This quote from Duncan shows the frustration that he and other British had towards the colonists for questioning anything that England does. They viewed themselves as completely superior, so anything out of line triggered them. Cora's response reminds him of the hardships the colonist experienced to settle there as well as what it costed them. This is a good example of the tensions that started to arise between the colonists and the British; where they are starting to form their own identity without the authority of England.

• This wasn't related to any readings for this movie, but signs of colonists revolting was brought up during the lecture that week.

In these couple lines, Webb is basically saying that the King would accept the colonists defense against any "savage" attack, especially if they were brought on by the French. This portrays the small conflicts that were going on between the French and the British where the colonists would have to pay. Comparing this to readings of that time, these events seem historically accurate as there were raids carried out by Native Americans on their former lands.

• Calloway, First Peoples, 168-169

This matched with what actually happened to Fort William Henry, where the French put constant pressure on the fort. The French knew that the British had to give in eventually, and when supplies ran low they did.

• Greig Santos-Buch. 2024. “Understanding the Siege and Battle at Fort William Henry.” May 15, 2024.

Gifts and trade were one of, if not the most popular ways of diplomacy between the natives and the British/French. This quote by Hawkeye describes that, but instead he talks about how the use of these gifts manipulate tribes into giving up their cultural values. This portrays colonialism in one of it's worst forms, where it tears apart tribes and lands.

• Calloway, World Turned Upside Down, 140

Magua's line's here represents the culture disconnect from the Natives and the British. One of these biggest contrasts are the gender roles, where women in British culture relied on men more than in Native cultures. This also speaks to the very negative view that lots of tribes had on the British.

• “The British Era.” n.d. Minnesota Historical Society.

The romantic and deeply mythological lines here serve to help understand the historical context they were in. Lots of captures by Indians were treated harshly, but some, especially women and children, weren't as they might have been adopted into the tribe's culture. Here is a specific saying that is only known in Hawkeye's culture, a sign that he is not only opening up to Cora but maybe wants her to learn his culture. Also, it is believed that the Natives had a deep respect for nature and that the elements were connected with them spiritually.

• Calloway, First Peoples, 169-170

Obviously this is a TV show, it this does happen to so many women with families. I am very strong willed and would not put up with being so submissive or subtle. People and children need to be aware of people’s feelings. And as a parent and wife you need to be strong enough emotionally to communicate that.

Very true. At the end of the day we are all human. Sometimes we just have to be more aware of how we interact with other cultures, because we would not want to come across as rude or insensitive.

This is very true! Especially when going to big cities it’s easy to be disgusted at the homelessness and drug use, it’s hard to not make faces or want to help. In bigger cities it’s more common to hide your facial expressions and just look at the ground instead of a more blunt approach.

This is a very interesting example of how much power the internet itself holds. The fact that K-pop fans managed to crash an entire software is quite amazing. Similar to the fact that 4chan users arranged a whole rendezvous for single men just to laugh at them, but for a greater cause. K-pop has only grown since 2020, which may very well eventually raise the question of what other tricks they have up their sleeve.

Another funny instance of this I thought of were the sugarfree Haribo Gummy Bears. The sweetener used in the bears were actually laxatives, and so online users went ahead and posted all types of wild parody stories of them eating the bears and suffering immense digestive pain from destroying the toilet to going to the hospital. While parody reviews definitely have a humor factor, I also think that they have the potential to make or break a product based on the overall perceived notion of the parody reviews collective.

I have seen this being done on yelp by people who really disliked a restaurant they went to. They will post photos of the food purposefully shading it in a bad light and then write a sarcastic review giving the restaurant 5 stars. While it isn't nice of them to be ingenuine I think it isn't completely harmful as the restaurant still gets 5 stars from them and benefits from that.

It's dangerous to see how for the sake of entertainment or trying to make other people think one is witty that they are playing along with unethical actions

Trolling has its pros whether it be bringing people together, creating communities (ex people who like trolling those indian scammer call centers) and it can surely get a laugh out of people (which I think is the main reason people troll)

I feel like this example of trolling is more of a form of digital protest as they leveraged the disruption of this troll to actually support a cause. By using trolling in a way to overwhelm platforms with random content, it helps protestors fight against the surveillance and cooperate actions that social media websites take, and this can be a very effective from of activism.

Unable to produce children

High school Bio then were two types of survived ship curve to increase population

make (something, typically an undesirable situation or an unfounded belief) continue indefinitely.

Ignore this highlight

I feel like people would pay more attention to what is taught in schools if they read this sentence. Not only because they would learn that Hitler got his idea for concentration camps from the United States but because of the statement, "Ironically, Adolf Hitler displayed more knowledge of how we treated Native Americans than American high schoolers today who rely on their textbooks." This is a scary thought, In a sense, Loewen is highlighting that a terrible man in history, who is not an American, knows more about American history than kids sitting in a classroom in the United States. I feel like this statement alone sums up just how much the education system keeps from us. It kind of makes me mad to think that horrible dictator knew more about my country than I do. I feel like more people should feel this way and make sure a change happens for future generations.

This is a great start to how we should think about the information that is exposed to students. The main problem that we see from this book is that the education system does not think students are capable of forming their own opinions about history. I feel like that is why a lot of the history we learn in high school is 'watered down," so to speak. I believe the education system can make up for its mistakes by doing its research. The problem is that we do not know if everyone who is in charge of what is required for students to learn understands what information is being left out. They simply could be going with what they know. The other problem is that, sadly, there are people in charge of the education system who know exactly what FACTS they are withholding. Hopefully, deceiving of what truly happened in our country's history will change shortly.

This statement shocked me. While we didn't go into deep detail about the Louisiana Purchase, it was emphasized that the United States bought the land from France. It is disappointing that this piece of history was withheld from my education. Loewen is correct when discussing how the Indians were portrayed in our education. All my life, I thought the stories of Native Americans not understanding land ownership were true. Because, sadly, those stories made sense. Native Americans were painted as people who just lived by the land and had no rule of law. However, Loewen gives us many examples of how the education system failed us by not debunking that statement,

First Instance: * Thesiger J. suggested sellers' terms "shall prevail" bound future dealings, implying buyer agreement. * Rejected on appeal to prevent unfair imposition of terms by offerors. Second Suggestion (Professor Guest): Divergence in reply leads to a counter-offer; acceptance could be implied from silence. * Concerns raised by Bridge L.J. and Lord Denning about the implications of this approach. Example Concern: * If a counter-offer significantly undercuts the original offer (e.g., £72,000 vs. £60,000), sellers may not expect a response, aligning with traditional commercial expectations.

A definite and seasonable expression of acceptance or a written confirmation which is sent within a reasonable time operates as an acceptance even though it states terms additional to or different from those offered or agreed upon, unless acceptance is expressly made conditional on assent to the additional or different terms

1. Timely acceptance valid even with different terms unless: Acceptance conditions on agreeing to additional terms.
2. Additional terms: Considered proposals for the contract. Become part of the contract between merchants unless: Offer limits acceptance to original terms. Terms materially alter the contract. Objection is given promptly.

formation of accpt

NCC can refer to content-specific NCC or full NCC.

Disorder of consciousness in which patients retain autonomic reflexes and the ability to spontaneously open their eyes despite being completely unresponsive in every other way.

An area of the posterior cerebral cortex spanning the temporal, parietal, and occipital lobes. There is a strong possibility that it is the location of the full and content specific NCC. Content specific NCC are populations of neurons associated with specific perceptual experiences (e.g., facial recognition).

An experimental model in which groups that provide subjective reports of what they perceive (a presented stimulus) are compared with groups that do not provide such reports. Instead, we attempt to determine what the latter group is perceiving via physiological measures (neuroimaging technology). This method allows the NCC to be distinguished from activity which relates to them.

In behavioral paradigms, consciousness is evaluated using verbal reports or physical responses indicating the perception of a presented stimulus. The accuracy of these reports/responses is unreliable when said stimuli are dubiously perceptible, because it is hard to tell whether the subject actually perceived the stimulus or simply guessed its position correctly. Forced-choice procedures can determine the subject's objective awareness of the stimulus by minimizing their subjective bias.

This quote shows how the executions reinforced the oppressive power supporting slavery. Also, the idea that racism was further developed due to the treat of rebellion.

Test

It seems that you're able to make notes on commits. Since a commit can be referenced by a tag, or branch, you can make notes on those, too -- kind of.

I have wondered if git's notes function would allow one to keep some kind of issue management system within the repository itself.

The passage effectively highlights the similarities in Hoover and Al Smith's platforms but could expand on why religion and Prohibition became focal points of the 1928 campaign. Adding more context about Hoover’s rise as Coolidge’s natural successor and his public service background would strengthen the analysis.

Despite underlying issues in both industrial and agricultural sectors, many Americans in 1929 and 1930 remained optimistic about a quick economic recovery. President Herbert Hoover reinforced this belief by confidently declaring in 1930 that “the depression is over,” underestimating the severity of the crisis.

Despite efforts by Populists and Progressives, economic inequality widened in the early 20th century, with wealth concentrating among the rich. Conservative policies in the 1920s, including tax cuts and deregulation, fueled this divide, benefiting the wealthiest Americans far more than the general population.

The Stock Market Crash was important because it marked the sudden collapse of an inflated economy fueled by speculation, leading to widespread financial panic. This crash triggered a chain reaction of bank failures, mass unemployment, and economic hardship that defined the Great Depression in the U.S. and globally.

Though the romance between Pocahontas and John Smith never happened, Pocahontas might have influenced the fate of John Smith. According to the Park Service, a theory is Pocahontas placed her head upon his when the Chief was about to bash his head open, but this has been debated for years.

McClurken, Jeff. “Pocahontas.” University of Mary Washington, September 3rd, 2024.

“Pocahontas: Her Life and Legend.” National Park Service, September 4, 2022. https://www.nps.gov/jame/learn/historyculture/pocahontas-her-life-and-legend.htm

This line shows the English's lack of empathy for anyone or anything due to the selfish drive to get rich. They believed that because they came there they are now the rulers of the land and the Natives are just uncivilized savages, which is far from the truth.

McClurken, Jeff. “Pocahontas.” University of Mary Washington, September 3rd, 2024.

When John asked Pocahontas about the gold, as shown in these lines, she thought he was talking about corn. Native Americans did not see gold as something of value, they valued resources like crops and animal skins. They lived by what they needed, not what they wanted. The British were after what they wanted, a material that was given value, not inherently valuable.

McClurken, Jeff. “Pocahontas.” University of Mary Washington, September 3rd, 2024.

These lines further explore the conceptions the British people had about Natives. The British believed that their way of life was superior. Though they took it upon themselves to rob and fight the natives because of the natives' skilled way of the land. They had captivated the land, and the British just replied on trade, occasionally, and taking it from them. Yet, they thought their way of life was more "civilized".

McClurken, Jeff. “Pocahontas.” University of Mary Washington, September 3rd, 2024.

This excerpt of the script shows the context in which Natives have knowledge of the land and how to benefit from it. Unlike the Natives, the British did not understand how to captivate the potentials of the land. They assumed the land would already be rich in nutrients, and didn't prepare to be laborers.

McClurken, Jeff. “Pocahontas.” University of Mary Washington, September 3rd, 2024.

This excerpt from the script portrays the English view on Native Americans during this time. They referred to them as "savages" and treated them with no respect. Native Americans were seen as an obstacle to the "get rich quick" goal they were pursuing.

McClurken, Jeff. “Pocahontas.” University of Mary Washington, September 3rd, 2024.

This excerpt of the script further proves the point that the most important goal the British had was to get rich and claim their riches in Britain. Colonizing wasn't necessarily a goal at first.

McClurken, Jeff. “Pocahontas.” University of Mary Washington, September 3rd, 2024.

When the English arrived, the Native Americans were already fighting amongst themselves. They understood the land and its advantages and disadvantages, unlike the British.

McClurken, Jeff. “Pocahontas.” University of Mary Washington, September 3rd, 2024.

The filmmakers tried to portray the ideals the British had when setting sail to the Americas. The Script indicates the Virginia Company believed the land would be filled with gold and rich with valuable resources, yet this was far from the truth.

McClurken, Jeff. “Pocahontas.” University of Mary Washington, September 3rd, 2024.

'scrap' tnv amd replace them by 'reasonable implementation'

This sentence that I highlighted the growth of colonial settlements into powerful self sustaining places. The statement "warring against Native Americans" refers to the constant conflict between European settlers and the indigenous people which were often fought over land and resources.This reflect the mindset of the colonist who viewed the land as theirs regardless of native being there prior

The sentence that I highlighted the growth of colonial settlements into powerful self-sustaining places. The statement "warring against the Native American" refers to the constant conflict between the European settlers and the indigenous people which often fought over land and resources. This reflects the mindset of colonist who viewed the land as their own prior to the Native American being there before them. The phrase 'subduing internal upheaval' probably refers to both the political and social struggles with the colonies.

