 (~2% of input) and no enrichment for SRSF2, SRSF1 or other proteins that we have tested.

4) Since the “35kDa” protein is so engrained with the history of this antibody and our results were most consistent with the idea that this protein is SRSF7 rather than anything else, we insert a degron tag to SRSF7. If the hypothesis is true, then we expect a shift of the SC-35 band, concomitant to the shift in SRSF7, which is indeed the case. This is not proof that SC-35 doesn’t recognize any other protein but it does provide very strong evidence (combined with the other two experiments) that the 35kDa band detected by SC-35 in immunoblots is in fact SRSF7.

5) We then show, by TagGFP2 insertion into the SRRM2 locus, that SC-35 mAb can recognize SRRM2 specifically on immunoblots, and furthermore truncations beyond a certain point completely eliminates this signal. We also show later that siRNA mediated KD of SRRM2 also leads to the elimination of the signal from immunoblots (Supplementary Figure 9).

6) Combining the results so far, we address the issue of immunofluorescence, i.e. which protein or proteins are responsible for this signal. We think there are two possible scenarios that could both be true based on the presented evidence so far:

a. This signal is mainly, if not entirely, originates from SRRM2. b. The signal is a combination of SRRM2, SRSF7 and/or other SR-proteins that the SC-35 might be cross-reacting.

7) We then take advantage of our cell lines with SRRM2 truncations. These truncated SRRM2 version are not recognized by SC-35 mAb on immunoblots, therefore it is reasonable to suspect that they will not be recognized by SC-35 mAb in immunofluorescence as well.

8) If scenario (b) is correct and nuclear speckles are still intact in these cells (which we show that they are indeed intact, judged by SON, RBM25 and SRRM1 stainings Fig. 3A-B), then we would expect either no change in SC-35 signal, or a somewhat reduced signal. We see a complete loss of signal.

9) Being extra careful with this result, we also mix the control cell line and SRRM2-truncated cells and image them side-by-side to address any issues related to imaging settings etc. There is no detectable SC-35 signal in truncated cells.

10) We also show that the 35kDa band is still unchanged in SRRM2 truncated cells (Figure 2E), showing that SRSF7 itself is not affected in these cells.

These results, combined together, show that SC-35 signal in immunofluorescence originates from SRRM2, and any other signal potentially contributed by other proteins are below the detection of immunofluorescence microscopy.

Reviewer #2:

This study reports important evidence that the widely-used SC-35 antibody primarily recognizes SRRM2 rather than the assumed SRSF2. The manuscript provides several lines of evidence supporting this conclusion, and the work has broad impact on the field of nuclear structure and function as this antibody is the most common marker for the major nuclear component, nuclear speckles.

The one concern with the manuscript is the interpretation of some of the previous literature and understanding in the field.

First, since the 1990s it has been widely known that the SC-35 mAb has very limited specificity for denatured proteins and was not suitable for immunoblots (see abcam page for ab11826). Indeed, the assumption has always been that it recognizes a folded epitope. Therefore, the use of western blots to conclude anything about the specificity of this antibody is inappropriate.

Secondly, it has also been previously documented that this antibody has cross-reactivity with SRSF7 (i.e. 9G8; Lynch and Maniatis Genes Dev 1996).

Third, most SR proteins are not abundantly observed in tryptic MS due to high cleavage of RS domains. This is particularly true of SRSF2, which has a highly "pure" RS domain (i.e. all RS repeats) that encompasses almost half of the total protein. SRRM2, on the other hand, has much more complex and degenerate RS domains that encompass a much smaller percentage of the total protein. SRRM2 is also 10x the size of SRSF2. Thus, given equal molar amounts of SRSF2 and SRRM2, one would expect at least 20x the number of peptides and much more complete coverage of SRRM2 vs. SRSF2. Therefore, while the subsequent immunoblot in Figure 1C is compelling evidence that SRRM2 is precipitated with the SC-35 antibody, while SRSF2 is not, the IP-MS data alone is not strong proof that the SC35 mAb primarily recognizes SRRM2 rather than SRSF2. The text should be revised accordingly.

Finally, the abstract implies that the demonstration of SON as a central component of speckles is new ("elusive core"). As appropriately referenced in the text, this is not the case, rather SON is often used as a marker for nuclear speckles, and SON has long been considered to be part of the core of speckles, as knock-down has been documented by several groups to disrupt speckles. The wording in the abstract should therefore be more parsimonious.

With all due respect to all previous researchers that have used mAb SC35 and published their results, we think that the specificity issue has become unnecessarily convoluted due to the initial inaccurate characterization. Abcam’s recommendations highlight the issue in an interesting way. In the old marketing images, abcam shows a single band in a total lysate prepared from HEK293 cells: https://www.abcam.com/ps/products/11/ab11826/reviews/images/ab11826_49518.jpg

However, producing such an image, in our experience as we have also reported in the manuscript, is only possible under non-ideal western-blotting conditions i.e. when the transfer is not adequate to reveal proteins with large molecular weights. Intriguingly, a customer (not us) complains about an improper WB result obtained with this antibody (with a 2-star rating):

https://www.abcam.com/sc35-antibody-sc-35-nuclear-speckle-marker-ab11826/reviews/68414?productWallTab=ShowAll

It looks like an unexplainable high-molecular smear without the information that we provide in our manuscript, but in light of it, it’s clear that protein stained here is SRRM2.

In our experience the antibody works perfectly fine for western blotting, and very specifically and robustly reveals SRRM2 at ~300kDa, as long as the immunoblotting conditions are optimized for large proteins. We also show that bulk of the signal around 35kDa originates from SRSF7, however as indicated by the other reviewer’s comments, and also previous research, the antibody probably cross-reacts with other proteins as well with varying degree.

In this sense, the antibody can be used for immunoblotting, but pretty much any result obtained from such an experiment must be verified with an independent antibody or independent methods, which we did in this manuscript.

The SC35 mAb is actually suitable for western blotting if the gel running and transfer conditions are carefully performed to have SRRM2: a) enter the gel and b) transferred properly to the membrane. Under conditions where SRRM2 is just not entering the gel (due to high percentage gels, or gels with too much bis-acrylamide), or doesn’t get transferred to a membrane (non-ideal buffer conditions, protein stuck in stacking part and cut away etc.), we have seen the unspecific bands, but we had to use the most sensitive detection reagents at hand to see those, so they are rather weak. We have provided a detailed explanation to what these conditions are in the methods section of our manuscript, but briefly: running the gel slowly allowing the protein to enter in the gel and transferring overnight with CAPS buffer were key to get the western blot working. As we have shown in Figure 2C and 2E, the majority of signal detected comes from SRRM2. The unspecific binding of SC35 mAb could only be scored if the above-mentioned conditions were not met.

We believe what made matters historically worse has been the use Mg++ precipitation that enriches many SR proteins, but actually completely depletes SRRM2 (Blencowe et al. 1994 DOI: 10.1083/jcb.127.3.593, Figure 5, https://pubmed.ncbi.nlm.nih.gov/7962048/ ). When we’re sure that SRRM2 is in the gel though, it just shines as a single band. So in conclusion, SC-35 is reasonably specific to SRRM2, especially in immunofluorescence, but it certainly cross-reacts with other SR-proteins, especially when SRRM2 is missing for technical or biochemical reasons.

We will update in the manuscript for the corresponding section by citing earlier studies reporting the specificity issues of mAb SC35.

We absolutely agree that IP-MS data alone is not enough to conclude that SC-35 recognizes SRRM2, or whether it is the primary target or not. The overwhelming amount of SRRM2 peptides detected, in addition to the overwhelming amount of total peptide counts from SRRM2 does strongly suggest that it is the case, which we then followed up by IP-western blotting which controls for relative input, and the various experiments shown in later figures.

We have looked at our MS results and found out that:

SRSF2 was detected with 4 unique peptides with an MS/MS count of 5 and a sequence coverage of 29% (intensity 3E+07), whereas SRRM2 was detected with 227 unique peptides with an MS/MS count of 3317 and a sequence coverage of 61.9% (intensity 2E+11).

These numbers show a 6600 times higher intensity for SRRM2 (not normalized). As the identification and abundance of different peptides/proteins can by dramatically different in MS, it is indeed correct that one should be careful with such comparisons. The only way would be to use peptide standards for both proteins and record standard curves, then a real quantitative comparison would give the true numbers. Hence, we will revise the wording of that section.

Finally, as the reviewer has pointed out, we have not shown that speckles can be reformed by introducing ectopically expressed SON/SRRM2 into cells which now appear not to have nuclear speckles. This would indeed be the formal proof showing that SON/SRRM2 are not just necessary but also sufficient to form nuclear speckles. Such an experiment is quite challenging due to the length of these proteins and difficulty in establishing conditions where one can express these proteins, but not overexpress them which leads to round-up speckles (as shown and discussed by Belmonte lab). Therefore, we will change the title to “SON and SRRM2 are essential for the formation of nuclear speckles” to better reflect our conclusions.

We really did try to be clear and just about the previous literature around SON. Indeed, it is clear that SON is a crucial part of NS, likely the most important component for the integrity of speckles. However, in all of these previous studies, RNAi-mediated depletion of SON, without exception, leaves behind spherical bodies that are strongly stained with mAb SC35, that also harbor other NS-markers (which we also show). This is of course not new, as we also appropriately cited previous work, however being able to dissolve these “left-over” speckles by co-depletion of SRRM2, and perhaps more importantly by deletion of the SRRM2’s C-terminal region is indeed novel.

In essence, our results show that in the absence of SON, as shown by previous work as well, NS-associated proteins are still able to organize themselves into nuclear bodies, indicating that either all other SR-proteins without the need of another organizer clump together, or another factor (or factors) is still acting as an organizer. When we remove the C-terminus of SRRM2, which we show is the primary target of SC-35, which strongly stains these left-over nuclear bodies in the absence of SON, then deplete SON, all NS markers that we could find become diffuse, indicating that nuclear speckles no longer exist, or become too small to be detected or classified as “nuclear bodies”. Co-depletion of SON and SRRM2 leads to the same phenotype, but co-depletion of SON and SRRM1 (or RBM25) doesn’t, leaving behind spherical nuclear speckles that harbor SRRM2 which are no different than SON KD cells.