The mentality of “I hit the game winning shot because I’m the best” vs “I missed the game winning shot because you didn’t get me the ball fast enough” Fascinating to see these examples from life be put into a structured explanation.have a coworker who, if there is a mistake, will always be the last to get back to the office and then proceed put the blame on someone else. If the job is completed on time, looks good and mistake free he will be the first one in the office reporting to the boss how good he did on the job.

This fundamental attribution error seems to be a rabbit hole our society is falling down. I had always referred to it as victim culture. We as a society are unable to see how our external attribution to a situation plays a role in the outcomes in life. Everything bad that happens to us is because somebody else is bad and out to get me. It is much easier to blame someone else, for reasons your assuming, then it is to realize the mistakes that were made.

Producer closes their channels. Consumers should exit when the channel they're polling on is closed.

Receive on closed channel's fine.

Since I was younger I’ve known that we have beliefs that are instilled dingo us, knowingly and unknowingly. The part that is always difficult for me is how do I notice them, and how do I change them. One of the Schemata I have realized is the way I perceive money. I have always felt like I never had enough, and it was going to run out. However, after 10 years of working, I have never gone without, been homeless or starved. I recognize the schema now I am focused on changing that database.

There is something similar in the psychology realm called Baader-Meinhoff. This is the phenomenon where you buy something, say a new silver Toyota Camery. All of a sudden it feels like every car you are seeing on the roads is a silver Toyota Camery. This happened to me when I bought my new truck, it made me think I made a good decision because of how many other people had the same truck. I wasn’t looking for the truck but it seemed like it was the only vehicle on the road that ever caught my attention.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

Summary:

The Notch signaling pathway plays an important role in many developmental and disease processes. Although well-studied there remain many puzzling aspects. One is the fact that as well as activating the receptor through trans-activation, the transmembrane ligands can interact with receptors present in the same cell. These cis-interactions are usually inhibitory, but in some cases, as in the assays used here, they may also be activating. With a total of 6 ligands and 4 receptors, there is potentially a wide array of possible outcomes when different combinations are co-expressed in vivo. Here the authors set out to make a systematic analysis of the qualitative and quantitative differences in the signaling output from different receptor-ligand combinations, generating sets of "signaling" (ligand expressing) and "receiving" (receptor +/- ligand expressing cells).

The readout of pathway activity is transcriptional, relying on the fusion of GAL4 in the intracellular part of the receptor. Positive ligand interactions result in the proteolytic release of Gal4 that turns on the expression of H2B-citrine. As an indicator of ligand and receptor expression levels, they are linked via TA to H2B mCherry and H2B mTurq expression respectively. The authors also manipulate the expression of the glycosyltransferase Lunatic-Fringe (LFng) that modifies the EGF repeats in the extracellular domains impacting their interactions. The testing of multiple ligand-receptor combinations at varying expression levels is a tour de force, with over 50 stable cell lines generated, and yields valuable insights although as a whole, the results are quite complex.

Strengths:

Taking a reductionist approach to testing systematically differences in the signaling strength, binding strength, and cis-interactions from the different ligands in the context of the Notch1 and Notch 2 receptors (they justify well the choice of players to test via this approach) produces a baseline understanding of the different properties and leads to some unexpected and interesting findings. Notably:

-                Jag1 ligand expressing cells failed to activate Notch1 receptor although were capable of activating Notch2. Conversely, Jag2 cells elicited the strongest activation of both receptors. The results with

Jag1 are surprising also because it exhibits some of the strongest binding to plate-bound ligands. The failure to activate Notch1 has major functional significance and it will be important in the future to understand the mechanistic basis.

-                Jagged ligands have the strongest cis-inhibitory effects and the receptors differ in their sensitivity to cis-inhibition by Dll ligands. These observations are in keeping with earlier in vivo and cell culture studies. More referencing of those would better place the work in context but it nicely supports and extends previous studies that were conducted in different ways.

-                Responses to most trans-activating ligands showed a degree of ultrasensitivity but this was not the case for cis-interactions where effects were more linear. This has implications for the way the two mechanisms operate and for how the signaling levels will be impacted by ligand expression levels.

-                Qualitatively similar results are obtained in a second cell line, suggesting they reflect fundamental properties of the ligands/receptors.

We appreciate the positive and constructive feedback.

Weaknesses:

One weakness is that the methods used to quantify the expression of ligands and receptors rely on the co-translation of tagged nuclear H2B proteins. These may not accurately capture surface levels/correctly modified transmembrane proteins. In general, the multiple conditions tested partly compensate for the concerns - for example, as Jag1 cells do activate Notch2 even if they do not activate Notch1 some Jag1 must be getting to the surface. But even with Notch2, Jag1 activities are on the lower side, making it important to clarify, especially given the different outcomes with the plated ligands. Similarly, is the fact that all ligands "signalled strongest to Notch2" an inherent property or due to differences in surface levels of Notch 2 compared to Notch1? The results would be considerably strengthened by calibration of the ligand/receptor levels (and ideally their sub-cellular localizations). Assessing the membrane protein levels would be relatively straightforward to perform on some of the basic conditions because their ligand constructs contain Flag tags, making it plausible to relate surface protein to H2B, and there are antibodies available for Notch1 and Notch2.

We agree that mCherry fluorescence does not provide a direct readout of active surface ligand levels. As the reviewer points out, the ability of Jag1 to activate Notch2 demonstrates that expressed Jag1 is competent for signaling. Further, in some cases, Jag1-Notch2 activation can be comparable to Dll1-Notch2 activation (Figure 2A). Following the reviewer’s suggestion, we performed a Western blot for multiple expression levels for each of three surface ligands (Dll1, Dll4, Jag1) (Figure 2—figure supplement 2). This blot revealed a signal for surface expression of Jag1. Interpretation is complicated by the expected dependence of the efficiency of surface protein purification on the number of primary amines in the protein, which varies among these ligands, and qualitatively correlates with the staining intensity. While this makes quantitative interpretation difficult, this result further supports the notion that Jag1 is present on the cell surface. Finally, we note that high signaling activity need not, in general, directly correlate with surface expression levels. In fact, one study showed an example in which increased ligand activity occurred with decreased basal ligand surface levels (Antfolk et al., 2017). While one would ideally like to know all parameters of the system, including surface protein levels, rates of recycling, etc. the perspective taken here is that the net effect of these many post-translational processing steps can be subsumed into the overall relationship between the expression of the protein (which, in our case, is read out by the co-translational reporter) and its activity, which is relevant for the behavior of developmental circuits, among other systems. To address this comment, we now explicitly mention the limitation of mCherry as a proxy for surface protein, and add a reference to previous work highlighting the relationship between surface levels and ligand activity.

In terms of the dependence of signaling on Notch levels, the metric of signaling activity used here is explicitly normalized by the mTurquoise co-translational reporter of Notch expression to account for differences in receptor expression across receiver clones. We have added a new figure to show the variation in expression (Figure 1—figure supplement 1A) and to demonstrate this normalization (Figure 1—figure supplement 5). Having said that, as the reviewer correctly points out, we cannot directly address the dependence on surface receptor levels with mTurquoise alone. To address this comment, we have added a figure that shows cotranslational and surface receptor expression for a subset of our receiver clones (Figure 1—figure supplement 1B). Although antibody binding strengths may vary, it appears unlikely that higher surface levels could explain most ligands’ preferential activation of Notch2 over Notch1, since Notch2 levels were lower than Notch1 levels in both surface expression and cotranslational expression.

Cis-activation as a mode of signaling has only emerged from these synthetic cell culture assays raising questions about its physiological relevance. Cis-activation is only seen at the higher ligand (Dll1, Dll4) levels, how physiological are the expression levels of the ligands/receptors in these assays? Is it likely that this would make a major contribution in vivo? Is it possible that the cells convert themselves into "signaling" and "receiving" sub-populations within the culture by post-translational mechanism? Again some analysis of the ligand/receptors in the cultures would be a valuable addition to show whether or not there are major heterogeneities.

The cis-activation results in this paper are, as the reviewer points out, conducted in synthetic cell culture assays. Cis-activation is observed across a large dynamic range of ligand expression, possibly including non-physiologically high levels. However, our previous work (Nandagopal et al, eLife 2019) showed that cis-activation does not require over-expression, as it occurred in unmodified Caco-2 and NMuMG cells with their endogenous ligand and receptor expression levels. As shown here in Figure 4B, cis-activation for Notch2 increases monotonically and is substantial even at intermediate ligand concentrations. In other cases, cis-activation is maximal at intermediate concentrations. We agree that the in vivo role remains unclear, and is difficult to determine due to the typical close contacts among cells in tissues. Therefore, these assays do not speak to in vivo relevance. Note that we can, however, rule out the possibility of trans signaling between well-mixed cell populations at these densities (Figure 4A).

It is hard to appreciate how much cell-to-cell variability in the "output" there is. For example, low "outputs" could arise from fewer cells becoming activated or from all cells being activated less. As presented, only the latter is considered. That may be already evident in their data, but not easy for the reader to distinguish from the way they are presented. For example, in many of the graphs, data have been processed through multiple steps of normalization. Some discussion/consideration of this point is needed.

We agree that in different experiments changes in a mean response can reflect changes in fraction of activated cells, or level of activation or some combination of both. In this work, most assays were conducted by flow cytometry, which provides a full distribution of cellular responses. We provided distributions for some experiments in the supplementary figures (i.e., Figure 4—figure supplement 1, and Figure 5—figure supplement 4). The sheer number of experiments and samples prevents us from displaying all underlying histograms. Therefore, we have provided all flow data sets in an extensive archive that is publicly available on data.caltech.edu (https://doi.org/10.22002/gjjkn-wrj28).

Impact:

Overall, cataloging the outcomes from the different ligand-receptor combinations, both in cis and trans, yields a valuable baseline for those investigating their functional roles in different contexts. There is still a long way to go before it will be possible to make a predictive model for outcomes based on expression levels, but this work gives an idea about the landscape and the complexities. This is especially important now that signaling relationships are frequently hypothesized based on single-cell transcriptomic data. The results presented here demonstrate that the relationships are not straightforward when multiple players are involved.

We appreciate this concise impact summary, and agree with its conclusions.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors extend their previous studies on trans-activation, cis-inhibition (PMID: 25255098), and cis-activation (PMID: 30628888) of the Notch pathway. Here they create a large number of cell lines using CHO-K1 and C2C12 cells expressing either Notch1-Gal4 or Notch2-Gal4 receptors which express a fluorescent protein upon receptor activation (receiver cells). For cis-inhibition and cis-activation assays, these cells were engineered to express one of the four canonical Notch ligands (Dll1, Dll4, Jag1, Jag2) under tetracycline control. Some of the receiver cells were also transfected with a Lunatic fringe (Lfng) plasmid to produce cells with a range of Lfng expression levels. Sender cells expressing all of the canonical ligands were also produced. Cells were mixed in a variety of co-culture assays to highlight trans-activation, cis-activation, and cis-inhibition. All four ligands were able to trans-activate Notch1 and Notch 2, except Jag1 did not transactivate Notch1. Lfng enhanced trans-activation of both Notch receptors by Dll1 and Dll2, and inhibited Notch1 activation by Jag2 and Notch2 activation by both Jag 1 and Jag2. Cis-expression of all four ligands was predominantly inhibitory, but Dll1 and Dll4 showed strong cis-activation of Notch2. Interestingly, cis-ligands preferentially inhibited trans-activation by the same ligand, with varying effects on other trans-ligands.

Strengths:

This represents the most comprehensive and rigorous analysis of the effects of canonical ligands on cis- and trans-activation, and cis-inhibition, of Notch1 and Notch2 in the presence or absence of Lfng so far. Studying cis-inhibition and cis-activation is difficult in vivo due to the presence of multiple Notch ligands and receptors (and Fringes) that often occur in single cells. The methods described here are a step towards generating cells expressing more complex arrays of ligands, receptors, and Fringes to better mimic in vivo effects on Notch function.

In addition, the fact that their transactivation results with most ligands on Notch1 and 2 in the presence or absence of Lfng were largely consistent with previous publications provides confidence that the author's assays are working properly.

We appreciate the thoughtful comments and feedback.

Weaknesses:

It was unusual that the engineered CHO cells expressing Notch1-Gal4 were not activated at all by co-culture with Jag1-expressing CHO cells. Many previous reports have shown that Jag1 can activate Notch1 in co-culture assays, including when Notch1 was expressed in CHO cells. Interestingly, when the authors used Jag1-Fc in a plate coating assay, it did activate Notch1 and could be inhibited by the expression of Lfng.

In our assays, we do in fact also see some signaling of Jag1 to Notch1, especially when dLfng is coexpressed (Figure 2—figure supplement 4, formerly Figure 2—figure supplement 3). While these levels are lower than those observed for other ligand-receptor combinations, they are significantly elevated compared to baseline. In specific natural contexts, it will be important to determine whether the weak but non-zero Jag1-Notch1 signaling acts negatively to suppress signaling from other ligands, or provides weak but potentially functionally important levels of signaling. Evidence for both modes exists in the literature. To address this, we have expanded the discussion of Jag1-Notch1 signaling and added references to other work on Jag1-Notch1 signaling to the Discussion section.

The cell surface level of the ligands was determined by flow cytometry of a co-translated fluorescent protein. Some calibration of the actual cell surface levels with the fluorescent protein would strengthen the results.

This issue was also raised by Reviewers #1 and #3. Please see responses to Reviewer #1, above.

Reviewer #3 (Public Review):

Summary:

This manuscript reports a comprehensive analysis of Notch-Delta/Jagged signaling inclusive of the human Notch1 and Notch2 receptors and DLL1, DLL4, JAG1, and JAG2 ligands. Measurements

encompassed signaling activity for ligand trans-activation, cis-activation, cis-inhibition, and activity modulation by Lfng. The most striking observations of the study are that JAG1 has no detectable activity as a Notch1 ligand when presented on a cell (though it does have activity when immobilized on a surface), even though it is an effective cis-inhibitor of Notch1 signaling by other ligands, and that DLL1 and DLL4 exhibit cis-activating activity for Notch1 and especially for Notch2. Notwithstanding the artificiality of the system and some of its shortcomings, the results should nevertheless be a valuable resource for the Notch signaling community.

Strengths:

(1)  The work is systematic and comprehensive, addressing questions that are of importance to the community of researchers investigating mammalian Notch proteins, their activation by ligands, and the modulation of ligand activity by LFng.

(2)  A quantitative and thorough analysis of the data is presented.

Weaknesses:

(1) The manuscript is primarily descriptive and does not delve into the underlying, mechanistic origin or source of the different ligand activities.

We agree that the goals of this paper were largely to discover the range of signaling modes that occur. A mechanistic analysis would be beyond the scope of this work, but we agree it is an important next step.

(2) The amount of ligand or receptor expressed is inferred from the flow cytometry signal of a co-translated fluorescent protein-histone fusion, and is not directly measured. The work would be more compelling if the amount of ligand present on the cell surface were directly measured with anti-ligand antibodies, rather than inferred from measurements of the fluorescent protein-histone fusion.

This issue was also raised by Reviewers #1 and #2. Please see responses to Reviewer #1, above.

(3) It would be helpful to see plots of the raw activity data before transformation and normalization, because the plots present data after several processing steps, and it is not clear how the processed data relate to the original values determined in each measurement.

We included examples showing how raw data is processed in Figure 4—figure supplement 1 and Figure 5—figure supplement 4. The sheer number of experiments precludes including similar figures for all data sets. However, all raw and processed data and data analysis code is publicly available at (https://doi.org/10.22002/gjjkn-wrj28).

(4) The authors use sparse plating of engineered cells with parental (no ligand or receptor-expressing cell to measure cis activation). However, the cells divide within the cultured period of 22-24 h and can potentially trans-activate each other.

If measured cis-activation signal arises solely from trans-activation, then the measured cis-activation signal per cell should increase with cell density, since trans-activation per cell does depend on cell density (Figure 4A). However, for the strongest cis-activators (Dll1- and Dll4-Notch2), signaling magnitude is similar when these cells are cultured sparsely or at confluence, which would otherwise allow efficient trans signaling (Figure 5A). Thus, for Dll1- and Dll4-Notch2 receivers, total signaling strength per cell depends little or not at all on the opportunity to signal intercellularly. Moreover, cis-activation signal for the Dll1- and Dll4-Notch2 combinations exceeded the maximum trans-signaling levels we could achieve for the same receivers when cis-ligand was suppressed (Figure 4B). These results argue that cis interactions dominate signaling in this context. However, we have not ruled out the possibility that trans-signaling between sister cells after division contributes to the comparatively weak cis-activation observed for Notch1 receivers.

Reviewer #1 (Recommendations For The Authors):

As outlined in the public review, there is a question of whether the nuclear H2B accurately reflects the surface levels of the transmembrane proteins (ligand and receptor). Clearly, it would not be feasible to check levels in all of the experimental conditions, but some baseline conditions should be analyzed.

We addressed this above.

Reviewer #2 (Recommendations For The Authors):

(1)  As mentioned above, it was unusual that Jag1 did not activate Notch1 in co-culture assays, but did activate Notch1 in plate-coating assays. The authors should add some text to the Discussion to explain why they think this is happening in their engineered cells. One possibility is that the CHO cells express Manic fringe (Mfng) which is known to reduce Jag1-Notch1 activation. Data for Mfng levels in CHO cells were not included in Supplemental Table 2. Knocking down all three Fringes in CHO cells might increase Jag1-Notch1 activation.

This is already addressed in a sentence in the results: “Strikingly, while Jag1 sender cells failed to activate Notch1 receivers above background (Figure 2D), plate-bound Jag1-ext-Fc activated Notch1 only ~3-fold less efficiently than it activated Notch2 (Figure 3B-D). This suggests that the natural endocytic activation mechanism, or potential differences in tertiary structure between the expressed and recombinant Jag1 extracellular domains, could play roles in preventing Jag1-Notch1 signaling in coculture.” Regarding the point about Mfng, we added a note to Supplementary Table about other CHO-K1 expression data.

(2) Figure 1-supplemental figure 1: Both the Notch1-Jag1 and Notch1-Jag2 cells show high expression of Jag1 in low 4epi, but any higher concentration reduces to control levels. How much of a problem is this for interpreting your data?

This was not the ideal behavior, but by binning cells by co-translational reporters for ligand expression, we were able to obtain enough cells in intermediate bins. (Note: Figure 1—figure supplement 1 is now Figure 1—figure supplement 2.)

(3)  Figure 1C legend: Are these stably-expressing cells or Tet-off cells? Please state in legend.

The figure legend has been updated.

(4)  Figure 1E: How long is the knockdown of Rfng and Lfng effective? Does it affect the expression of Lfng later?

siRNA effects generally last for at least 72-96 hours, so we do not anticipate this being an issue.

(5) Page 9: "Lfng significantly decreased trans-activation of both receptors by Jag1 (>2.5-fold)". If there is no Jag1-Notch1 activation, how can Lfng decrease trans-activation?

We added a note in the main text to clarify that while Jag1-Notch1 signaling is relatively low, it can still be detectably decreased.

(6) Figure 4A legend: Please define what "2.5k ea senders and Rec" means. In the text, it says "To focus on cis-interactions alone, we then cultured receiver cells at low density, amid an excess of wildtype CHO-K1 cells" (page 14).

This was clarified in the text.

(7)  Page 14: "By contrast, Notch2 was cis-activated by both Dll1 and Dll4, to levels exceeding those produced by trans-activation by high-Dll1 senders (Figure 4B, lower left)." Where is the trans-activation data? 4B, lower right?

We updated this reference in the main text.

(8)  Page 16: "For Notch2-Dll1 and Notch2-Dll4, single cell reporter activities correlated with cis-ligand expression, regardless of whether cells were pre-induced at a high or low culture density (Figure 4D)." It appears that Notch2-Dll1 has lower Notch activation at sparse culture than confluent.

We agree that the level signaling is lower in sparse compared to confluent on average. This is explained by the sensitivity of the Tet-OFF promoter to culture density (Figure 4—figure supplement 2). However, the key point of this experiment is the positive correlation, which is consistent with cis-activation, and inconsistent with the pre-generation of NEXT hypothesis diagrammed in Figure 4C, which would not be expected to produce such a correlation.

(9a) For the creation of the C2C12-Nkd cells: Has genomic sequencing been done to confirm editing of Notch2 and Jag1 loci?

We confirmed the knockdown but did not do genomic sequencing.

(9b) The gel in Figure 7-Supplement 1C is not adequate for showing loss of Jag1. It should be repeated.

In this case, we have only the single gel. We added a note in figure legend that no duplicate was performed.

(10) Figure 7A: Which Fringes are expressed in C2C12 cells? You should provide a rationale for knocking down just Rfng.

Figure 7—figure supplement 1A shows the levels of expression in C2C12. Note that Mfng is not highlighted because its levels were undetectable.

(11) Figure 7-Supplement 1D: This is confusing. Notch2 levels are not reduced in the left panel, and Notch1 and Notch2 levels are not reduced in the right panel?

C2C12-Nkd cells exhibit reduced levels of Notch1 and Notch3. This can be seen in Figure 7—figure supplement 1A. Panel D presents the results of additional siRNA knockdown, performed to prevent subsequent up-regulation of Notch1 and Notch3 during the assay. These knockdown results were variable, as shown. The Notch2 siRNA knockdown was not essential for these experiments, but performed despite very low levels of Notch2 to begin with. In the revision, we have added this note to the Methods.

Reviewer #3 (Recommendations For The Authors):

(1) The results section of the manuscript is very dense and difficult to follow, as are the figure legends.

We appreciate the criticism, and regret that it is not easier to read in its current form.

(2) The authors could emphasize areas of concordance with published results (where available) to place their artificial, engineered system into a better biological context. Are there any examples of studies in whole organisms where cis-activation plays a role?

We are not aware of examples of cis-activation in whole organisms at this point.

(3) How do the authors rationalize the different responses of Notch1 to cell-presented Jag1 as opposed to immobilized Jag1, where its signal strength is second in rank order on a molar basis?

This comment was addressed above in response to the first recommendation from Reviewer #2.

It is also difficult to understand Figure 2_—_figure Supplement 3B, in which it appears that Jag1 induces a Notch1 reporter response when LFng is knocked down (dLfng), and how those data relate to the inactive response to Jag1 shown in the main figures.

The issue here is a difference of normalization. Figure 2A in the main text is normalized to the sender expression level, i.e. relative signaling strength. By contrast, Figure 2—figure supplement 4B (previously Figure 2—figure supplement 3B) shows absolute signaling activity, which can appear higher because it does not normalize for ligand expression. For Jag1-Notch1 signaling in particular, substantial signaling required very high levels of Jag1. We have added a new figure to demonstrate these two types of normalization (Figure 2—figure supplement 1A).

See the Authr response image 1 below for a direct comparison of these two normalization modes using data from both Figure 2A and Figure 2—figure supplement 4B. Note how the Jag1-Notch1 signaling activities that are nonzero in the top plot go to zero in the bottom plot as a result of normalizing the values to ligand expression.

Author response image 1.

Comparison of normalization modes in Figure 2A and Figure 2—figure supplement 4B (formerly 3B).

Normalized trans-activation signaling activities for different ligand-receptor combinations (with dLfng only), either with further normalization to ligand expression (bottom row) or without further normalization (top row). Normalized signaling activity is defined as reporter activity (mCitrine, A.U.) divided by cotranslational receptor expression (mTurq2, A.U.), normalized to the strongest biological replicate-averaged signaling activity across all ligand-receptor-Lfng combinations in this experiment. Saturated data points, defined here as those with normalized signaling activity over 0.75 in both dLfng and Lfng conditions, were excluded. Colors indicate the identity of the trans-ligand expressed by cocultured sender cells. Error bars denote bootstrapped 95% confidence intervals (Methods), in this case sampled from the number of biological replicates given in the legend—n1 (for Notch1) or n2 (for Notch2). See Methods and Figure 2A caption for more details. Note that the only difference between this figure and the new Figure 2—figure supplement 1A is that this figure additionally includes the Jag1-high data from Figure 2—figure supplement 4B.

script>

eLife Assessment

This valuable study significantly enhances our understanding of how various ligands and receptors interact within the Notch signaling pathway. By developing novel cell-based assay systems, the authors systematically analyzed the effects of different ligand-receptor combinations on pathway activation. The convincing data reveal intriguing and unexpected differences and provide a foundation for interpreting Notch signalling in both normal and disease-related contexts.

Reviewer #1 (Public review):

Summary:

The Notch signaling pathway plays important roles in many developmental and disease processes. Although well-studied there remain many puzzling aspects. One is the fact that as well as activating the receptor through a trans-activation, the transmembrane ligands can interact with receptors present in the same cell. These cis-interactions are usually inhibitory, but in some cases, as in the assays used here, they may also be activating. With a total of 6 ligands and 4 receptor there are potentially a wide array of possible outcomes when different combinations are co-expressed in vivo. Here the authors set out to make a systematic analysis of the qualitative and quantitative differences in the signaling output from different receptor ligand combinations, generating sets of "signaling" (ligand expressing) and "receiving" (receptor +/- ligand expressing cells).