Reviewer #3:

Nuclear speckles in the last several years have attracted significant attention for their association with transcriptionally active chromosome regions (after largely being ignored by most for the previous 20 years). Overwhelmingly, a single monoclonal antibody has been used as a marker for nuclear speckles for several decades.

This manuscript now argues convincingly that the main target that is recognized by this monoclonal antibody is not SRSF2 (SC35) as long thought, but rather SRRM2. The authors thus clarify a vast literature, while also focusing attention on the very large protein SRRM2 that in many ways resembles another nuclear speckle protein, SON. Both have huge IDRs and unusual RS repeats, while SON has been proposed to act as a scaffold for many SR-containing proteins, which is likely also true for SRRM2, by extension. Moreover, the manuscript provides a convincing explanation for why the target of this antibody was previously misidentified, by showing a lesser cross-reaction with SRSF7, of similar MW to SC35.

Finally, the manuscript suggests that SON and SRRM2 together help nucleate nuclear speckles, as a double KD, or a SON KD in a background of a truncated SRRM2, leads to loss of nuclear speckle-like staining of other proteins normally enriched in nuclear speckles (RBM25, SRRM1, PNN). The authors go on to suggest that this double KD approach will now provide an important means of disrupting nuclear speckles to aid in functional studies.

Interestingly, some of the results of this manuscript actually are already confirmed or consistent with previous literature. For example, a cited paper describes changes in Hi-C compartmentalization patterns after "elimination" of nuclear speckles- actually, they performed a SRRM2 KD and showed loss of SC35 staining, which is now explained as simply due to the KD that they performed. More recently, a new proteomics study of nuclear speckles (Dopie et al, JCB, 2020: https://doi.org/10.1083/jcb.201910207) reported both SON and SRRM2 as the two most highly enriched nuclear speckle proteins, with enrichment scores similar to each other but more than twice that of all other speckle proteins. Moreover, this same paper also did a SRRM2 KD and observed loss of anti-SC35 staining but not SON staining.

Overall, I found this manuscript of significant interest for people in the nuclear cell biology field and technically thorough and well done. I just had one issue and one point to make in my main comments, plus some minor points.

1) The evidence that nuclear speckles are nucleated by SON and SRRM2 is based on the dispersion of staining of nuclear speckle proteins RMB25, SRRM1, and PNN. However, an alternative explanation is that some other protein(s) nucleates nuclear speckles, while these other nuclear speckle proteins bind to SON and SRRM2, and are therefore enriched in nuclear speckles. To eliminate this concern, the authors could show that SON and/or SRRM2 do not bind to these proteins- for instance using co-IP or other methods. Of course, it could be that such binding or scaffolding of nuclear speckle proteins is how they form nuclear speckles. But just one protein that is not bound by SON and SRRM2 but still stains nuclear speckles after the double KD would be inconsistent with their hypothesis. Therefore, if they do find that all these proteins bind SON and/or SRRM2 they could simply discuss this as a scaffolding mechanism but qualify their conclusion based on the alternative explanation described above.

2) In our lab we have not been comfortable using the kinase manipulations, discussed in this paper, to eliminate nuclear speckles for experimental purposes because the cells appear very sick after these manipulations. For other reasons, we also tried a double SON and SRRM2 KD. Our experience is that the cells after this double KD were also not very normal. If the authors are suggesting the SON and SRRM2 double KD as an experimental tool to disrupt nuclear speckles in order to access nuclear speckle function, then it would be valuable for them to indicate cell toxicity, etc. Many SR-protein KDs for example do not allow selection of stable cells. What about this double KD?

The first point of Reviewer #3 has been addressed above in response to the Reviewer #2.

We have stated that our work identifying SON and SRRM2 as the elusive core of nuclear speckles paves the way to study the nuclear speckles under physiological conditions. Here, we have used the cells 24 hours after transfection (~18 hours of knock-down) as the primary reason being that SON-KD caused a mitotic arrest if the cells were kept longer in culture. This was reported earlier in Sharma et al MBC 2010. There was no additional severity in the phenotype when the SON-KD was combined with SRRM2-KD, therefore we believe the arrest phenotype we scored is mainly due to depletion SON. In this sense, double-depletion of SON and SRRM2 can be used to study the effects of loss of NS (transcription, post-transcriptional, topological), but certainly within a time-frame of around 24 hours in cells that haven’t gone through mitosis. We will clarify this statement in the revised manuscript to avoid any misunderstanding as pointed by the reviewer. Faster depletion strategies, and/or a system where cells are mitotically arrested would be required to observe long term effects more reliably.

Reviewer #3:

General assessment:

In this research article, authors claim that HIP1 plays an important role in promoting the proliferative ability of prostate cancer cells by HIP1-STAT3-GDF15 signaling axis activation. HIP1 overexpression increased STAT3 signaling in response to FGF2 receptor activation and increased GDF15 transcription. The increase in GDF15 protein secretion was dependent on HIP1 and STAT3 expression and was shown to have paracrine growth-promoting effects. Although some of the information is new, the relevance and importance of this information is inconclusive and not supported from the data presented in this article.

Major Comments:

This paper needs a substantial amount of revision, as indicated below.

A. Novelty:

HIP-1 has been extensively studied in cancer including prostate cancer (Rao et al., 2002). Its role in STAT3 signaling has also been demonstrated (Hsu et al, 2015). This study is not very novel.

B. Major comments:

1) Figures 1A, S1: Changes in p-AMPK1α, and p-Akt are very profound in this array, however, the authors indicate that "By contrast to our validation of STAT3 phosphorylation by Western blotting, it was not possible to detect increased levels of p-AMPK1α (T174), p-Akt (S473) or p-PLC-γ1 when we attempted to validate these by blotting (Supplementary Figure S1D-F)." Why do the authors think this is happening? Did the authors use the same experimental conditions for the array and validation experiments? These apparent discrepancies need further clarification.

2) Figure 1E: the authors show that shHIP1#2 caused a modest knockdown of HIP1, while shHIP1#1 induced a dramatic reduction in HIP1 protein level, however, both the shRNAs significantly inhibited pSTAT3 to the same extent. This indicates that total knockdown (KD) of HIP1 is not necessary to completely shut-down the activity of pSTAT3. How does this translate to the biological functions of HIP1?

3) How come DMSO treatment blocks the phosphorylation of ERK1/2 in lane 2 of Fig 1(F)?

4) Figure S1F: pSTAT3 western blot: the authors should indicate which band they considered positive for p-STAT3; if it's the lower band why was there no activity in lane 4?

5) Fig 2A and 2B should be repeated in HIP1 knockout cells.

6) What is the endogenous level of HIP1 and GDF15 in prostate cancer cell lines vs. normal prostate epithelial cells? Why was HIP1 overexpressed in LNCaP cells? Was the level of HIP1 expression low in LNCaP and PNT1A, when compared in a panel of prostate cancer cell lines? Did the authors observe any differential expression of HIP1 and GDF15 in hormone sensitive vs. hormone resistant prostate cancer cells?

7) GDF15 is a very ambiguous biomarker of cancer as its levels are even higher in the case of mental disorders including psychosis (for reference https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5554200/ ). And from this study, it is not even clear that the GDF15 upregulation is just one of the several outcomes of the activation of this signaling axis or if it is the only consequence of this signaling axis to promote the growth of cancer cells by increasing paracrine signaling. An experiment in GDF15 knockout cells/mice can document the role of this axis in a more precise manner.

8) It has been shown that wt p53 significantly reduces STAT3 tyrosine phosphorylation and inhibits STAT3 DNA binding activity in prostate cancer cell lines that express both constitutively active STAT3 and mutant p53 protein. The authors have claimed that the increase in STAT3 phosphorylation is due to HIP1 expression. All three of the cell lines evaluated in this paper have different p53 status and show differences in expression of activated STAT3. Is the expression of HIP1 independent of the status of p53?

9) Figure 3: Does STAT3 silencing (siRNA/stattic) downregulate HIP1, and does this decrease STAT3 activation over time? Also, does STAT3 silencing or treatment with WP1066 inhibit HIP1-induced tumor growth in vivo?

10) The role of GDF15 in prostate cancer is likely stage specific. It may promote early stages of tumorigenesis, but suppress the progression of advanced prostate cancers. The authors claim that HIP1 overexpression is mediated by stat3 activation, which leads to increased secretion of GDF15. Does expression of HIP1 correlate with the expression of GDF15 and does this also associate with stage-specific progression of prostate cancer?

11) How was cellular transformation studied and confirmed? Did HIP1 cause transformation of normal prostate cells?

12) Fig 1B: HIP1 western blot is not clear, please quantify 1C, 1D, 1E.

13) Most of the studies are done only in one cell line which is not adequate.

14) What is the clinical relevance of this study? The authors should study clinical samples along with multiple cell lines.

15) Several of the Western blot figures need better quality blots; Figs 1E (FGFR), S2C (all).

It’s important to note that just like the Declaration of Independence, the similarity is that both authors use lists to make their points and in this reading, the author is pointing out how men treated women, and the whole reading is basically about the many things that He has done to her.

Reviewer #1:

Summary:

In this study, the authors seek to determine patterns of KRAB-ZFP family evolution and identify the factors that drive those patterns. To do so, they first annotated KRAB-ZFP genes in the human genome and determined the age of these genes in four different ways: orthology, divergence age of full protein, KRAB, and ZnF domain respectively. They found that age estimates based on the KRAB domain and Zinc finger array were older and younger, respectively, relative to full-length or orthology-based estimates of divergence, and that many human KRAB-ZFPs emerged in the eutherian common ancestor. They also determined that older KRAB-ZFPs were more likely to have variant, disordered KRAB domains, and that zinc finger arrays were most variable at the residues directly in contact with DNA. By reanalyzing existing data, the authors claim that most KRAB-ZFPs bind to non-TE regions, and that many KZFP genes are expressed during early embryonic development. They show correlative evidence that KRAB-ZFPs are capable of positively regulating gene expression, and functionally validate a single candidate gene of a KZFP using reporter gene assays. Based on this evidence, they propose a 2-way model of evolution of KRAB-ZFP evolution, where older KRAB-ZFPs are more likely to have non-TE silencing roles and thus have different patterns of evolution compared with younger KRAB-ZFPs.

General Comments:

While the subject of KRAB-ZFP family evolution is of interest, the data and conclusions the authors present in this manuscript are mostly confirmatory. Nearly every major conclusion of the paper, including the 2-way model of KRAB-ZFP evolution, has been extensively documented before by the Trono lab (Imbeault, et al. 2017 Nature; Helleboid, et al. 2019 EMBO J; Ecco, et al. 2017; Pontis, et al. 2019), many of which the authors cite. The conclusion that older KZFPs gained new functions not related with TEs repression (such as imprinting regulation or meiotic hotspot determination) is already well established knowledge, which goes together with the model of higher purifying selection of the zinc finger array to retain the binding specificity, while the KRAB domain loses interaction with KAP1. Furthermore, the fact that KZFPs don't only bind to TEs has also been already reported by Imbeault et al. that originally provided the datasets re-analyzed in this manuscript.

The functional validation of ZNF611 binding to one of its target sequences is welcome and adds another example of a KRAB-ZFP that might have positive transcription regulatory function, however it is only a single KRAB-ZFP in a single assay. The finding that a KRAB-ZFP is capable of activating gene expression is also confirmatory (Ye at al. 2004; Frietze et al. 2010; Hallen et al. 2011).

There is value in replicating existing research, but the article is not written with that in mind. One contrast with previous studies is that their reanalysis of existing ChIP-seq data showed KRAB-ZFPs primarily bind to non-TE regions. However, these findings are based on thin evidence. It is not enough to say that a KRAB-ZFP mostly binds non-TE regions because >50% of its binding sites are outside of a TE. Rather, more quantitative statistics, such as enrichment or depletion of binding in a given genomic compartment compared to a random expectation is required. Additionally, there is no evidence such as heatmaps or metaplots over a subset of peaks to further demonstrate that the peaks identified in the new analysis are any better than the previous analysis. The authors argue that the more significant p values of their peaks are indicative of better peak calls, but there is no formal comparison of true/false negative rate (such as at known binding sites). Furthermore, many TEs, which are poorly mappable, will have less significant p values simply because fewer unique reads are mapped there relative to unique sequences. More careful analysis will be needed to assess these claims.

Finally, the paper itself is hard to read and the logic is difficult to follow, often due to a lack of sufficient detail. The methodology is also light on details, making it challenging to understand exactly what the authors did or did not do (see specific examples below). Additionally, the figures (especially Figure 1, Figure 3A, and Figure 4) are difficult to read and understand as currently presented.

Specific Comments:

1) The title and abstract make it clear that the authors are trying to argue that noncoding sequence contributes to rapid evolution of the KRAB-ZFP family. While this is possibly true, the authors' data, which is limited to a phylogenetic analysis of a single gene (using methodology that does not work well for highly repetitive sequences such as the KRAB-ZFP C2H2 zinc finger array) and its potential binding site. Much more analysis (such as selection analysis of more KRAB-ZFPs and their predicted or empirically determined binding sites) is required to make this claim.

2) Page 4, lines 66-70: The authors present the two possible models of KRAB-ZFP evolution (ie: arms race/domestication model) as if they are mutually specific, when most argue they would not be. Also, the authors state: "and (2) the domestication model (Ecco et al., 2017; Pontis et al., 2019), in which KZFPs regulate domestication of TEs instead of restraining the transposition potential of TEs". This should be rephrased, because in most of the cases reported, the "domesticated TEs" have lost transposition potential and only regulatory and protein coding sequences got domesticated with new functions. If the authors were referring to the adaptation of KZFPs to non-TE related functions, this cannot be called domestication, since KZFP genes are already from the host.

3) Page 5, lines 91-93: Here and throughout the authors use language such as "later" or "earlier" which is confusing - these should be replaced with "younger/more recent" and "older".

4) Page 6, lines 111-115: This section is highly speculative and should be moved to discussion.

5) Page 6 line 122: The authors do not define, here or in the methods, what constitutes a "variant" KRAB domain.

6) Page 7 lines 129-133: The authors only inferred their conclusion, yet they state that their result is consistent with a previous study. No real evidence is provided there.

7) Page 7 line 138-140: The authors say that the data suggests variant KRAB domains were formed gradually rather than in a burst, but their analysis is not sufficient to conclude this. Also, the only conclusion that can be drawn from Figure 2A is that the KZFPs that were clustered as "vKRAB" are on a separated branch in the tree on the left. This would mean that early in evolution some KZFP got a "vKRAB" and subsequently this gene underwent duplication and diversification, like all the other KZFP genes with "sKRAB" did.

8) Page 9 lines 189-193: Does the 90% cited here refer to 90% of the ~50% that are called as "tending to bind non-TE sequence" or 90% of all KZFPs? Regardless, this point is very misleading: the fact that less than 50% of the binding sites of a KZFP is not found to overlap TEs does not mean that the KZFP only binds to non-TEs. Some of this non-TE binding could also be an artifact of overexpression, which has not been considered but which has been well documented (for example ZFP809, Macfarlan Lab, and PRDM9 Simon Myers lab).

9) Lines 196-197, the authors state that they randomly selected 30 KZFPs. The authors should state in a supplementary figure which KZFPs were selected and, among them, what is the percentage of KZFPs that bind or not to TEs according to the analysis performed in the original paper (Imbault et al. 2017) and in this manuscript.

10) Page 11 line 230: Here and throughout the rest of the document the authors use the acronym "PCGs" without defining it (outside a figure legend).

11) Page 11 lines 234-237: Here the authors cite their use of pLI, RVIS, Shet, and dN/dS values as evidence of purifying selection. Of those, only dN/dS measures purifying selection, and the authors do not specify whether the dN/dS values they obtain are statistically significant evidence of purifying selection relative to a neutral model (likely the case when only considering chimp-human, as the authors do). Moreover, while the other measures do suggest some constraint, the differences between the KZFP-TE and KZFP-nonTE protein coding genes is very subtle. Also, they don't provide any explanation as to why, according to their claim, there should be less purifying selection for the KZFPs involved in mesoderm differentiation. Thus, the authors should temper their claims or else omit this data.

12) Page 11 lines 249-251: It is not clear how the author defined genes as transcription factors (they also do not define the acronym), or why they included them in the analysis. Additionally, the authors say that the divergence time of KZFPs is correlated with expression level but does not provide correlation values or the significance of these correlations.

13) Page 12 lines 266-268: This is not surprising, since TEs are generally silenced, while the rest of the genes can be either active or silent, so comparison of accessibility of cumulative TEs versus non-TEs will inevitably show open chromatin for non-TEs.

14) Page 13 lines 280-286: Here the authors try to draw conclusions from comparing chromatin accessibility of binding sites in ESCs and 293T cells and conclude that because they are more accessible in ESCs that suggests that KRAB-ZFPs activate in conditions. In reality, it is difficult to compare epigenetic states across cell lines, especially in undifferentiated vs differentiated, making it almost impossible without genetic manipulation to determine that KRAB-ZFPs are the cause of these differences.

15) How were the target genes selected for qPCR validation among the KZFP targets? In Fig.5 suppl. 3 the authors show that there is a fraction of genes that is only accessible in ESCs, but there is also a similar number of genes that is accessible both in ESCs and HEK293T cells, so the authors could have tried to validate some of those in both cell lines...Also, if the KZFPs are responsible for the target genes activation, why overexpression did not activate genes that are repressed in HEK293T cells? The ChIP-exo dataset used here (from Imbeault et al. 2017) was obtained from overexpression of the KZFPs in HEK293T cells, so obviously the proteins could bind to these genes in this cell line. This would rather suggest that if it's true that the tested KZFPs can promote transcriptional activation, this might be a secondary effect, since it might rely on something else making the genes already accessible and expressed in ESCs.

16) Page 16 lines 367-373: The conclusions that can be drawn from the ZNF611 reporter assay and associated evolutionary analysis are minimal. First, the authors cloned in a large chunk of DNA (1.2kb) rather than just the predicted binding site. This is mitigated somewhat by the deletion, but the deletion construct also deletes sequence upstream of the binding sites making the results hard to interpret. Additionally, the evolutionary analysis is very weak - traditional methods to generate phylogenetic trees do not work well for repetitive sequences, such as the ZnF arrays, and the bootstrap values on the tree are poor. There is almost no experimental methodology on how the tree was generated, how the authors overcame these issues, and how the authors identified the orthologous binding site in different species.

17) Page 18 lines 417-424: There is not enough detail about how the human KRAB-ZFPs were identified. Bare minimum, the authors need to report thresholds used to determine if a protein's domains scored high enough to be either a KRAB or C2H2 ZF domain.

18) Page 19: Given the highly repetitive nature of KRAB-ZFPs, it is not sufficient to use the homology estimations from Ensembl to identify orthologous proteins. Other methods, such as synteny, should be used to confirm orthologs. Additionally, the authors identify homologs between different KRAB domains based on %identity, but this will likely give spurious results, as functional domains do not evolve neutrally and often have high similarity across proteins due to functional constraint. Regarding the phylogenetic analysis, there is again not enough detail to explain how the authors overcome issues with alignments and low bootstrap values - additionally, they did not perform a model test prior to constructing the tree, which can impact the final results.

19) Page 22 lines 517-520: The authors do not elaborate why they chose FC > 1.1 or FC < 0.9 to call differentially expressed genes

20) Page 23 lines 529-532: How the authors performed the gene ontology enrichment/depletion analysis is not clear. For example, if the authors indeed prefiltered their list to remove genes that have no GO terms, that would bias the results.

21) Page 24 lines 552-554: For the non-targeting siRNA, it is unclear whether this is a scramble or targeting another gene (such as GFP)?

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Reply to the reviewers

We would like to thank the reviewers for their comments and suggestions. Our responses to them are listed below. We are hopeful that they will be satisfied with our responses and the changes we made in the revised version of the manuscript.