The readout of pathway activity is transcriptional, relying on the fusion of GAL4 in the intracellular part of the receptor. Positive ligand interactions result in proteolytic release of Gal4 that turns on expression of H2B-citrine. As an indicator of ligand and receptor expression levels, they are linked via TA to H2B mCherry and H2B mTurq expression respectively. The authors also manipulate expression of the glycosyltransferase Lunatic-Fringe (LFng) that modifies the EGF repeats in the extracellular domains impacting on their interactions. The testing of multiple ligand receptor combinations at varying expression levels is a tour de force, with over 50 stable cell lines generated, and yields valuable insights although as a whole, the results are quite complex.

Strengths:

Taking a reductionist approach to test systematically differences in the signaling strength, binding strength and cis-interactions from the different ligands in the context of the Notch1 and Notch 2 receptors (they justify well they choice of players to test via this approach) produces a baseline understanding of the different properties and leads to some unexpected and interesting findings. Notably:<br /> - Jag1 ligand expressing cells failed to activate Notch1 receptor although were capable of activating Notch2. Conversely, Jag2 cells elicited the strongest activation of both receptors. The results with Jag1 are surprising also because it exhibits some of the strongest binding to plate bound ligands. The failure to activate Notch1 has major functional significance and it will be important in future to understanding the mechanistic basis.<br /> - Jagged ligands have the strongest ciis-inhibitory effects and the receptors differ in their sensitivity to cis-inhibition by Dll ligands. These observations are in keeping with earlier in vivo and cell culture studies. More referencing of those would better place the work in context but it nicely supports and extends previous studies that were conducted in different ways.<br /> - Responses to most trans-activating ligands showed a degree of ultrasensitivity but this was not the case for cis-interactions where effects were more linear. This has implications for the way the two mechanisms operate and for how the signaling levels will be impacted by ligand expression levels.<br /> - Qualitatively similar results are obtained in a second cell line, suggesting they reflect fundamental properties of the ligands/receptors.

Weaknesses:

One weakness is that the methods used to quantify the expression of ligands and receptors rely on co-translation of tagged nuclear H2B proteins. These may not accurately capture surface levels/correctly modified transmembrane proteins. In general, the multiple conditions tested partly compensate for the concerns - for example as Jag1 cells do activate Notch2 even if they do not activate Notch1 some Jag1 must be getting to the surface. But even with Notch2, Jag1 activities are on the lower side, making it important to clarify, especially given the different outcomes with the plated ligands. Similarly, is the fact that all ligands "signalled strongest to Notch2" an inherent property or due to differences in surface levels Notch 2 compared to Notch1?.. The results would be considerably strengthened by calibration of the ligand/receptor levels (and ideally their sub-cellular localizations). Assessing the membrane protein levels would be relatively straightforward to perform on som eof the basic conditions because their ligand constructs contain Flag tags, making it plausible to relate surface protein to H2B, and there are antibodies available for Notch1 and Notch2

In the revised version this has been addressed to some extent. A figure showing the relationship between co-translated mTurquiose and surface receptor expression for some clones (Figure 1-figure supplement 1B) goes some way to address the concerns that differences in Notch1 and Notch 2 could be due to the receptor levels. The data analyzing surface ligand levels is more equivocal, (a Western blot for biotinylated surface proteins), as the levels detected vary substantially between Dll1 and Dll4 (the latter barely detectable). But as a signal for surface expression of Jag1 was obtained this rules-out one concern that this ligand was failing to reach the surface. A discussion of the caveats of the approach is warranted, to make clear the limitations.

Cis-activation as a mode of signaling has only emerged from these synthetic cell culture assays raising questions about its physiological relevance. Cis-activation is only seen at the higher ligand (Dll1, Dll4) levels, how physiological are the expression levels of the ligands/receptors in these assays? Is it likely that this would make a major contribution in vivo? Is it possible that the cells convert themselves into "signaling" and "receiving" sub-populations within the culture by post-translational mechanism. Again some analysis of the ligand/receptors in the cultures would be a valuable addition to show whether or not there are major heterogeneities.

It is hard to appreciate how much cell to cell variability in the "output" there is. For example, low "outputs" could arise from fewer cells becoming activated or from all cells being activated less. As presented, only the latter is considered. That maybe already evident in their data, but not easy for the reader to distinguish from the way they are presented. For example, in many of the graphs, data have been processed through multiple steps of normalization. Some discussion/consideration this point is needed.

Impact:<br /> Overall, cataloguing of the outcomes from the different ligand-receptor combinations, both in cis and trans, yields a valuable baseline for those investigating their functional roles in different contexts. There is still a long way to go before it will be possible to make a predictive model for outcomes based on expression levels, but this work gives an idea about the landscape and the complexities. This is especially important now that signaling relationships are frequently hypothesised based on single cell transcriptomic data. The results presented here demonstrate that the relationships are not straightforward when multiple players are involved.

Reviewer #2 (Public review):

Summary:

In this manuscript the authors extend their previous studies on trans-activation, cis-inhibition (PMID: 25255098) and cis-activation (PMID: 30628888) of the Notch pathway. Here they create a large number of cell lines using CHO-K1 and C2C12 cells expressing either Notch1-Gal4 or Notch2-Gal4 receptors which express a fluorescent protein upon receptor activation (receiver cells). For cis-inhibition and cis-activation assays, these cells were engineered to express one of the four canonical Notch ligands (Dll1, Dll4, Jag1, Jag2) under tetracycline control. Some of the receiver cells were also transfected with a Lunatic fringe (Lfng) plasmid to produce cells with a range of Lfng expression levels. Sender cells expressing all of the canonical ligands were also produced. Cells were mixed in a variety of co-culture assays to highlight trans-activation, cis-activation, and cis-inhibition. All four ligands were able to trans-activate Notch1 and Notch 2, although Jag1 transactivated Notch1 weakly. Lfng enhanced trans-activation of both Notch receptors by Dll1 and Dll4, and inhibited both receptors by Jag 1 and Jag2. Cis-expression of all four ligands were predominantly inhibitory, but Dll1 and Dll4 showed strong cis-activation of Notch2. Interestingly, cis-ligands preferentially inhibited trans-activation by the same ligand, with varying effects on other trans-ligands.

Strengths:

This represents the most comprehensive and rigorous analysis of the effects of canonical ligands on cis- and trans-activation, and cis-inhibition, of Notch1 and Notch2 in the presence or absence of Lfng so far. Studying cis-inhibition and cis-activation is difficult in vivo due to the presence of multiple Notch ligands and receptors (and Fringes) that often occur in single cells. The methods described here are a step towards generating cells expressing more complex arrays of ligands, receptors and Fringes to better mimic in vivo effects on Notch function.

In addition, the fact that their transactivation results with most ligands on Notch1 and 2 in the presence or absence of Lfng were largely consistent with previous publications provides confidence that the author's assays are working properly.

Weaknesses:

In the original version, there was a major concern about quantifying the amount of Notch receptors and ligands on the cell surface (especially Jag1) based on total fluorescence. The authors have added data to demonstrate that most of the receptors and ligands are on the cell surface, allaying most of these concerns.

The author is right people are already unsure about how to treat animals, fetuses, and others, so adding AI could make things even more confusing. We should figure out our current ethical problems before worrying about how to treat AI.

The Design Policy of the Excluded Middle could hold back AI research by preventing us from creating systems that might help us learn about real sentience. How can we be careful with ethics while still trying out new AI ideas?

Shouldn’t we focus on improving the treatment of known sentient beings before worrying about the moral status of potential sentient AI?

If a sentient AI like Robot Alpha existed, it would make us rethink how do we treat AI? It would be the same as a person or maybe you'd think of it as a pet or something.

How so? The author should have explained this answer in better detail.

I don't think AI will ever be a sentient or alive being

Is consciousness the only thing that enables real beings and AI to have emotions? Or can you still be conscious and remain emotionless?

If people believe some AI is sentient and it has caused such implications as someone taking their own life due to a toxic relationship, why would scientists and roboticist continue to advance AI to have such an affect on someone's life. Why wouldn't we limit it's capabilities and prevent events like this from happening.

Connect Cephalus’s criticism of those who think of living well in terms of pleasure (329a-b (Greek)) with Aristotle’s first criticism of the view that living well/eudaimonia is pleasure (NE I.4 1095a18-26 (Greek), I.5 1095b14-22 (Greek)). (Christiana Olfert)

Connect Cephalus’s criticism of those who think of living well in terms of pleasure (329a-b (Greek)) with Epicurus’s argument that pleasure is “our first and kindred good” (Letter to Menoeceus, DL X.128-9 (Greek)). (Christiana Olfert)

Connect Cephalus's perspective on living well from the end of his life (329a ff. (Greek)) with Aristotle's view of happiness as being defined over a "complete life" (NE I.7 1098a18-20 (Greek)), and his questions about whether one's happiness can be settled before (or even after) death (NE I.10-11 (Greek))

This is a great example of a specific prompt that you can give AI

Policymakers need to move fast to set clear rules for AI, making sure it's safe and beneficial for everyone while encouraging new ideas.

This is interesting because it uses AI to help teachers and students learn better, making lessons more personal and saving time in classrooms all over the country.

AI can take care of boring tasks, so teachers can focus more on their students and working with each other.

Enhanced Capabilities with Plugins: The introduction of plugins allows ChatGPT to interact with online content, perform advanced mathematical functions, and access a variety of external services, broadening its utility.

Code Interpreter Features enable ChatGPT to analyze data, create charts, solve mathematical problems, edit files, and develop hypotheses, making it a powerful tool for data analysis and problem-solving.

AI can also unintentionally repeat unfair stereotypes from the data it learns from, which can lead to harmful biases in the content it produces. We need to ultimately slow down on our production of AI before more things get out of hand

Just like Robinhood did in the GameStop Story

By annoying all knowledge, we evolve the annotation into WISDOM

I love that Gien uses this term of TRAILBLAZING :-)

Brehon Law is the same

This is the CORE of Dialogue

Winning arguments is a core source issue that is a problem - this is why DIALOGUE is much more effective

We too note that most issues are not problems, they are symptoms. This is why we synthesised an action learning process which we now call Cohesion Circle - to get to the source of the symptom

I was told that THIS nasty little problem if ours is a parasite from Sirius lodged in our limbic brains millennia ago

We see this not as consensus, rather as COHERENCE

I wonder where Dan is in 2024. We would love to connect with him to discover such

In another ceremony, I have been shown that there is a parasite in our limbic systems - I was told that some people know it s ID (Jung)

I have been shown in sacred ceremony that this is the case. That there are an unlimited number of parallel universes operating in real time in one 3D space which we call earth.

And which other parallel universes call something else in their dimensions.

Style

Style: bringing back a memory. Structure: nostalgia

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make one up for hyperpost hp

Personally I have found that one of the most common kinds of trolling found is when people comment something sarcastically or ingenuine on a post where someone was hoping to be taken seriously. I find this form of trolling to me very discouraging and harmful.

I know many comments here talk about how much of an outrage making the blue checkmark purchase-able, and in doing so, making trolling way easier than before, however, I would like to add on the ethical POV about verification, identity and authenticity: monetizing one of the only ways to verify an important figure's identity online is definitely a morally wrong decision. When taking a step back and looking at the situation, is a bit more money worth all the problems that arise??

Trolling has become very common on social media. And I can't lie; I've been on both sides of the stick during my time on social media. But I realized that I gain absolutely nothing from it, and if someone has to receive happiness from ruining someone else's happiness, then that person isn't in a good spot mentally.

Regarding this issue, I believe the main objectives of trolling are roughly two. Firstly, trolling may be aimed at attracting the attention of internet users and gaining more followers for one's account. Specifically, many account operators will post controversial topics to attract more attention to their works. Some people may follow the operators' accounts to engage in arguments in the controversial topic. When the operators think their accounts have gained enough followers, they may delete the previous controversial topic and change the account's nickname, posting advertisements or other works to earn money. Secondly, trolling may also be aimed at diverting public attention. For example, when a star has negative news, it is very likely that the star's company will troll in order to protect the star's reputation. The company will register many accounts to post controversial topics to gain popularity, while diverting people's attention away from the star.