REVIEWER #1

Reviewer #1 (Evidence, reproducibility and clarity (Required)): In this manuscript, Ameen and colleagues report the results of a multidimensional proteomic analysis which combined quantitative proteomics, phosphoproteomics and N-terminomics in an effort to identify neuronal proteins displaying altered abundance or modifications by proteolysis and/or phosphorylation following an excitotoxic insult. Excitotoxicity is known to initiate by over-activation of ionotropic glutamate receptors which allows an increase in intracellular Ca2+ , ultimately leading to activation of proteases. The analysis revealed that glutamate treatment for up to 240 min did not significantly affect the abundance of neuronal proteins but caused dramatic changes in the phosphorylation state of many neuronal proteins. Based upon the phosphopeptides and neo-N-peptides, which contain the neo-N-terminal amino acid residue generated through proteolytic cleavage of intact neuronal proteins during excitotoxicity, the authors identified the proteins that undergo phosphorylation, dephosphorylation and/or enhanced proteolytic processing in excitotoxic neurons. By combining different software packages, they found that these modified proteins form complex interactions that affect signaling pathways regulating survival, synaptogenesis, axonal guidance and mRNA processing. These data suggest that perturbations in the aforementioned pathways mediate excitotoxic neuronal death. Then, the authors showed by Western blot analysis that CRMP2, a crucial regulator of axonal guidance signaling, exhibited enhanced truncation and reduced phosphorylation at specific sites upon glutamate treatment. These events may contribute to injury to dendrites and synapses associated with excitotoxic neuronal death. Furthermore, the authors showed that calpains are responsible for the proteolytic processing and cathepsins for enhanced degradation of proteins during excitotoxicity. Blockage of calpain-mediated cleavage site of the tyrosine kinase Src during excitotoxicity confers neuroprotection in an in vivo model of neurotoxicity. In that regard, over twenty protein kinases are predicted to be activated in excitotoxic neurons. Collectively, this study contributes to the construction of an atlas of phosphorylation and proteolytic processing events that occur during excitotoxicity and as such they can be targeted for therapeutic purposes.

**Comments** Comment: The identification of potential calpain cleavage sites in neuronal proteins modified during excitotoxicity is an interesting finding of the study. However, the atlas presented appears to miss components such as Kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), a protein recently shown to be cleaved by calpain during excitotoxicity (López-Menéndez et al, 2019, Cell Death and Disease 10, 535).

Response: The calpain cleavage site of neuronal ARMS/KIDINS220 was mapped to the peptide bond between Asn-1669 and Arg-1670 (Gamir-Morralla, et al. (2015) Cell Death & Diseases 6, e1939). The cleavage is expected to generate two truncated fragments – one of ~185 kDa and another of ~10 kDa at the N-terminal and C-terminal sides, respectively of the cleavage site. Our TAILS analysis failed to detect the 10 kDa fragment which contains the neo-N-terminus generated by calpain cleavage. Here are the possible explanations:

The neo-N-terminus of the 10 kDa C-terminal fragment is unlikely to be observed in our experiment as the TAILS method relies on the production of peptides by trypsin. The 10 kDa fragment has Arginine as the first amino acid which means that the N-terminal peptide released and isolated by the TAILS method would be a single amino acid. In their publication, Gamir-Morralla, et al. showed that the total levels of both intact and degraded ARMS/Kidins220 decreased as a result of ischemic cerebral stroke, suggesting degradation rather than proteolytic processing to generate stable truncated fragments as the final outcome of calpain cleavage of ARMS/Kidins220 (Figure 2b of the publication by Gamir-Morralla, et al.). The TAILS method predominantly detects proteolytic processing whereas degradation can be more difficult to capture. Degradation often results in peptides containing less than 5-6 amino acids that are difficult to align with a single protein or result in transient peptide that may not be detectable in neurons at 240 min after glutamate treatment. **Overall, it is possible that Kidins220 is generated but was undetected by the TAILS approach.

Comments: The CRMP2 antibody (Cell Signalling, 35672) used for western blots (figure 5D, also figure S11) and immunofluorescence (figure 5E) is problematic. Copied from https://www.cellsignal.com/products/primary-antibodies/crmp-2-d8l6v-rabbit-mab/35672: Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding lle546 of human CRMP-2 protein. The truncated CRMP2 (figure 5D) studied in the whole section (residues 1-516 or 1-517, ~57kDa) cannot be recognized by this monoclonal antibody. The detected band with the red letters in figure 5D might represent another cleavage product. In any case, asking Cell Signalling for more information about the exact immunogen might help, but since it's monoclonal and derived from residues surrounding lle546 it's very hard to include residues before aa516 and the unique epitope recognition upstream of aa516. The whole result section and discussion has to be reconsidered. Alternatively another antibody can be used to repeat those experiments in order to support the hypothesis. Time and resources are very familiar to authors since they have to repeat their previous work with a new antibody. Finally, there are no "western blot" and "immunofluorescence" methods for CRMP2.

Response: We would like to apologise for incorrectly listing the catalogue number of the anti-CRMP2 antibody purchased from Cell Signalling technology. Rather than the rabbit monoclonal anti-CRMP2 antibody (Cell Signalling, Cat#: 35672), we used the polyclonal anti-CRMP2 antibody (Cell Signalling, Cat#9393) to perform all the Western blot and immunofluorescence analysis in this paper. The e-mail confirming the purchase of this antibody is appended. According to the vendor, the antibody was raised by immunizing rabbits with a synthetic peptide derived from the human CRMP2 sequence. We decided to order this antibody because Zhang, et al. (Sci Rep. 2016; 6: 37050) reported that it could detect the truncated CRMP2 fragments generated by calpain cleavage in primary cortical neurons in vitro in response to axonal damage.

*The procedures of Western blot and immunofluorescence detailing the correct CRMP2 antibody descriptions are added in the revised version of the submitted manuscript.

*

Comment: The truncated DCLK1 bands detected in figure S8B cannot be attributed to the proteolytic processing of DCLK1 at the sites described: T311↓S312, S312↓S313 and N315↓G316 (predicted M.W. of the (C-terminal) products: 48.7-49.1kDa (figure S8A) which is very close to be well-separated with conventional PAGE). The number and the separation of the bands suggest other cleavage sites. Response: We agree with the reviewer’s comment that conventional SDS-PAGE cannot differentiate the proteolytic products generated by cleavage at the three sites identified by TAILS. Furthermore, the TAILS methods could not detect all peptides generated by a protein during proteolysis. Therefore, validating our results with a Western blot experiment may reveal unidentified peptides in certain cases. We have now added the following statement in the revised manuscript to reflect the presence of other cleavage sites: “Besides detecting the 50-56 kDa truncated fragments, the antibody also cross-reacted with several truncated fragments of ~37-45 kDa. These findings suggest that DCLK1 underwent proteolytic processing at multiple other sites in addition to the three cleavage sites identified by our TAILS analysis.”

Comment: Could the striking observation that almost all proteolytic processing during excitotoxicity is catalyzed by calpains and/or cathepsins have derived (partially) from unspecific targets of calpeptin such as a subset of tyrosine phosphatases (Schoenwaelder and Burridge, 1999: approx. 1h treatment of fibroblasts with approx.. 10x less concentration) or other(s)? Response: Schoenwaelder and Burridge (1999, JBC 274:14359) reported that calpeptin exhibits both protease inhibitor as well as a protease inhibitor-independent activities in fibroblasts. Besides inhibiting calpains and cathepsins, they demonstrated that calpeptin could selectively inhibit a subset of membrane-bound tyrosine phosphatases. Since the TAILS method monitored the protease inhibitor activity of calpeptin, the proteolytically processing events mitigated by calpeptin in neurons during excitotoxicity are likely attributed to its protease inhibitor activity. Additionally, Schoenwaelder and Burridge reported this unconventional protease inhibitor-independent activity of calpeptin in fibroblasts. Since the protein tyrosine kinases expressed in neurons and fibroblasts are different, it is unclear if calpeptin can also exert such activity in neurons.

Comment: Describing the final part of figure 4C the authors suggest that "Liver kinase B1 homolog (LKB1), CaM kinase kinase β (CaMKKβ) and transforming growth factor‐β‐activating kinase 1 (TAK1) are the known upstream kinases directly phosphorylating T172 of AMPKα to activate AMPK (Herrero-Martin et al., 2009; Woods et al., 2005; Woods et al., 2003). Our findings therefore predict activation of these kinases during excitotoxicity (Figure 4C)." The first question arising here is whether these three kinases are the only ones know to phosphorylate AMPKα. Even if this is true, it is highly speculative to suggest that the findings of the present study predict the activation of these kinases during excitotoxicity, without providing the necessary experimental data, since the increased phosphorylation of AMPK may be an indirect effect of the reduced function of a phosphatase. Thus the proposed model does not hold. Response: Agree. We have therefore revised our interpretation of the results to reflect this possibility. The Revised sentence on page 13 reads “**Liver kinase B1 homolog (LKB1), CaM kinase kinase β (CaMKKβ) and transforming growth factor‐β‐activating kinase 1 (TAK1) are the known upstream kinases directly phosphorylating T172 of AMPKα to activate AMPK (Herrero-Martin et al., 2009; Woods et al., 2005; Woods et al., 2003), while a member of the metal-dependent protein phosphatase (PPM) family could dephosphorylate T172 of AMPK in cells (Garcia-Haro et al., 2010). Our findings therefore predict activation of these kinases and/or inactivation of the PPM family phosphatase in neurons during excitotoxicity (Figure 4C).”

Additionally, we also deleted the schematic diagram depicting the possibility of activation of LKB1, CaMKKβ and TAK1 in Figure 4 of the revised manuscript.

__**Minor points**

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Minor Comment: Highlights could present the key points of the study in a more straightforward manner. Response: Agree. We have edited the highlights in our revised manuscript to make them more straightforward.

Minor comment: Figure 4A is too complicated. Proteins considered as hubs of signaling pathways in neurons should be somehow highlighted to distinguish them.

Response: Agree. We have now highlighted the signalling hubs by shading them in green in the revised figure. As we merged figures 2 and 4 of the original manuscript, these signalling hubs are presented in Figure 2B of the revised manuscript.

Minor Comment: The analysis of proteins with enhanced truncation and reduced phosphorylation such as CRMP2 and DCLK1 is fragmented. In addition, the authors should mention the criteria based on which these proteins were selected for further analysis.