While most things have two sides, trolling does not seem to have its positive side. There are many users or bots on the internet today who constantly post controversial topics or posts, such as those related to gender opposition; racism, or spreading false information to defame others. Their purpose is simply to bring negative emotions and arguments to internet users. I think that most social media platforms should ban accounts that continuously post controversial topics in order to stop the spread of negative emotions.

I feel like one of the goals of trolling is to manipulate emotions by introducing and using fake content, which then often results in frustration or chaos on this social media pages which can create attention. Trolling does its part in truly undermining genuine conversation between users and it can also turn these social media pages and their comment sections into hostile enviroments.

In some cases, there are may be other reasons that causes people to engage trolling behaviors. People might troll to fit in with a certain online community or group of friends who engage in similar behaviors. Or they may use trolling to get more subscribes and pursue profits.

One example of Punishment via Trolling that comes to mind is during 2021, Texas opened an abortion reporting site. However, TikTokers from Texas started broadcasting to the fans to overload the website with thousands of fake messages, complete with local zip codes. In this way, people were encouraged to troll in the name of punishing anti-abortion services.

Directionality doesn't really matter if you can see both notes at once (e.g. side by side in Obsidian panes; an array of paper cards on your table).

Though, if time is your fundamental ordering pattern, you could use old-to-new flow whereby new stuff preferentially appends to old notes before they're codified as new/standalone/etc. notes. See also: TheSephist's incremental notes

I think the underlying point is one of common law-like precedence -- some (set of) note N, constrains the semantic/action/application/etc. space of (novel) (set of) note N'. Though, I'm not a mathematician...

Citation doesn't have to be hard either -- Zotero immediately comes to mind

Mostly communicative efficiency :-)

Equiano's description of how Africans were kidnapped into slavery is reminiscent of the scene in Amistad when Cinquè is abducted into slavery. Like in Equiano's writing, slavers went into villages and took those they wanted as slaves. African Slavers kidnapping people is a darkly interesting part of the Atlantic Slave-trade, as Africans almost worked as middle-men by kidnapping people and selling them to the Europeans.

Spielberg, Steven, Debbie Allen, Colin Wilson, David Franzoni, Morgan Freeman, Nigel Hawthorne, Anthony Hopkins, et al. Amistad. Universal City, CA: DreamWorks Home Entertainment, 1999.

Equiano's depiction of slave suicide is much like what is depicted in the film. In the film, we see slaves commit suicide that would rather die than see the conditions that faced them in North America. Amistad takes this a step further when it shows slaves being thrown off the ships when rations began to get low on the ship.

Spielberg, Steven, Debbie Allen, Colin Wilson, David Franzoni, Morgan Freeman, Nigel Hawthorne, Anthony Hopkins, et al. Amistad. Universal City, CA: DreamWorks Home Entertainment, 1999.

This description of the slave ships is much like how the film depicts the conditions experienced on the Amistad. What makes Equiano's work compliment the film well is how he fills in the blanks the viewer doesn't see in the film. In this, Equiano describes the smells, the noises, the screams of death. By giving a personal account of these conditions, it makes what the viewer witnesses in Amistad much harder to watch. As John M. said in our weekly discussion, "not only is it important for depictions of the Middle Passage to be spread by films like Amistad, it is crucial that they actually be ACCURATE."

Spielberg, Steven, Debbie Allen, Colin Wilson, David Franzoni, Morgan Freeman, Nigel Hawthorne, Anthony Hopkins, et al. Amistad. Universal City, CA: DreamWorks Home Entertainment, 1999.

Equiano writes about the feelings of terror and fear felt at seeing white slavers for the first time, while Amistad illustrates how these feelings turned into anger that led Cinqué to lead a revolt killing the crew of the Amistad.

Spielberg, Steven, Debbie Allen, Colin Wilson, David Franzoni, Morgan Freeman, Nigel Hawthorne, Anthony Hopkins, et al. Amistad. Universal City, CA: DreamWorks Home Entertainment, 1999.

This point really struck me as something that must've been universally felt by all Africans before they traveled to North America. As Equiano described, the feeling of being awestruck at the scale of European ships (which many Africans probably had never seen.) and how that feeling of awestruck turned into terror at what came next.

The unrealistic belief that African slaves could return home is visible in both Equiano's writings, and the movie. However, Equiano realized quite early on that he would never be able to make it home. Conversely, the Amistad slaves hoped that they would be able to steer their ship home. Interestingly, Equiano and Cinqué have two vastly different "endings." Upon his freedom, Equiano stayed in England, where he eventually died seemingly making no attempt to return to Africa, while Cinqué returned back to Africa following his freedom, where he died.

McClurken, Jeff. "Lecture on Amistad." at University of Mary Washington, September 24,2024.

Being ripped from one's family was all too common when Africans were taken as slaves. Like Equiano's work suggests, those that were taken together were even separated from their loved-ones. What's more, Equiano is later briefly reunited with his sisters, albeit for a short time. This was incredibly uncommon as families were usually torn apart permanetly.

Like Equiano wrote about earlier he writes about how slaves that came together on the ships were still torn from their families and sold. Unlike Equiano, Cinqué was taken from his family that remained in Africa while he was taken to North America.

Its interesting how hazing is assumed to be physcially abusing usually but there is also a digital version of it which both cause damage

I find it interesting that there is a distinct difference between Trolling and actual hate speech. Because trolling is intended to invoke a reaction out of users while hate speech is actual hate. I think ethically we need to draw the line on how far we can go while trolling so that people don't get the wrong reaction with sesitive topics.

In this class, I keep finding out new things about things I thought I already knew about. Similar to social media before the internet, I never thought of trolling prior to social media. But it makes sense because trolling doesn't even have to be only on social media; it can be in person or on different forms of the internet. I would think trolling started when the internet came out and people realized they could say things anonymously.

Trolling for newbies highlights the dynamics of online communities, where experienced users assert their status by tricking newcomers. This reinforces group identity through shared knowledge, but it also fosters exclusion and creates barriers for those trying to join, shaping the early culture of internet message boards with elitism.

What causes women to be treated unfairly in society? We can always see women being criticized online, from their appearance to their figure to their personality, and in many dirty jokes, women's privacy is the most ridiculed. However, people on the Internet have magnified this misogyny by taking advantage of the "anonymous nature" of the Internet.

I have actually witnessed a lot of trolling online and I realised the trollers want a reaction out of you. Even if its positive or negative they just want a reaction. A good way to stop trollers is to agree with them show a general stance and not show any emotional reaction to there comments

I have heard that it takes two to tango. When one party keeps trying to cause a riot, others will ignore him and he will lose interest in causing a riot. But is this really useful on the Internet? Some people are constantly venting their negative emotions in life through the Internet and spreading negative and pessimistic comments. Our disregard may make them more rampant, so there needs to be specific regulations to reduce the occurrence of such behavior.

How can we use the power of pauses in our conversations to create more meaningful connections with others?

Recognizing the rituals involved in ending conversations is essential for nurturing relationships. Thoughtful closures, whether in person or online, reinforce our connection and ensure that both parties feel valued and respected. These small gestures can significantly enhance the quality of our interactions.

It's remarkable how small, everyday interactions can significantly impact our relationships and well-being. By approaching conversations with openness and affirmation, we not only enhance our connections but also create an environment where individuals feel valued and understood.

How can we foster environments—both in-person and online—where emotional and social support can thrive to improve overall mental health?

In what ways can we enhance communication and community support to help individuals realize their abilities and improve their overall well-being?

What steps can communities take to reduce stigma and provide support for the one in five individuals facing mental health challenges?

This is cool because I know some people like to work ahead when they don't have any homework to work on.

I think this is a smart decision because with AI and cheating you never actually learn anything.

Im not sure if i want to look forward to this or not because I dont really know what it is.

I'm looking forward to doing this assignment because I need to learn to be safer when lifting.

GlobalID::Locator.locate_signed params

How does one share annotations?

Annotations via hypothes.is, but also secondary notes? Not sure.

Quizzes are weekly

Notes taking technique

As societies grow, trust becomes harder to establish, making group identity more crucial. People often rely on shared values, cultural markers, or common experiences to determine who belongs. The need to define "we" versus "not-we" becomes a way to manage trust and maintain social cohesion in complex, diverse communities.

I think some of them may not take nihilistic philosophy. A lot of trolling are hired for specific reason, which can be used to infamise their employers' rivals. There are also trolling serve for political ideas. So they may be trolling on purpose.

A consequentialist perspective that aligns politcally with the protest could believe that any harm done by the trolling would be outweighted by the impact the protest had, therefor believing it to be ethical. A Kantian perspective believe that acting disingenuously or maliciously, regardless of reason, is always unethical.

specific examples show why the topic matters and show that Honore is aware of what his audience values background info

Résumé de la vidéo [00:00:00][^1^][1] - [00:23:24][^2^][2]:

Ce webinaire présente Panoramax, une infrastructure numérique qui pourrait surpasser Google StreetView. Il explore les fonctionnalités, les avantages et les contributions de divers intervenants.

Moments forts: + [00:00:00][^3^][3] Introduction et objectifs * Présentation de Panoramax * Comparaison avec Google StreetView * Objectifs du webinaire + [00:00:32][^4^][4] Intervenants et perspectives * Présentation des intervenants * Différentes perspectives sur Panoramax * Rôle des collectivités et des contributeurs + [00:01:20][^5^][5] Utilisation et contributions * Utilisation par la ville de Bayonne * Contributions à OpenStreetMap * Besoins et avantages pour les collectivités + [00:06:00][^6^][6] Fonctionnalités techniques * Comparaison avec Mapillary * Décentralisation et souveraineté des données * Fonctionnalités spécifiques de Panoramax + [00:12:00][^7^][7] Gouvernance et financement * Financement par la DINUM et l'IGN * Contributions multiples et partenariats * Rôle de la communauté et des acteurs publics

Résumé de la vidéo [00:23:26][^1^][1] - [00:46:45][^2^][2]:

La vidéo présente le projet Panoramax, une infrastructure numérique collaborative pour la collecte et l'utilisation de photos, visant à dépasser les limitations de Google StreetView.

Moments forts: + [00:23:26][^3^][3] Introduction du projet Panoramax * Problèmes avec Mapillary après son rachat par Meta * Avantages de Panoramax pour la contribution de photos * Démarche collaborative pour la collecte de photos + [00:25:00][^4^][4] Mise en œuvre du projet * Utilisation d'une GoPro pour la collecte de photos * Participation des collègues de différents services * Intégration avec le SIG web Easy Go + [00:30:00][^5^][5] Avantages de Panoramax * Photos de lieux non couverts par Google * Mise à jour régulière des images * Participation de divers services et citoyens + [00:35:00][^6^][6] Utilisation par Geovélo * Cartographie des aménagements cyclables * Collecte de photos immersives le long de la Loire à Vélo * Intégration des photos dans les outils internes + [00:40:00][^7^][7] Perspectives et développement futur * Collaboration avec les collectivités * Utilisation des photos pour la mise à jour des données * Démonstration de l'intégration des photos dans OpenStreetMap

Résumé de la vidéo [00:46:47][^1^][1] - [01:10:47][^2^][2]:

Cette partie du webinaire présente Panoramax, une infrastructure numérique collaborative pour la collecte et l'utilisation de photos géolocalisées. Les intervenants discutent des défis techniques, des cas d'usage variés, et de la gouvernance communautaire du projet.

Moments forts: + [00:46:47][^3^][3] Géolocalisation et qualité des photos * Développement d'une application mobile pour corriger les écarts de géolocalisation * Acceptation de toutes les photos géolocalisées sur Panoramax * Tri des photos pour caractériser leur qualité + [00:48:13][^4^][4] Utilisation des photos pour divers usages * Vérification de l'état des routes et des arrêts de bus * Identification et classification des panneaux routiers * Analyse des façades et des bâtiments + [00:50:01][^5^][5] Gouvernance et communauté * Projet initié par OpenStreetMap France et l'IGN * Plus de 36 millions de photos et 500 contributeurs * Forum des géocommuns pour échanger et voter sur les priorités + [00:55:00][^6^][6] Financement et priorités * Importance de convaincre les financeurs avec des retours utilisateurs * Souplesse dans le fonctionnement pour s'adapter aux besoins * Défis potentiels liés aux indicateurs d'impact + [01:03:01][^7^][7] Détection automatique et IA * Détection de panneaux de signalisation et signalisation horizontale * Collaboration avec des universités et des chercheurs * Développement d'une application mobile pour faciliter la contribution

Résumé de la vidéo [01:10:50][^1^][1] - [01:30:35][^2^][2]:

Cette vidéo présente Panoramax, une infrastructure numérique qui pourrait surpasser Google StreetView. Les intervenants discutent de l'intégration de Panoramax dans divers outils SIG (Systèmes d'Information Géographique) et de ses applications pratiques.

Moments forts: + [01:10:50][^3^][3] Introduction et intégration * Discussion sur l'intégration de Panoramax * Utilisation dans les outils SIG * Exemple de stationnement PMR + [01:13:02][^4^][4] Fonctionnalités interactives * Visualisation des données en temps réel * Mise à jour des données de stationnement * Navigation et revue thématique + [01:15:00][^5^][5] Questions et réponses * Débat sur l'intégration des photos * Possibilité de pointer vers des séquences de photos * Partage de ressources et documentation + [01:18:20][^6^][6] Utilisation pratique * Expériences avec OpenStreetMap * Comparaison avec Google Maps * Développement de l'interface utilisateur + [01:23:04][^7^][7] Perspectives futures * Intégration dans les services de l'IGN * Projets de modélisation 3D * Expansion internationale et collaboration

Adding your personal experience to support a point or introduce a new angle to the conversation can be a valuable contribution to the discussion and support your stand.

I like the concept of the "believing game" where I take the shoes of the original author and write from his perspective fairly, even if planning to disagree with his thoughts later.

It's important to summarize other views before presenting your own for two reasons: one is to understand other perspectives and be in a position to give your "say," and the other is that it's expected as a way of strengthening your arguments in an academic conversation.

Level of dissatisfaction * desirability of proposed change * practicality moet groter zijn dan de personal cost of changing

eLife Assessment

This important study uses in vitro and in vivo methods to identify HpARI proteins from H. polygyrus as modulators of the host immune system. The data from comprehensive approaches for investigating differential roles of HpARI proteins are convincing. This paper is relevant to those who investigate host-pathogen interactions at the systems and molecular levels.

Reviewer #1 (Public Review):

Colomb et al have further explored the mechanisms of action of a family of three immunodulatory proteins produced by the murine gastrointestinal nematode parasite Heligmosomoides polygyrus bakeri. The family of HpARI proteins binds to the alarmin interleukin 33 and depending on family members, exhibits differential activities, either suppressive or enhancing. The present work extends previous studies by this group showing the binding of DNA by members of this family through a complement control protein (CCP1) domain. Moreover, they identify two members of the family that bind via this domain in a non-specific manner to the extracellular matrix molecule heparan sulphate through a basic charged patch in CCP1. The authors thus propose that binding to DNA or heparan sulphate extends the suppressive action of these two parasite molecules, whereas the third family member does not bind and consequently has a shorter half-life and may function via diffusion.

Reviewer #2 (Public Review):

Colomb et al. investigated here the heparin-binding activity of the HpARI family proteins from H. polygyrus. HpARIs bind to IL-33, a pleiotropic cytokine, and modulate its activities. HpARI1/2 has suppressive functions, while HpARI3 can enhance the interaction between IL-33 and its receptor. This study builds upon their previous observation that HpARI2 binds DNA via its CCP1 domain. Here, the authors tested the CCP1 domain of HpARIs in binding heparan sulfate, an important component of the extracellular matrix, and found that 1/2 bind heparan, but 3 cannot, which is related to their half-lives in vivo.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review): 

Summary: 

Colomb et al have further explored the mechanisms of action of a family of three immunodulatory proteins produced by the murine gastrointestinal nematode parasite Heligmosomoides polygyrus bakeri. The family of HpARI proteins binds to the alarmin interleukin 33 and depending on family members, exhibits differential activities, either suppressive or enhancing. The present work extends previous studies by this group showing the binding of DNA by members of this family through a complement control protein (CCP1) domain. Moreover, they identify two members of the family that bind via this domain in a non-specific manner to the extracellular matrix molecule heparan sulphate through a basic charged patch in CCP1. The authors thus propose that binding to DNA or heparan sulphate extends the suppressive action of these two parasite molecules, whereas the third family member does not bind and consequently has a shorter half-life and may function via diffusion. 

Strengths: 

A strength of the work is the multifaceted approach to examining and testing their hypotheses, using a well-established and well-defined family of immunomodulatory molecules using multiple approaches including an in vivo setting. 

Weaknesses: 

There are a few weaknesses of the approach. Perhaps some discussion and speculation as to how these three family members might operate in concert during Heligmosomoides polygyrus bakeri infection would help place the biology of these molecules in context for the reader, e.g. when and where they are produced. 

We agree that the roles of these proteins during infection requires further study and is not fully elucidated in infection here. We have added further discussion to the manuscript on their potential roles during infection (track changes manuscript, lines 277 – 283).

Reviewer #2 (Public Review): 

Summary: 

Colomb et al. investigated here the heparin-binding activity of the HpARI family proteins from H. polygyrus. HpARIs bind to IL-33, a pleiotropic cytokine, and modulate its activities. HpARI1/2 has suppressive functions, while HpARI3 can enhance the interaction between IL-33 and its receptor. This study builds upon their previous observation that HpARI2 binds DNA via its CCP1 domain. Here, the authors tested the CCP1 domain of HpARIs in binding heparan sulfate, an important component of the extracellular matrix, and found that 1/2 bind heparan, but 3 cannot, which is related to their half-lives in vivo. 

Strengths: 

The authors use a comprehensive multidisciplinary approach to assess the binding and their effects in vivo, coupled with molecular modeling. 

Weaknesses: 

(1) Figure 1C should include Western. 

We apologise for this oversight, and now include an uncropped western blot image as a Figure 1, Figure Supplement 1.

(2) Figure 1E: Why does HpARI1 stop binding DNA at 50%? 

It is currently unclear why HpARI1 does not bind to all DNA in the EMSA assay, however this was our repeated finding. With our revised findings we can now state definitively that HpARI1 has a lower affinity for HS compared to HpARI2, and in each of our assays (EMSA (Fig 1D-E), size exclusion chromatography (Fig 4A), HS-bead pull-down (Fig 4B), lung cell surface binding (Fig 4C) and ITC (Fig 4D)) HpARI1 always shows a weaker response compared to HpARI2. We hypothesise that HpARI1 binds more weakly to DNA/HS to allow it to diffuse further from the site of deposition, but we have yet to demonstrate this during infection. We add further discussion of this point (track changes manuscript, lines 262 – 266).

(3) ITC binding experiment with HpARI1? Also, the ITC results from HpARI2 do not seem to saturate, thus it is difficult to really determine the affinity. 

We have now included HpARI1-HS ITC, and re-ran the HpARI2 experiment to saturation (Fig 4D-E).

(4) It would be helpful to add docking results from HpARI1. 

We have now included HpARI1-HS docking, in Figure 5B.

(5) Some conclusions are speculative and need to remain in the Discussion. e.g.: a) That HpARI3 may be able to diffuse farther 

We have rewritten these points to remove the speculation on localisation from the abstract (lines 18-19) and introduction (line 78).

b) That DNA/HS may trap HpARI1/2 at the infection site. 

Likewise, these points have been rewritten in the abstract and introduction as above, and we have made it clearer that this is a model that we are proposing in the discussion (line 277-283).

Reviewer #1 (Recommendations For The Authors): 

The paper is well-written and the data well-presented. I have one small comment that the authors may like to consider. In the discussion, second paragraph, line 17, perhaps, "evolved" rather than "developed". 

Thank you for this suggestion, we have made this change (line 248).

Some argue that while transplantation can help, it may not address the root causes of coral decline, such as water quality degradation or rising ocean temperatures. Without mitigating these broader issues, coral transplants may simply fail over time. However, others see transplantation as a crucial stopgap measure to preserve key species and habitats while long-term solutions are developed

The reality is that restoration efforts, while beneficial, may never fully return coral reefs to their pristine state. This is due to the complex interactions between species, environmental conditions, and historical degradation. As a result, conservationists now focus on creating resilient reefs that can survive in future conditions rather than merely replicating past ecosystems

Coral reefs are known as the "rainforests of sea", these are the biodiversity hotspots that support a vast range of marine species. However, their rapid decline due to climate change, overfishing, and pollution has created an urgent need for restoration strategies. Scientists and conservationists now face the challenge of reversing damage to these ecosystems, which are vital for both marine life and coastal human populations.

The Kellogg's incident, in which the general public used electronic means to interfere with the company's operations, is a fascinating illustration of collective resistance in the digital age. It, in my opinion, represents society's reaction to large businesses abusing their power. But this kind of "data poisoning" begs the moral dilemma of how to define an appropriate mode of dissent. Although flooding the hiring process with phony applications paralyzes it, it may have unintended consequences for other parties, therefore a deeper analysis of the boundaries of technical confrontation is required.

Social media collects user data not only to improve the user experience, but also for compelling business purposes. This makes me wonder: if data collection becomes more widespread, do we have appropriate control over what data is used? While users often implicitly agree to data collecting tactics when they sign up, this does not mean they fully understand how the data will be used. As a result, this behavior is not always desired and may occasionally go against the user's wishes.

eLife Assessment

This is an important study on the damage-induced checkpoint maintenance and termination in budding yeast that provides novel and convincing evidence for a role of the spindle assembly checkpoint and mitotic exit network in halting the cell cycle after prolonged arrest in response to irreparable DNA double strand breaks (DSBs). The study identifies particular components from these checkpoints that are specifically required for the establishment and/or the maintenance of a cell cycle block triggered by such DSBs. The authors propose an interesting model for how these different checkpoints intersect and crosstalk for timely resumption of cell cycling even without repairing DNA damage that has been revised by addressing the bulk of the reviewers' comments to the first version of the manuscript.

Reviewer #1 (Public review):

Summary:

In their manuscript, Zhou et al. analyze the factors controlling the activation and maintenance of a sustained cell cycle block in response to persistent DNA DSBs. By conditionally depleting components of the DDC using auxin-inducible degrons, the authors verified that some of them are only required for the activation (e.g., Dun1) or the maintenance (e.g., Chk1) of the DSB-dependent cell cycle arrest, while others such as Ddc2, Rad24, Rad9 or Rad53 are required for both processes. Notably, they further show that after a prolonged arrest (>24 h) in a strain carrying two DSBs, the DDC becomes dispensable and the mitotic block is then maintained by SAC proteins such as Mad1, Mad2 or the mitotic exit network (MEN) component Bub2.

Strengths:

The manuscript dissects the specific role of different components of the DDC and the SAC during the induction of a cell cycle arrest induced by DNA damage, as well as their contribution for the short-term and long-term maintenance of a DNA DSB-induced mitotic block. Overall, the experiments are well described and properly executed, and the data in the manuscript are clearly presented. The conclusions drawn are generally well supported by the experimental data. Their observations contribute to drawing a clearer picture of the relative contribution of these factors to the maintenance of genome stability in cells exposed to permanent DNA damage.

Weaknesses:

The main weakness of the study is that it is fundamentally based on the use of the auxin-inducible degron (AID) strategy to deplete proteins. This widely used method allows an efficient depletion of proteins in the cell. However, the drawback is that a tag is added to the protein, which can affect the functionality of the targeted protein or modify its capacity to interact with others. In fact, three of the proteins that are depleted using the AID systems are shown to be clearly hypomorphic, and hence their capacity to induce a strong checkpoint response might be compromised. A corroboration of at least some of the results using an alternative manner to eliminate the proteins would help to strengthen the conclusions of the manuscript.

Reviewer #2 (Public review):

Summary:

The manuscript analyzes and attempts to discriminate genetic requirements for DNA damage-induced cell cycle checkpoint induction, maintenance, and adaptation in budding yeast bearing one or two unrepairable DNA double strand breaks using auxin-induced degradation (AID) of key DNA damage response (DDR) factors. The study paid particular attention to solving a puzzle regarding how yeasts bearing two unrepaired DNA breaks fail to engage in "adaptation" whereas those with a single unrepairable break eventually resume cell cycling after a prolonged (up to 12 h) G2 arrest.

The key findings are: 1. Genetic requirements for the entry and the maintenance of DDC are separable. For instance, Dun1 is partially required for the entry but not the DDC maintenance whereas Chk1 is only required for maintenance. 2. Cells with two unrepairable breaks respond to DDR only up to a certain time (~12-15 h post damage) and beyond this point, depend on spindle assembly checkpoint (SAC) and mitotic exit network (MEN) to halt cell cycling. 3. The authors also propose an interesting concept that the location of DNA breaks and their distance to centromeres are important factors dictating the effect of SAC/MEN on the duration of cell cycle arrest after prolonged arrest (and cells become "deaf" to persistent arrest signals) and yeast's adaptability following DNA damage. The results provide most compelling evidence to date on the role of SAC/MEN in DNA damage response and cell cycle arrest albeit its impact might be limited to the handful of model systems due to the vastly different centromeric elements and far larger chromosome sizes in metazoan cells. The study albeit briefly discussed the basis of transitions from entry, maintenance, and adaptation ( ex. changes in centromeric architectures), it does not offer detailed explanations or a testable hypothesis to this topic.