Response: IPA analysis revealed synaptogenesis and axonal guidance as the top-ranked perturbed canonical signalling pathways governed by neuronal proteins undergoing significantly increased proteolytic processing and altered phosphorylation. As CRMP2 and DCLK1 are the key players in these pathways, they were chosen for further biochemical analysis to validate the TAILS results. To address this point, we added a few statements in the sections describing results of biochemical analysis of CRMP2 and DCLK1 in the revised manuscript. The additional sentences on page 13 now read “IPA analysis of the significantly modified neuronal proteins identified in our study predicted perturbation of signalling pathways governing axonal guidance and synaptogenesis in neurons during excitotoxicity (Figure S7). Since CRMP2 (also referred as DPYSL2) is a key player in neuronal axonal guidance and synaptogenesis (Evsyukova et al., 2013) and it underwent significant changes in phosphorylation state and proteolytic processing (Figures 5A and S7), it was chosen for validation of our proteomic results.” The additional sentences on page 15 read ”Similar to CRMP2, DCLK1 is also a key player in regulation of axonal guidance and synaptogenesis (Evsyukova et al., 2013). Since our TAILS results revealed significant proteolytic processing of DCLK1 (Figure S8A), it was chosen for validation of our proteomic results.”

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Minor comment: The potential therapeutic relevance of phosphorylation and proteolytic processing events that occur during excitotoxicity can be further explored. Response: Thanks for the suggestion. We have added a paragraph describing the additional evidence that protein kinase inhibitors and cell-permeable inhibitors blocking calpain cleavage of specific neuronal proteins as potential neuroprotectants to reduce brain damage induced by ischemic stroke. The additional sentences near the end of the Discussion section (page 25) now read “Since CRMP2 is key player in axonal guidance and synaptogenesis revealed by our proteomic analysis as the most perturbed cellular processes in excitotoxicity, blockade of its cleavage to form the truncated CRMP fragment is another potential neuroprotective strategy. Indeed, a cell-permeable Tat-CRMP2 peptide encompassing residues 491-508 close to the identified cleavage sites of CRMP2 could block calpain-mediated cleavage of neuronal CRMP2 and protect neurons against excitotoxic cell death (Yang et al., 2016)**.”

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The additional paragraph at the end of the Discussion section (page 25) now reads: “Besides the neuronal proteins undergoing enhanced proteolytic processing during excitotoxicity, protein kinases predicted by our phosphoproteomic results to be activated during excitotoxicity are also targets for the development of neuroprotective drugs. For example, our results demonstrated significant activation of neuronal AMPK during excitotoxicity, suggesting that aberrant activation of AMPK can contribute to neuronal death. Of relevance, small-molecule AMPK inhibitors could protect against neuronal death induced by ischemia in vitro, and brain damages induced by ischemic stroke in vivo. Likewise, inhibitors of Src and other Src-family kinases were known to protect against neuronal loss in vivo in a rat model of in traumatic brain injury (Liu et al., 2008a; Liu et al., 2017). Future investigation of the role of the excitotoxicity-activated protein kinases in excitotoxic neuronal death will reveal if small-molecule inhibitors of these kinases are potential neuroprotective drug candidates.”

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Minor comment: I am sorry but I could not find Figure 8, which is supposed to show the "In vivo model of NMDA neurotoxicity" (please, see page 30).

Response: Our apology for the mistake. This should be Figure 6 of the revised manuscript.

Minor comment: Introduction: O'Collins et al., 2006; Savitz and Fisher, 2007; both references are missing.

Response:* This was an oversight from our part and the references have been added to the revised manuscript.**

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Minor comment: Figure S1A-B: vehicle treatment time course is needed. Response: All neurons were cultured in neurobasal media for seven days. The control neurons were incubated in culture media while we started treating the other neurons with glutamate for MTT and LDH assay. The additional paragraph describing the design of the cell viability/death assays in page 32 reads “Primary cortical neurons were incubated for 480 min with and without the addition of 100 μM of glutamate. The control neurons were incubated for 480 min in culture medium. For neurons treated with glutamate for 30 min, 60 min, 120 min and 240 min, they were pre-incubated in culture medium for 450 min, 420 min, 360 min and 240 min, respectively prior to the addition of glutamate to induce excitotoxicity. For neurons treated with glutamate for 480 min, they were treated with glutamate just after seven days of culture in neurobasal media.”

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Minor comment: Figure 5E: Control close-up is missing. Response: A close-up view of the control neurons is now provided in Figure 4E of the revised manuscript.

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Minor comment: "Moreover, the number of CRMP2-containing dendritic blebs in neurons at 240 min of glutamate treatment was significantly higher than that in neurons at 30 min of treatment (inset of Figure 5E)." Such a statistic is not shown in the graph. Response: The statistical analysis results are now added to the revised manuscript in Figure 5E.

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Minor comment: "Consistent with this prediction, our bioinformatic analysis revealed that the identified cleavage sites in most of the significantly degraded neuronal proteins during excitotoxicity are mapped within functional domains with well-defined three-dimensional structures (Figures 6A)." Authors might mean figure S12A? Response: Correct. Our apology for the mislabelling. This has been corrected to “S12A”in the revised manuscript.

Minor comment: "Neuronal Src was identified by the three criteria of our bioinformatic analysis to be cleaved by calpains to form a stable truncated protein fragment during excitotoxicity (Figures 6A and Table S6)." Authors might mean figure 6D?

Response: Correct. Our apology for the mislabelling. Since we merged figures 2 and 4 of the original manuscript. This has been corrected to now read “(Figure 5D)” on page 18 of the revised manuscript.

Minor comment: Figure 2B: Clusters 1, 3, 4 and 6 do not follow treatment trends homogenously in all time points. For example in cluster 1 there is a phosphopeptide following the pattern 1, 0, -1 and another one following the pattern 0, 1, -1, which is actually a very different pattern even if the end value is stable (-1). The first example could belong to the cluster 6 as well, while the second example to cluster 5. Please elaborate on the rationale behind the categorization. Is there any other clustering method that can be used without making the categorization more complicated? Response: Since we merged Figures 2 and 4 of the original manuscript. This comment relates to the right panel of Figure 2A of the revised manuscript. The rationale behind the categorization of the phosphopeptides into six clusters was based upon the patterns of changes of their abundance (i.e. average of log-2 normalized z-score of phosphopeptide intensity) in three sample groups. **We calculated the number of permutations where the number of sample groups in set (n) = 3 (i.e. Control neurons, neurons of 30 min glutamate treatment and neurons of 240 min glutamate treatment) and number of sample groups in each permutation (r) = 3 (i.e. all three sample groups should be present in each permutation). Hence the number of permutations is 6. The six clusters refer to the six possible permutations of the patterns of abundance changes of the identified phosphopeptides rather than the end results.

Minor comment: A problem of the manuscript is its length and lack of coherence. Apart from presenting the data from the proteomics, phosphoproteomics and N-terminomics analyses, the authors focus on several different proteins to perform validation experiments and further characterize the biological significance of their modification. Because these proteins do not fall on the same pathway, the authors end up presenting several independent stories that complicate the reader. Response: We agree that proteins that do not operate in the same signalling pathway were chosen for further biochemical analysis. Their choice was justified because they are key players in the most perturbed canonical signalling pathways identified by bioinformatic analysis with the IPA software. We agree that this may complicate the reader. However, it also helps to illustrate that excitotoxic neuronal death is a complicated cell death process caused by dysregulation of multiple neuronal proteins which regulate different cellular processes.

Minor comment: Moreover, it is necessary for the authors to restructure their introduction, and avoid over-representing previous research on nerinetide, which is not used anywhere in the manuscript. Instead, the introduction must be more focused to better capture the necessity and essence of the present study. Response: We agree. Based on the reviewer’s comments, we decided to restructure the introduction by shortening the description of the results of Nerinetide research. Please refer to the track changes of the revised manuscript for the changes.

Minor comment: Taking into account figures 1 and S2 I understand that the authors combined samples of neuronal cell cultures (treated or not with Glu) with samples from mouse brains (that have undergone ischemic stroke/TBI or sham operation). If this is the case, why did the authors do that? How did they combine the different samples? And why this is not mentioned anywhere is the main text? Response: For a data-independent acquisition (DIA) based mass spectrometry experiment, it is essential we generate a library of identifiable peptides first using a standard data-dependent acquisition (DDA) approach. For the DIA type experiment to work, the identified peptides have to be in that library first. Excitotoxicity is a major mechanism of neuronal loss caused by ischemic stroke and traumatic brain injury. We therefore included the brains of sham-operated mice, brains of mice suffering ischemic stroke and traumatic brain injury to construct the spectral libraries and that is why the library contains pooled samples from the representative samples. Pre-fractionation of the pooled peptides was also performed to increase the number of identifiable peptides and generate a deeper library.

• Once we generated that library, all samples are analysed individually as a separate DIA experiment. The DIA approach then makes use of the generated library for identification and quantitation. This methodology allows for deeper identification and lower number of missing values. These statements were added in the method section of the revised manuscript (page 33)*

Minor comment: Regarding figure 5D, the authors write in the main text "Consistent with our phosphoproteomic results, the truncated fragment CRMP2 fragments could not cross-react with the anti-pT509 CRMP2 antibody (Figure 5D)" In the upper blot the truncated CRMP2 fragment runs well below the 70 kDa marker. However, in the middle panel, where we see the blot with the phospho specific antibody, the respective area of the blot has been cropped, so we cannot see whether the truncated fragment cross-reacts with the phospho specific antibody. Response: The presentation of the western blots in Figure 5D in the revised manuscript are now less cropped and clearly demonstrate there is no cross reactivity of the phospho specific antibody with the truncated fragment. Please refer to the revised Figure 5 for the updated Western blot images.

Minor comment: It is strange that only 1 and 13 proteins showed significant changes in abundance at 30 and 240min respectively. Especially after 240min of glutamate treatment one could expect that many proteins should change in their levels, since the neurons are almost diminished by cell death at that point. How could the authors explain this phenomenon? Additionally, in their previous publication, they showed that much more proteins change significantly in abundance following glutamate treatment (at 30min and 240min).

Response: Even though our global spectral libraries contain over 49,000 identifiable peptides derived from 6524 proteins, only 1696 quantifiable proteins were identified in the DIA mass spectrometry analysis (Figure 1) because we used stringent criteria for their identification: (i) false discovery rate of We agree with the reviewer that many more proteins are expected to change their abundance at 240 min as significant cell death was detected. However, if we had used less stringent false discovery rates of their identification and quantification, included proteins with just one unique identified peptide and lowered the threshold of abundance fold changes, many more proteins with significantly changed abundance would be detected. But we preferred to use these stringent criteria to ensure a high confidence in our identification of neuronal proteins undergoing significant changes during excitotoxicity.*

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In agreement with the low number of neuronal proteins exhibiting significant changes in abundance reported in this manuscript, our previously published study (Hoque, et al. (2019) Cell Death & Diseases) detected only 26 neuronal proteins undergoing changes in abundance. Hence, we disagree with the reviewer that our previous publication reported much more proteins undergoing changes in abundance in excitotoxicity.

Reviewer #1 (Significance (Required)): Comment on significance: The manuscript delivers a large amount of data, regarding changes in the proteome, the activation of specific kinases, phosphatases, as well as the molecular pathways that are activated at distinct time points of excitotoxicity. This information could be used in future studies to validate and develop potential therapeutic strategies that could protect against neuronal loss in various neurological disorders. Response: We are excited that Reviewer #1 felt that this large amount of generated data will be useful for subsequent studies to validate and develop novel therapeutic strategies.

Comment on significance: The same group has very recently published a work very similar to the particular manuscript (Hoque et al. Cell Death and Disease, 2019). In their previous publication, the authors cover a large part of their current objectives. They performed again a proteomic and phosphoproteomic analysis of mouse primary cortical neurons treated with glutamate for distinct time points, in their aim to identify changes in expression and phosphorylation state of neuronal proteins upon excitotoxicity. Apart from the N-terminome, which they investigate in their current manuscript, the proteomic and phospho proteomic analysis are very similar. As such, and because of the fact that the current manuscript is very extensive, the authors should consider to minimize it, and include only their novel findings (changes in the N-terminome, the involvement of specific kinases that contribute to excitotoxic neuronal death, the regulatory mechanism of CRMP2, etc).

Response: Since the coverage of phosphoproteins undergoing changes in neurons during excitotoxicity identified in the current study is much higher than that of phosphoproteins identified in our previously published study, we prefer to retain the description of the phosphoproteomic findings in this manuscript. Nonetheless, we agree that the manuscript needs to be shortened. Our suggestions to shorten the manuscript are listed below:

1. Move the description and results of global proteomic analysis to supplementary information. Since we made the same observation that only a small number of neuronal proteins undergo significant changes in abundance during excitotoxicity in our previously published study, moving the global proteomic analysis results away from the main text will not adversely impact the quality of the presentation.
2. For the description of how we classified the identified N-terminal peptides as those derived from degradation and those derived from proteolytic processing, we would like to move it to the supplementary information. Comment on significance: The authors should describe in a simpler way the proteomic and bioinformatics analyses they are using in the manuscript. It is difficult to understand the methodology used if you are not an expert in proteomics and bioinformatics. My suggestion is to revise their text and make it simpler and more concise. Response: We agree with this criticism. As we are not allowed to make a major revision of the manuscript at this stage, the revised manuscript contains only minor revisions that addresses all of the comments and suggestions provided by the two reviewers. Further changes will be added in the next revised version. Our suggestions to further restructure the manuscript are listed below:

Figure S5 depicting the rationale for classification of N-terminal peptides as products of degradation and those of proteolytic processing will be moved to the main text. The description of the rationale in the main text will be revised to help readers who are not experts in proteomics to better understand the rationale. A diagram depicting the workflow of our TAILS method will be added as a supplementary figure. For bioinformatic analysis of the proteomic results, we will provide in the supplementary information the definition of the following terms relevant to Ingenuity Pathway Analysis and PhosphoPath analysis of the perturbed biological processes and signalling pathways: (a) Canonical Signalling Pathways, (b) Cellular Processes and (c) Interaction Networks. A short description of how their identification benefits the mapping of the neurotoxic signalling networks in neurons will be provided in the supplementary information.

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REVIEWER #2

Reviewer #2 (Evidence, reproducibility and clarity (Required)): Comment: In this article, Ameen and collaborators identify the modified proteins during neuronal excitotoxicity by using an in vitro model in which mouse primary cortical neurons are treated 30 and 240 min with 100 µM Glutamate. They use different approaches: a quantitative label-free global and phospho-proteomic methods and a quantitative N-terminomic procedure called Terminal Amine Isotopic Labelleling of Subtrates (TAILS). Results show that 240 min glutamate has minimal impact on protein abundance (13 neuronal proteins show significant changes) but enhance a modification of phosphorylation state and proteolysis of nearly 900 proteins. A significant part of these proteins are involved signalling pathway involved in cell survival, synaptogenesis and axonal guidance.

The paper is globally well written and experiments are convincing. The methodology and the analysis are well described and well explain. The text and each figure are clear and accurate. However, I have just one comment that needs answers and/or clarifications. Thanks for your work. Response: We appreciate the compliment provided by this reviewer on our submitted manuscript.

**Minor comment:**

Minor comment: Primary neurons are used at DIV7 and it has been shown that at DIV7 the percentage of astrocytes is relatively low, however astrocytes plays a key role in glutamate recapture and release. It will be relevant to know the percentage of glial cell in the culture model of the authors and how astrocytes are involved in glutamate recapture and also in excitotoxicity.

Response: The compositions of the DIV7 cultures are: 94.1+/- 1.1 % neurons, 4.9%+/-1.1% astrocytes, and *

Reviewer #2 (Significance (Required)):

Comment on significance: Excitotoxicity is a cell death process involved in many neurological disorders. However, nowadays, there are no existent FDA-approved pharmacological agents targeted to protect against excitotoxicity leading to neuronal death. A better comprehension of excitotoxicity is required to improve prevention, therapy and reparation following the disease.

With this work, the authors highlighted modified proteins in excitotoxic neurons. Interestingly, few of these proteins are involved in cell survival, mRNA processing or axonal guidance. This atlas of phosphorylation and proteolytic processing events during excitotoxicity permit the identification of new therapeutic targets such as calpain-mediated cleavage of Src kinase. This atlas will interest a lot of team working on neurological disorders such as Alzheimer disease, Parkinson disease or stroke. It will permit to better characterize cellular/molecular events involved in neuronal loss and will permit to find new therapeutic targets. Response: In response to this comment and a similar comment by Reviewer 1, we expanded the discussion to include the potential therapeutic values of our findings.

Comment on significance: My field of expertise: Stroke, cell death, excitotoxicity, signalling pathways and molecular targets, autophagy. I don't have sufficient expertise to evaluate proteomic analysis.

Response: No response is needed.

Bacon has the ability to show stops and silence of various kinds in his work of arts. He wants to make something special so it's unlike other paintings. Bacon wants to create something that has depth and complexity, something that gives other's a sensation without them getting too bored or looking into it too much. Instead of wanting a story in his pieces, he says that he wants people to just stick to the facts rather than making a whole story. What would've been the outcome if Bacon had decided not to implement stops and silence of various kinds in the process of his paintings?

Mera Lee-Penehira's view

This happens a lot

I love how here, humankind contains elements of the earth itself. "Fragments related to the skies", "blended with streams of rain", how beautiful! Maybe this is why we feel such a pull to nature, it's like a call back home. Also, made in the "image of the all-controlling gods" reminds me of Christianity, where it's stated that mankind is made in the image (with the breath) of God. Another correlation between two creation myths!

It's interesting how the beginning of the text involves a direct acknowledgement of and address to the gods, as if this way he can channel their power and speak in truth to their story. The word "attempt" highlights how the speaker has created a hierarchy between himself and the gods, putting himself, a human storyteller, far below that of the gods. That in spite of whatever reputation he may have, any story he tells is just a feeble attempt to capture the greatness of the gods' true story.

I think it's interesting that in this rendition Zeus became king by getting the trust of other immortal beings instead of just being "the strongest." I think you could draw parallels between Zeus' rise to to power with that of different heroes through-out Greek mythology (Odysseus comes to mind).

Right at the beginning, it's obvious that this was meant to be performed orally, not just read. In fact, this first line in particular seems to have many similarities to Slam Poetry, like the introduction stated. Specifically, the use of alliteration, such as in "broad-bosomed Earth", "secure seat", and "dark dim" generate a sort of rhythm that draws the reader to the words and the picture they attempt to create. Perhaps what's most intriguing to me are the uses of assonance and consonance alongside this alliteration. One of these examples being "inhabit the peaks of snow-capped Olympus". Here, the majority of words contain a "p" or "b" sound at the start, end, or beginning. These poetic devices continue throughout the poem, and as someone who adores Slam Poetry, I am drawn in.

It's really interesting that there are different ways that beings come to be in mythology. It's not just the reproduction of a male and female figure but rather has more to do with the power and the substantial position that the beings hold.

interesting point. I would like to know what my peers think of this? In Belize, I don't know that we have a hispanic heritage month. Our independence was from the British and it's the 21st of September.

I think his answer is very interesting. It's crazy to think that for some there is some sort of privilege behind this identity. I kind of agree with his answer. I don't feel like the identity is inclusive enough. What is it that actually makes us latinx? I may be labeled latina based on just looks, when my experiences make me feel otherwise.

Wow, this is something that I have never heard in my 22 years of life, but I just think it's laughable when you realize that some of these social constructions made by society are just crazy. I didn't know that there was a difference between a wealthy male and a wealthy female when we're simply talking about a piece of green paper.

quotes with confidence of fixing and improving AIs

He asks rhetorical questions here in order to show how interpretive this album is. The concept of folklore is that you can interpret it how you like, just like actual folklore. Do you want Taylor to be part of the love triangle? or do you want the characters to be separate from her entirely? it's up to you.

Essentially this is saying that anything not proven is just an assumption and not a fact. It's yet to be verified.

He just wanted the Natives Americans to continue on the path of their of destruction as he thought that was the dance was for.