Overall, the conclusion of the study is well supported by the elegant set of genetic experimental data and employed multiple readouts on DDC factor depletion on checkpoint integrity and cell cycle status. Although the study simply measures Rad53 phosphorylation as the primary metric to assess checkpoint status, it successfully demonstrated how the signaling is modified through the different stages and that eventually cells become recalcitrant to DDC signaling after a prolonged arrest. The results are clear, and rigorously tested and carefully interpreted with good discussion on the possible limitations. The revision provided detailed responses to the reviewers' comments and addressed a few key concerns, one of which is universally raised by the reviewers on the full functionality of AID tagged DDC factors, by simply expressing excess Rad9-AID to restore more normal looking checkpoint response. It will be interesting if the excess expression of other DDC factors could overcome suboptimal checkpoints in cells after 24 h post damage.

Reviewer #3 (Public review):

Summary:

The DNA damage checkpoint (DDC) inhibits the metaphase-anaphase transition to repair various types of DNA damage, including DNA double strand breaks (DSBs). One irreparable DSB can maintain the DDC for 12-15 hours in yeast, after which the cells resume the cell cycle. If there are two DSBs, the DDC is maintained for at least 24 hours. In this study, the authors take advantage of this tighter DDC to investigate whether the best-known proteins involved in establishing the DDC are also responsible for its long-term maintenance during irreparable DSBs. They do this by cleverly degrading such proteins after DSB formation. They show that most, but not all, DDC proteins maintain the cell cycle block. Interestingly, DDC proteins become dispensable after 15 hours and the block is then maintained by spindle assembly checkpoint (SAC) proteins.

Strengths:

The authors have engineered a tight yeast system to study DDC shutdown after irreparable DSBs and used it to address whether checkpoint proteins (DDC and SAC) contribute to the long-term maintenance of DSB-mediated G2/M block. The different roles of Ddc2, Chk1 and Dun1 are interesting, while the fact that SAC overtakes DDC after 15 hours is intriguing and highlights how DSBs near and far from centromeres can have a profound impact on cell adaptation to DSBs. In their revision, the authors have now improved the Rad9-AID methodology to place Rad9 in the context of DDC adaptation, as well as widening the association between adaptation and proximity to centromeres.

Weaknesses:

Some of the results they present essentially confirm their own previous findings, albeit with a tighter strain design for long-term arrest. Conclusions about the maintenance of G2/M in several mutant combinations could have been strengthened by adding simple microscopy experiments with DAPI staining. No clear mechanism for how depletion of Bub2, but not Bfa1, can relieve the G2/M (metaphase) block is given.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Recommendations For The Authors):

To hopefully contribute to more strongly support the conclusions drawn by the authors, I am including a series of concerns regarding the manuscript, as well as some suggestions that could be useful to address these issues:

(1) The main results of this study derive from the use of auxin-inducible degron (AID)-tagged proteins. Despite the great advantages of the AID strategy to conditionally deplete proteins, the AID tag can affect the normal function of a protein. In fact, some of the AID-labeled DDC components generated in this work are shown to be hypomorphic. Hence, the manuscript would have benefited from the additional confirmation of some of the observations using a different way to eliminate the proteins (e.g., temperature-sensitive mutants).

Most ts mutants are also hypomorphic; hence we don’t see there is much advantage to their use. The addition of the AID to these proteins alone does not interfere with the ability to sustain checkpoint arrest as demonstrated in Figure S1. Instead we found that by overexpressing Rad9-AID we could demonstrate that inactivating Rad9 after 15 h behaved the same way as the inactivation of Ddc2, significantly strengthening our finding that the DDC checkpoint becomes dispensable while the SAC takes over. 

(2) In cells depleted of Rad53-AID, the deletion of CHK1 stimulates an earlier release from a mitotic arrest induced by two DSBs (Figures 2D and 3C). Likewise, the authors claim that a faster escape from the cell cycle block can also be observed when upstream factors such as Ddc2, Rad9, or Rad24 are depleted in the absence of CHK1 (Figures 2A-C and Figures 3D-F). However, this earlier release from the cell cycle arrest, if at all, is only slightly noticeable in a Rad9-AID background (Figures 2B and 3E). In this sense, it is also worth pointing out that Rad9-AID chk1Δ (Figure 3E) and Rad24-AID chk1Δ (Figure 3F) cells were only evaluated up to 7 h, while in all other instances, cells were followed for 9 h, which hinders a fair assessment of the differences in the release from the cell cycle arrest.

As noted above, we have now been able to examine Rad9 over the long-time frame.

(3) Although only 25% of the cells depleted for Dun1 remained in G2/M arrest 7 h following the induction of two DSBs, it is shocking that Rad53 was nonetheless still phosphorylated after the cells had escaped the cell cycle blockage (Figure 4A).

This persistence of Rad53 phosphorylation is also seen with the inactivation of Mad2, allowing escape in spite of continued Rad53 phosphorylation.

(4) Generation of Rad9-AID2 and Rad24-AID2 strains did not fully restore the function of these proteins, since most cells had adapted 24 h after induction of two DSBs (Figure S1C). Nonetheless, Rad9-AID2 and Rad24-AID2 are still likely more stable than their AID counterparts, and hence the authors could have instead used the AID2 proteins for the experiments in Figure 2 to better evaluate the role of Rad9 and Rad24 in the maintenance of the DDC-dependent arrest.

We note again that we have found a way to study Rad9 up to 24 h. 

(5) Deletion of BFA1 has been shown to promote the escape from a cell cycle arrest triggered by telomere uncapping (Wang et al. 2000, Hu et al. 2001, Valerio-Santiago et al. 2013). Likewise, while cells carrying the cdc5-T238A allele cannot adapt to a checkpoint arrest induced by one irreparable DSB, BFA1 deletion rescues the adaptation defect of this mutant CDC5 allele (Rawal et al., 2016). The authors show how, using AID-degrons of Bfa1 and Bub2, that only Bub2, but not Bfa1, is required to maintain a prolonged cell cycle arrest after the induction of two DSBs. To reinforce this point, and as shown for mad2Δ cells (Figure S6A), the authors could perform a complete time course using both the Bfa1-AID and a bfa1Δ mutant to demonstrate that they do indeed show the same behavior in terms of the adaptation to a two DSB-induced cell cycle arrest.

We thank the reviewer for noting these other instances where bfa1D promoted an escape from arrest. We tested a 2-DSB bfa1 deletion, data has been added to Figure S9E-F. We did not observe a difference in the percentage of cells escaping arrest between the 2-DSB bfa1 deletion and the 2-DSB BFA1-AID strains.

(6) Bypass or adaptation of a checkpoint-induced cell cycle arrest in S. cerevisiae often leads to cells entering a new cell cycle without doing cytokinesis and, hence, to the accumulation of rebudded cells. However, the experiments shown in the manuscript only account for G1 or budded cells with either one or two nuclei. Do any of the mutants show cytokinesis problems and subsequent rebudding of the cells? If so, this should have been also noted and quantified in the corresponding assays.

In the cases we have studied we have not seen instances where the cells re-bud without completing mitosis (at least as assessed by the formation of budded cells with two distinct DAPI staining masses). In the morphological assays we have done, we score the continuation of the cell cycle by the appearance of multiple buds, G1, and small budded cells. In our adaptation assays when cells escaped G2/M arrest they formed microcolonies indicating no short-term deficiency in cell division.

(7) The location of the DSB relative to the centromere of a chromosome seems to be a factor that determines the capacity of the SAC to sustain a prolonged cell cycle arrest. The authors discuss the possibility that the DSB could somehow affect the structure of the kinetochore. Did they evaluate whether Mad1 or Mad2 were more actively recruited to kinetochores in those strains that more strongly trigger the SAC after induction of the DSBs?

We have not attempted to follow Mad1/2 recruitment. ChIP-seq could be used to monitor Mad1/2 localization at the 16 centromeres in response to DSBs and the spread of g-H2AX across the centromere. Our previous data showed that g-H2AX could spread across the centromere region and could create a change that would be detected by Mad1/2.  This change does not, however, affect the mitotic behavior of a strain in which the H2A genes have been modified to the possibly phosphomimetic H2A-S129E allele.

(8) The authors could speculate in the discussion about the reasons that could explain why the DDC is required for the maintenance of checkpoint arrest at early stages but then becomes dispensable for the preservation of a prolonged cell DNA DSB-induced cycle arrest, which is instead sustained at later stages by the SAC.

Our suggestion is that cells would have adapted, but modification of the centromere region engages SAC.

Finally, some minor issues are:

(1) The lines in the graphs that display the results from adaptation assays (e.g., Figures 1B and 1E) or cell and nuclear morphology (e.g., Figures 1D and 1G) are too thick. This makes it sometimes difficult to distinguish the actual percentages of cells in each category, particularly in the experiments monitoring nuclear division.

Fixed

(2) While both the adaptation assay and the analysis of nuclear division in Figures 1E and 1G, respectively, show a complete DDC-dependent arrest at 4h, the Western blot in Figure 1F suggests that Rad53 is not phosphorylated at that time point. Do these figures represent independent experiments? Ideally, the analysis of cell budding and nuclear division, which is performed in liquid cultures, and the Western blot displaying Rad53 phosphorylation should correspond to the same experiment.

Cell budding in liquid cultures and adaptation assays were performed in triplicate with 3 biological replicates and the collective results are shown in each graph showing the percentage of large-budded cells. Western blot samples were collected in each liquid culture experiment. The western blot in 1G is a representative western blot.

(3) It is somewhat confusing that the blots for the proteins are not displayed in the same order in Figures 2A (Rad53 at the top) and 2B or 2C (Rad53 in the middle).

Fixed.  We place Rad53 – the relevant protein - at the top.

Reviewer #2 (Recommendations For The Authors):

(1) Yeast with the two breaks responds to DNA damage checkpoint (DDC) until sometimes 4-15 h post DNA damage. Since the auxin-induced degradation does not completely deplete all the tagged proteins in cells, the results should be more carefully considered and not to interpret if the checkpoint entry or maintenance depends on each target protein's ability to induce Rad53 phosphorylation. It should be theoretically possible if checkpoint maintenance requires only a modest amount of checkpoint factors especially because the experiments involve the induction of one or two DSBs. The low levels of DDC factors may be insufficient for Rad53 activation but could still be effective for cell cycle arrest. Indeed, the Haber group showed that the mating type switch did not induce Rad53 phosphorylation but still invoked detectable DNA damage response. To test such possibilities, the authors might consider employing yet another marker for DDC such as H2A or Chk1 phosphorylation besides Rad53 autophosphorylation. Alternatively, the authors might check if auxin-induced depletion also disrupts break-induced foci formation for checkpoint maintenance or their enrichment at DNA breaks using ChIP assays at various points post-damage.

DAPI staining of Ddc2-AID cells show that when IAA is added 4 h after DSB induction (Figure S3A), cells escape G2/M arrest as evidenced by the increase in large-budded cells with 2 DAPI signals, small budded cells, and G1 cells. Overexpression of Ddc2 can sustain the checkpoint past 24 h, but without SAC proteins like Mad2 they will eventually adapt (Figure S6B).

That Rad9-AID or Rad24-AID in the absence of added auxin (but in the presence of TIR1) is unable to sustain arrest suggests to us that low levels of Rad9 or Rad24 are not sufficient to maintain arrest.  As the reviewer notes, normal MAT switching doesn’t cause Rad53 phosphorylation or arrest, though early damage-induced events such as H2A phosphorylation do occur.  But our point is that Rad9 or Ddc2 is needed to maintain arrest only up to a certain point, after which they become superfluous and a different checkpoint arrest is imposed. At that point apparently a low level of these proteins plays no obvious role.

(2) It is interesting that DDC no longer responds to the damage signaling after 15 h of DSB-induced prolonged checkpoint arrest after two DNA double-strand breaks. Is this also applicable to other adaptation mutants? The results might improve the broad impact of the current conclusions. It is also possible that the transition from DDC to SPC depends on simply the changes in signaling or in part due to the molecular changes in the status of DNA breaks or its flanking regions. Indeed, the proposed model suggests that the spreading of H2A phosphorylation to centromeric regions induces SAC and thus mitotic arrest. The authors could measure H2A phosphorylation near the centromere using ChIP assays at various intervals post-DNA damage. It is particularly interesting if depletion of Ddc2 at 15 h post DNA damage does not alter the level of H2A phosphorylation at or near centromere.