I think it's nice to have a calm cat that just patrols the area and does whatever he wants

I am very interested with this very first sentence. It interests me because I feel as though this gap is almost gone. i never think about how women get paid less than men because I feel as though we do not get paid less than men. I have never been in a working environment where men are paid more just because they are male. I feel as though I never have really listened or understood or read about this gender gap, so i am excited to see how the pay is different and why that is and how the future will hopefully be different and the male and females will have equal pay.

I find it amusing Herodotus makes it sound like the Persians absolutely love celebrating their birthdays. It's something small that you forget people have been doing for hundreds of years. It's not just a modern thing.

In 1.1 of Doing Philosophy, it talks about the scientific method and mentions that "Philosophy, like science, aims at solving problems and getting at the truth. Unlike science, however, philosophy is more concerned with explaining how it's possible for concepts to apply than how it's possible for events to occur. " (23) Therefore I think philosophy does get to achieve positive results like how other sciences do, it just goes even deeper.

Beliefs are not just about one person, it's about how that person's believes will affect others

In my view this practice is not just helpful but should be promoted throughout. As David talks about it in his TEd talk too that sharing education is a mutual support. We as teachers are always in constant search of material for our learners similarly we can contribute the same for our fraternity. It's like strengthening our self and evolving with the community. The resources will eventually benefit us all.

Interesting that instead of people admitting their beliefs are ultimately unjustified due of the lack of evidence, they instead try to explain their rationale -- is a direct example of "Just because someone consistently believes something doesn't mean that it's likely to be true"1. This scenario could also quickly become an example of the unacceptable premise "begging the question"2.

1) Theodore Schick Jr., Lewis Vaughn, How to Think About Weird Things: Critical Thinking for a New Age, (New York: McGraw-Hill Education, 2019), 84, e-book. 2) Theodore Schick Jr., Lewis Vaughn, How to Think About Weird Things: Critical Thinking for a New Age, (New York: McGraw-Hill Education, 2019), 51, e-book.

The fact that Chapelle is so famous, and his work consumed to such degree, has a lot more to say about the consumers than Chapelle himself. Just because David Chapelle said something does not mean I am going to agree with him merely based on the fact that it was a public figure who said it. His Netflix specials being so popular is clear evidence that this is comedy people like indulging in. Chapelle is one voice from the millions that have controversial opinions. So I consider that we should look at the people that are around us, and even at ourselves before condemning a stranger.

It's interesting, in the past I would have considered journaling about the project I just completed as useful notes to have for the future when I picked the project up again; but here the author focuses on the benefit of having your mind clear for your next project. Both are valuable, but I like the immediate benefit in the present, as it motivates to do it right now, instead of for some unsure reward in the future depending on if the project is ever picked up again.

just replace with

if not format_spec

(but add note that it's not None, but rather "")

Exactly... the viewer clicked on his face when deciding what to watch on Netflix... not much else needs to be said more than that. Although as I continue to read this article, my opinion or stance on a certain issue or joke may or may not change.

I also like how this writer distinguishes "successful comedy" from "offensive comedy." A joke can be successful in that it makes someone laugh, but that doesn't mean that it isn't offensive to others and therefore devoid of criticism because "it's just a joke."

As an influential public figure, Chappelle owes a certain responsibility to his audience. Normalizing a life-altering experience like sexual abuse just because "it happens to everyone" is a very dangerous sentiment. Just because it's something that happens often doesn't mean it should be ignored, dismissed or belittled. It is very dangerous considering that his thinking is obviously influencing his audience (who couldn't stop laughing); his lack of knowledge about power structures is very damaging.

"The Word, the Word Is an egg:" This means to me that like an egg, once it's cracked you can't un-crack it, just like when you say something there's no taking it back.

I create abstractions because I want the layers of my program to use simple concepts, in other words, concepts which hide lots of complexity. This is good when the code works as intended.

It may make tracing the execution of the program more difficult when something goes wrong.

It's interesting that this way of thinking is so new. I find it interesting how, before the 20th century, we seemed to generally only think of the mind as separate from the body (of course, this is simply what is implied by the text, not necessarily true of the whole world before the 20th century). Did everyone before this point just think of the mind like Spock, all logic and entirely separate from the illogic of the body?

LISTS: • Sequence of values just like strings • Values in the list are called elements or items • A list can contain a list within itself and is called nested list • Lists are mutable • In operator can be used in the same way as strings Map: - it’s a function that acts onto each of the elements in a sequence Reduce:-an operation which combines sequence of characters into a single value Filter:-an operation which acts upon few elements of the list ***If two objects are identical they are also equivalent but if they are equivalent, they are not necessarily identical

LISTS: • Sequence of values just like strings • Values in the list are called elements or items • A list can contain a list within itself and is called nested list • Lists are mutable • In operator can be used in the same way as strings Map: - it’s a function that acts onto each of the elements in a sequence Reduce:-an operation which combines sequence of characters into a single value Filter:-an operation which acts upon few elements of the list *If two objects are identical they are also equivalent but if they are equivalent, they are not necessarily identical

The scene I chose that very much stood out to me took place on page 32 in Act 1, where Quang is in a conversation with a man called Captain Chambers and his translator. The first aspect of this scene that I noticed was the obvious gibberish the American captain speaks. His dialogue includes lines like “Whoop whoop, fist bump. Mozzarella sticks, tator tot, french fry,” or “Shit-ass. USA! USA!” Firstly, I see this choice in language as a way of showing American culture stereotypes. The use of specific random words such as “french fry,” “USA! USA!” and “yella fella” are all phrases that I assume people associate with this view of America, where the Americans are seen as very stupid, brash, rowdy partiers. I also believe that this kind of diction emphasizes whose perspective we are looking at the story from. We talked about the classic “white straight male hero” character that is played time and time again in thousands of shows, and how we never see plays that depict important characters who are in the minority. In Vietgone, we are experiencing the play through the viewpoint of Vietnamese people, not the American soldiers, Hippies, and Redneck Bikers that we meet briefly. Therefore, with the Playwright choosing to show english language as a gibberish word vomit, it reinforces the importance and impact of making sure we have representation of everyone within theatre and society. The second thing I noticed in this scene is the contrast between what Captain Chambers speaks in relation to what his translator relays to Quang. For example when Captain Chambers says “Shit-ass” or “Fuck a duck, yella fella. Fuck a duck,” the actual translation is “I’m sorry” and “So sorry, bro. There’s just no way. Have a nice ride.” It is very interesting that the English language to Quang is depicted as very aggressive and hostile compared to it’s real meaning. I think it further reveals Quang’s strong dislike for America and his desire to stay in Vietnam. It is as if his hate for America is so strong that he is even subconsciously trying to further distance himself from the United States, by interpreting what Captain Chambers says with a hostile connotation. Lastly, within this scene there is a subsection within the conversation between the Captain and Quang:

Quang: I just airlifted three dozen civilians here in it, what do you mean I ‘don’t have a helicopter’? Captain Chambers: Splish Splash Translator: “We pushed it off the carrier to make room for incoming planes. Too many copters were coming at one time. We needed space so we pushed it off into the sea.” Quang: What NO! Captain Chambers: Shit-ass. USA! USA! Translator: “I’m so sorry about your loss, but there is no going back. You’re coming to America now.” Quang: I don’t want to go to America! Captain Chambers: Shit-ass Translator: I’m sorry

This part of the scene is a good insight into America’s involvement in the Vietnam War. It is a widespread opinion that the United States should have never gotten involved, as citizens believed there was no reason and were incredibly scared of it’s violence. America’s involvement proved unsuccessful as they lost to Vietcong with their efforts having no effect on the outcome in the end. I think that this passage is a good representation of that historical aspect of this play, where the Americans (or Captain Chambers in this example) found their way into the conflict and didn’t end up helping anyone. In this case, his help was actually detrimental to Quang’s plans, and yet Quang wasn’t given the choice to do anything about it. It was decided for him.

Just think it's interesting in this article that they continue to provide gendered examples per a man's and woman's experiences of happiness and that it is the woman suffering for a cause. Made me think of nuns, but maybe religious-based happiness is adjacent to this conversation.

building reflection as a habit is a process that takes time; it's something that some don't often 'just do' without some motivation

Pg 258

"I saw that the best thing I could do was get hold of a dictionary - to study, to learn some words. I was lucky enough to reason also that I should try to improve my penmanship."

I think it's very admirable for someone to really push themselves to learn something that they really do not know with just the help of inanimate objects. And in his case especially, he copied word after word to think about writing words and how to learn it.

What can be done to promote a peaceful and tolerant society, for starters we need to look at the bigger picture like what is happening right in front of us everyday (the pandemic, the killings daily, people are angry because nothing is being done. They are putting a band-aide on everything and saying it's getting better when it's not. Something that would be positive would be assisting people coming home from prison to get jobs without judgement this would help the recidivism rate. One thing I will say is that somethings people are becoming tolerant of are just insane to me for example trying to normalize pedophiles and consider it a mental health disorder!! I'm sorry but I can guarantee any sane person and parents won't be tolerant or peaceful about something so depraved and disgusting.

This shows that it is not just a few countries that need to do better, but that every country needs to do a little better. With just those counties under preforming on social progress it doesn't change much from the average score of the world and is still far from the Global Goal. It takes more than just doing better, too. The world needs to be actively using it's wealth for the people's well-being or order to get to our goal. This is a role for the whole world, not a select few countries because everyone can do better and there's always room for improvement.

Just because places are richer does not mean that their social progress is high. With wealth can come problems, as the speaker says. Economic growth helps poverty because a poor country won't be able to help it's poor citizens. In relation to problems like climate change, though, economic growth won't really be able to help much, which seems to be the same concept when looking at other Global Goals.

This just makes me think of how in fantasy video games they will do practically any ridiculous thing to their female characters EXCEPT make them not conventionally attractive. That’s just too far apparently. But it’s for art apparently so that makes them immune to criticism

Yes, it's not perfect English, but the point still gets across. Just like how my parents sometimes would speak to me in "broken" English I would always understand and there would be only a slim chance that I wouldn't understand.

It's interesting to me that they would mention this in a supreme court ruling. Usually this sort of thing is just assumed and never really written out for people to see.

"The opportunity to be truly recognized and genuinely considered as a community equal to the white race is more valuable than it just being a requirement by law that these rights are given to us. "

I think Washington is trying to say that it's not just the law that needs to be changed. Its people, their way of thinking, their regards towards the black community, etc. Washington is recognizing the need to prioritize the economy, and workforce in order for his community to establish their importance and contribution to society. In return, this establishment will allow them to move forward seeking more opportunity, rights, and power in the US.

Here W.E.B Dubois is showcasing some arguments or disagreements between himself and Washington. Washington reminds me of Martin Luther King and his peaceful protest and mild mannerism. Washington wanted for unity within the south between the whites and African Americans by working their way up to become a superior wealthy negro and in a ways "proving" their right to be free and active in the field and strong workers in their communities. Dubois on the other hand wanted all rights legally fought for and obtained. He believed in civil rights and laws that should be followed no mater your socio-class or status. so who is correct and who is not? I would say that it's all about how you understand what's going on and your opinion on what's right and whats wrong. I say that both were successful at achieving rights for African Americans during this time just with different strategies.

I recently came across a well known quote from Booker T. Washington that I think speaks volumes to what is going on right now in the US and what he spoke about in his speech in 1895. "A lie doesn't become truth, wrong doesn't become right, and evil doesn't become good, just because it's accepted by a majority" (Booker T. Washington). Just because someone says it louder and more often, doesn't mean it's true.

Here it is being stated that African Americans need to not only want the ability to be equal, for all laws of the land to apply to them but they should want the ability to know how to use these laws and privileges for their prosperity. It's not just important to be equal in written legislation but it's important to be able to to take what was given and turn it into further equality, more rights, more opportunities, and seek congressional positions of higher power.

I think it's easy to state that Booker T. Washington and W.E.B. Dubois had differing opinions on racial equality strides for African American's in the South. Washington preached racial cooperation by African American's in Dubois opinion "submitted" to whites. Washington saw things as hard work and education being a higher power over the active systemic racism at the time while Dubois saw African American's admitting to the conceived inferiority at the time. He believed African American's gave up political power, civil rights and higher education. Dubois believed Washington's propoganda fueled this loss. Washington fought for an African American to be treated more equally to their counterpart. In the quote of "cast down your bucket where you are" Washington is stating that Southern Whites had what they needed all along, they just never looked onto African American's as equal counterparts. It's interesting to see the differing outlook of the two but to also highlight the theme of both pieces as a fight for further racial equality.

Booker T. Washington and W.E.B DuBois both were great African American role models and leaders during their lives. Now given the asked audience some may lean their support and towards one over the other.

I personally can rationalize the concept behind Booker T. Washington's "The Atlantic Compromise" speech of 1895. I felt he had good intentions especially with the "Cast down your bucket" analogy and was trying his best to prove to the White Southerns that African Americans can be an asset and ally to them and not just as manual labor. Everyone has to earn their kept, work hard for what they want in life and this was no different then what Washington was trying to get the White man to understand. He offered a powerful speech that in my interpretation stated "give us a chance". Look not at our color but our ability to contribute to society. He focused on industrial learning and pitched that with industrial education that his people (African Americans) could also looked to as upstanding citizens of the community. He pitched the the idea that let us prove to you that we too should be given an opportunity. He was adamant for both whites and blacks to have an open mind of each other. He had humble intentions I feel; regardless of the criticism his speech received.

W.E.B DuBois, as stated from the NAACP (https://www.naacp.org/naacp-history-w-e-b-dubois/) was an activist, scholar, and global citizen. He was so well educated and served many roles through out his productive life. It's very admiring he was the first black person to received a PH-D ever especially at the elite Harvard University.<br> Dubois dedicated his life's work to bettering the lives for ALL African American's not just the one's who would be a better pillar of the community. The Famous "We hold these truths to be self-evident: That all men are created equal; that they are endowed by their Creator with certain unalienable rights; that among these are life, liberty, and the pursuit of happiness." (Source: W. E. Burghardt Du Bois, The Souls of Black Folk (Chicago, 1903) is a common passage in history that almost everyone has heard or read somewhere. It delivers a powerful message to all races not just those who feel they should be more prestigious than others. Although DuBois applauded Booker T. Washington's Atlantic Promise speech more criticism and anger I feel resulted. He did not agree with many of the things Washington stated because DuBois felt Washington was belittling the African American race stating we are not Negros we are people. He also mentions that industrial education was not the only answer. The fact that Booker T. Washington layed out things seemed like an insult to many African Americans. He places blame not on Washington but on the nation for how the world was towards his race.

A lot to take in, both men were hero's to many and paved the way for African American progress.

I am certain that these areas will contribute to the growth of social media

I struggle as a parent of a toddler (3yo) with the idea that we even use language like "curriculum" for what goes on in a child care center - but wouldn't it be great if these were considerations that could be more explicitly discussed for those settings? These caretakers are part of our family and they are integral to raising our kids - maybe it's just where I live it feels like critical race parenting questions just aren't explicit.

The word "temporal" is relative to time. Poetry having a "temporal dimension" means that it's content can pertain to current events. As discussed in class, time can be identified through what a poem tells, just like music.

Not even just their fellow psychology professors; I believe that society as a whole chooses not to recognize people's barriers because it's easier to judge that to accommodate.

I don't completely agree with this statement. Although the barriers that people go through are legitimate, some people with no "barrier" can be legitimately lazy. Being lazy is unwilling to do work or use energy. I am lazy sometimes when there are no barriers between me and doing work. Sometimes I would rather play games instead of doing work I need to do. And I know it's my fault, not any barriers that were placed in front of me.

It’s not that big of deal to him and he should just let it go.

My Mom works in human resources for a massive pharmaceutical company, so I've talked with her a lot about this hyperinflation of metrics that ultimately serves no purpose. It's very easy to get wrapped up in the numbers because the numbers are usually trustworthy. What's interesting to me here is that Wong and Gerras note that "few collectors... believe in the data they are collecting". It's gotten to the point where they are apparently just going through the motions and having enough data to check off their own boxes.<br> It seems to me that these requirements of soldiers in the field have gotten so burdensome that it may be becoming a tactical disadvantage. Instead of spending countless hours generating reports and checking in, those soldiers could be training for the next mission or preparing for contingencies. Statistics and data can be invaluable in certain circumstances, but the inflation of bureaucracy at all levels seems to always have a tendency to produce requirements that, in the end, have little to no benefit.

Aniyah Rucker

Why do you think she is questioning if she's really bubbly and happy? Do you think she is just using it as a disguise so that she won't be judged by the opposing race or do you believe that is the type of person she truly is and want to be?

We're meat bags with an organic computer in our heads, it doesn't have to be that heightened

not a problem that ppl are working to address, maybe because it's hard to prove that students aren't just guilty and deserving

Reviewer #3:

The methods used by the authors seem like potentially really useful tools for research on neural activity related to sequences of stimuli. We were excited to see that a new toolbox might be available for these sorts of problems, which are widespread. The authors touch on a number of interesting scenarios and raise relevant issues related to cross-validation and inference of statistical significance. However, given (1) the paucity of code that they've posted, and its specificity to specific exact data and (2) the large literature on latent variable models combined with surrogate data for significance testing, I would hesitate to call TDLM a "framework". Moreover, in trying to present it in this generic way, the authors have made it more difficult to understand exactly what they are doing.

Overall: This paper presents a novel approach for detecting sequential patterns in neural data however it needs more context. What's the contribution overall? How and why is this analysis technique better than say Bayesian template matching? Why is it so difficult to understand the details of the method?

Major Concerns:

The first and most important problem with this paper is that it is intended (it appears) to be a more detailed and enhanced retelling of the author's 2019 Cell paper. If this is the case, then it's important that it also be clearer and easier to read and understand than that one was. The authors should follow the normal tradition in computational papers:

Present a clear and thorough explanation of one use of the method (i.e., MEG observations with discrete stimuli), then present the next approach (i.e., sequences?) with all the details necessary to understand it.

The authors should start each section with a mathematical explanation of the X's - the equation(s) that describes how they are derived from specific data. Much of the discussion of cross validation actually refers to this mapping.

Equation 5 also needs a clearer explanation - it would be better to write it as a sum of matrices (because that is clearer) than with the strange "vec" notation. And TAUTO, TF and TR should be described properly - TAUTO is "the identity matrix", TF and TR are "shift matrices, with ones on the first upper and lower off diagonals".

The cross validation schemes need a clear description. Preferably using something like a LaTeX "algorithm" box so that they are precisely explained.

Recognizing the need to balance readability for a general reader and interest, perhaps the details could be given for the first few problems, and then for subsequent results, the detail could go into a Methods section. Alternatively, the methods section could be done away with (though some things, such as the MEG data acquisition methods are reasonably in the methods).

Usually, we think about latent variable model problems from a generative perspective. The approach taken in this paper seems to be similar to a Kalman filter with a multinomial observation (which would be equivalent to the logistic regression?), but it's unclear. Making the connection to the extensive literature on dynamical latent variable models would be helpful.

Minor concerns:

1) Many of the figures, and some of the text are from the 2019 Cell paper. The methods text is copied verbatim without citation.

2) The TLDM model is presented without context or comparison to other computational approaches employed to identify sequences. Is it also used in the 2016 Kurth-Nelson paper? How does it compare, e.g., to Bayesian template matching (in the case of hippocampal data)?

3) Cite literature from recent systems neuroscience using hidden Markov models and related discrete state space approaches on neural activity.

4) How does this method deal with a long sequence for which the intra-sequences have variance in their delta t's? Or data where the observations have some temporal lag relative to each other?

5) In the "sequences of sequences" section, the authors talk about combining states into meta states. But then the example they give, it appears they just use their vanilla approach. This whole section belongs in a different place than a "supplemental note". The data need proper attribution, an IACUC/ethics statement, etc.

6) While code can be useful, it is not archival in the same way equations are. Supplementary Note 1 should be in the Methods, and needs to be rewritten in such a way that it explains the steps (i.e., in an algorithm box) rather than just using code. Moreover, when the data generated via this code is used in the text, this section in the methods can be mentioned/linked.