Our previous data have suggested that the involvement of the SAC in prolonging DSB-induced arrest involved post-translational modification of centromeric chromatin such as the Mec1- and Tel1-dependent phosphorylation of the histone H2A (Dotiwala). In budding yeast there is also a similar DSB-induced modification of histone H2B (Lee et al.). To ask if there is an intrinsic activation of the SAC if the regions around centromeres were modified by checkpoint kinase phosphorylation, we examined cell cycle progression in strains in which histone H2A or histone H2B was mutated to their putative phosphomimetic forms (H2A-S129E and H2B-T129E).  As shown in Figure S11, there was no effect on the growth rate of these strains, or of the double mutant, suggesting that cells did not experience a delay in entering mitosis because of these modifications. We note that although histone H2A-S129E is recognized by an antibody specific for the phosphorylation of histone H2A-S129, the mutation to S129E may not be fully phosphomimetic. 

(3) It is puzzling why Rad9-AID or Rad24-AID are proficient for DDC establishment but cannot sustain permanent arrest in the two break cells. It appears Rad53 phosphorylation for DDC is weaker in cells expressing Rad9-AID or Rad24-AID according to Fig.2B and C even though their protein level before IAA treatment is still robust. This might also explain why the results of depleting Rad53 and Rad9 are very different. It also raises concern if the effect of Rad24 depletion on checkpoint maintenance is in part due to the weaker checkpoint establishment. It might be necessary to use the AID2 system to redo Rad24 depletion to exclude such a possibility.

We believe that the AID mutants are very sensitive to the low level of IAA present in yeast.  The instability of the protein is entirely dependent on the TIR1 SCF factor, so the proteins themselves are not intrinsically defective; they are just subject to degradation.  Overexpressing Rad9 allowed us to evaluate its role at late time points. 

(4) It is intriguing that the switch from DDC to SAC might take place at around 12 h when yeasts with a single unrepairable break ignore DDC and resume cell cycling (so-called "adaptation"). Since 4h and 15h are far apart and the transition point from DDC to SAC likely takes place between these two points, it will be very helpful to analyze and compare cell cycle exit after 24 h by treating IAA at multiple points between 4-15h.

When we add IAA to Mad2-AID and Mad1-AID 4 h after DSB induction, cells remain arrested for up to 12 h after DSB induction. At 15 h cells begin to exit checkpoint arrest indicating that the handoff of checkpoint arrest must occur between 12 to 15 h after DSB induction. If we degraded DNA damage checkpoint proteins at any point before Mad2, Mad1, and Bub2 begin to contribute to checkpoint arrest, then arrested cells will likely adapt in a similar manner to when IAA was added 4 h after DSB induction.

(5) Some of the Western blot quality is poor. For instance, in Figure 6C, Mad1-AID level after IAA addition is not compelling especially because the TIR level (the loading control) is also very low.

In Figure 6C, while the relative levels of TIR1 are similar in the IAA treated and untreated samples, there is no detectable amount of Mad1-AID in the IAA treated samples indicating that Mad1-AID was successful degraded with the AID system.

(6) Fig. 8 is complex. It might be helpful to define the different types of arrows in the figure. The legend also has a spelling error, Rad23 should be Rad24.

We’ve defined what each arrow means in the legend and corrected the spelling error in the figure legend.

Reviewer #3 (Recommendations For The Authors):

Major concerns:

Much of the manuscript states that two unrepairable DSBs lead to a long and severe G2/M arrest. Two main cytological approaches are used to make this statement: bud size and number on plates after micromanipulation (microcolony assay), and cell and nuclear morphology in liquid cultures. While the latter gives a clear pattern that can be assigned to a G2/M block as expected by DDC, i.e. metaphase-like mononucleated cells with large buds, the former can only tell whether cells eventually reach a second S phase (large budded cells on the plate can be in a proper G2/M arrest, but can also be in an anaphase block or even in the ensuing G1). The authors always performed the microcolony assay, but there are several cases where the much more informative budding/DAPI assay is missing. These include Dun1-aid and others, but more importantly chk1D and its combinations with DDC proteins. Incidentally, for the microcolony assay, it is more accurate to label the y-axis of the corresponding graphs (and in the figure legends and main text) with something like "large budded cells"; "G2/M arrested cells" is misleading.

Figures have been updated to more accurately reflect what we are measuring.

The results obtained with the Bfa1/Bub2 partner are intriguing. These two proteins form a complex whose canonical function is to prevent exit from mitosis until the spindle is properly aligned, acting in a distinct subpathway within the SAC that blocks MEN rather than anaphase onset. The data presented by the authors suggest that, on the one hand, both SAC subpathways work together to block the cell cycle. However, why does canonical SAC (Mad1/Mad2) inactivation not lead to a transition from G2/M (metaphase-like) arrested cells to anaphase-like arrest maintained by Bfa1-Bub2? Since Bfa1-Bub2 is a target of DDC, is it possible that DDC knockdown also inactivates this checkpoint, allowing adaptation? On the other hand, can the authors provide more data to confirm and strengthen their claim of a Bfa1-independent Bub2 role in prolonged arrest? Perhaps long-term protein localization and PTM changes. Bub2-independent roles for Bfa1 have been reported, but not vice versa, to the best of my knowledge.

In the mitotic exit network Bfa1/Bub2 prime activation of the pathway by bringing Tem1 to spindle pole bodies. Phosphorylation of Bfa1 causes Tem1 to be released and phosphorylate Cdc5 to trigger exit by MEN. It has been shown that DNA damage, in a cdc13-1 ts mutant, phosphorylates Bfa1 in a Rad53 and Dun1 dependent manner. This phosphorylation of Bfa1 could release Tem1 and prime cells to exit checkpoint arrest when cells pass through anaphase. Looking at Tem1 localization to spindle pole bodies and interactions with Bfa1/Bub2 in response to DNA damage might give insight into why cells don’t experience an anaphase-like arrest when they are released by either deactivation of the DNA damage checkpoint or SAC.

We have previously shown that a deletion of bub2 in a 1-DSB background shortens DSB-induced checkpoint arrest. Deletion of bfa1 in a 2-DSB background showed ~80-70% of cells stuck in a large-budded state as measured through an adaptation assay tracking the morphology of G1 cells on a YP-Gal plate and DAPI staining. Deletion or degradation of bfa1 might not release cells from arrest because the Mad2/Mad1 prevent cells from transitioning into anaphase. Our DAPI data for Bub2-AID shows an increase in cells with 2 DAPI signals (transition into anaphase) and small budded cells indicating that degradation of Bub2 is releasing cells into anaphase and allowing cells to complete mitosis.

Further suggestions:

It would be richer if authors could provide more than one experimental replicate in some panels (e.g., S1A,B; S4A; and S6B).

S1C confirms that Rad9-AID and Rad24-AID will adapt by 24 h even with the point mutant TIR1(F74G) which has lower basal degradation than TIR1. S4A has been updated with additional experimental replicates. The 48 h timepoint after DSB induction was to show the importance of Mad2 even when Ddc2 is overexpressed.

Figure 1: Rearrange figure panels when they are first mentioned in the text. For example, it makes more sense to have the plate adaptation assay as panel B for both 1-DSB and 2-DSB strains, budding plus DAPI as panel C, and Rad53 as panel D.

These figures have been rearranged in the order that they are mentioned in the paper.

Figure 5: Correct Ph-5-IAA in the Rad53 WBs (it should be 5-Ph-IAA).

This has been corrected.

Figure S2: The straight line under the "+IAA" text box is misleading. I think it should also cover the "-2" time point, right? Also, check the figure legend. Information is missing and does not correspond to the figure layout.

This has been corrected.

Figure S3: Perhaps "Cell cycle profile as determined by budding and DAPI staining" is a better and more accurate legend title.

The legend title has been updated to “Cell cycle profile as determined by budding and DAPI staining in Ddc2-AID and Rad53-AID mutants ± IAA 4 h after galactose.”

Figure S5: Detection of both Rad53 and Ddc2 in the same blot could lead to misinterpretation as hyperphosphorylated Rad53 appears to coincide with Ddc2 migration.

Figure S5A-B are representative western blots where Rad53 was probed to show activation of the DNA damage checkpoint by Rad53 phosphorylation. When measuring the relative abundance of Ddc2 we did not probe all blots for Rad53.

Table S1: Include the post-hoc test used for comparisons after ANOVA.

A Sidak post-hoc test was used in PRISM for the one-way ANOVA test. PRISM listed the Sidak post-hoc test as the recommended test to correct for multiple comparisons. A column has been added to S. Table 1 to show which post-hoc test was used.

Page 10, line 4: The putative additive effect of chk1 knockout with Dun1 depletion should also be compared to chk1 alone (in Figure 3A).

We address the additive effect of chk1 knockout with Dun1-AID depletion in a later section on Page 11, line 6. Since we had not explored possible effects from downstream targets of Rad53 for prolonging checkpoint arrest when Rad53 was depleted, we did not mention the effect of the chk1 knockout on Dun1 depletion.

Page 14, second paragraph, line 4: "Figure 6A-D", is it not?

Figure S6A is measuring checkpoint arrest in a deletion of mad2 in a 2-DSB strain. Figure 6A-D shows how degradation of Mad2-AID and Mad1-AID after the handoff of arrest causes cells to exit the checkpoint in a Rad53 independent manner.

Test de notifications Hypothesis 2

The long term emotional benefits ofpet oeneership became evident in the lower PTSD scores among pet owners over time. This suggests that pets may serve as a critical source of emotional support during the long-term recovery phase. Pets provide companionship, a sense of routine, and an emotional outlet, all of which contribute to reducing feelings of isolation and anxiety. The act of caring for a pet may also help restore a sense of purpose and normalcy in a post-disaster context. Moreover, interacting with pets has been shown to increase levels of oxytocin, a hormone associated with reducing stress and promoting feelings of well-being. In the context of prolonged recovery, where survivors may struggle with ongoing challenges like housing instability, financial difficulties, and rebuilding their lives, pets can offer consistency and unconditional love, which may buffer against the development of long-term psychological disorders like PTSD. This effect highlights the potential role of animal-assisted therapy or pet ownership as part of mental health interventions in disaster recovery programs.

The psychological. trauma and shock are so overwhelming that specific factors like pet ownership may not show a measurable impact on mental health outcomes. One potential explanation for the lack of a significant association between pet ownership and PTSD scores one month after the disaster is that all evacuees, regardless of whether they owned pets, were dealing with a similar level of acute stress and disruption. The chaos of the evacuation, uncertainty about future, and loss of property or loved ones likely overshadowed the nuanced emotional benefits that pets might offer. Furthermore, the conditions in evacuation shelters could have been so challenging for both humans and animals that any potential comfort a pet could provide was mitigated by the logistical difficulties of caring for them in such an environment. The delay in showing a positive effect on mental health suggests that pets may play a more substantial role in long-term recovery rather than in the immediate aftermath of a disaster.

The fact that pet acceptance was left to the discretion of individual shelter managers reveals the absence of a national or regional policy for handling animals in emergency situations. This lack of standardized protocol created a highly inconsistent experience for evacuees. In some shelters, animals were allowed but kept in separate areas, while in others, pets were outright refused. This inconsistency led to frustration, as many pet owners felt forced to make difficult choices between their own safety and their responsibility to their pets. The emotional toll on evacuees was exacerbated by the uncertainty of not knowing if their shelter would accept pets, causing additional anxiety during an already traumatic situation. This scenario highlights the need for comprehensive disaster planning that explicitly addresses pet welfare, ensuring uniformity and reducing the stress on pet owners during evacuations.

Has already helped those who have already integrated AI.

How it could be integrated and how it could boost productivity.

eLife Assessment

This important study examines the role of Microrchidia (MORC) proteins in the human malaria parasite Plasmodium falciparum. Solid experimental results, including genome editing and chromatin profiling methods (ChIP-seq and Hi-C), provide a comprehensive picture of the critical role MORC plays in shaping parasite chromatin. Depletion of MORC results in a lethal collapse of heterochromatin and parasite death, nominating the factor as a new target of antimalarial therapies.

Reviewer #1 (Public review):

Summary:

The authors investigated the function of Microrchidia (MORC) proteins in the human malaria parasite Plasmodium falciparum. Recognizing MORC's implication in DNA compaction and gene silencing across diverse species, the study aimed to explore the influence of PfMORC on transcriptional regulation, life cycle progression and survival of the malaria parasite. Depletion of PfMORC leads to the collapse of heterochromatin and thus to the killing of the parasite. The potential regulatory role of PfMORC in the survival of the parasite suggests that it may be central to the development of new antimalarial strategies.

Strengths:

The application of the cutting-edge CRISPR/Cas9 genome editing tool, combined with other molecular and genomic approaches, provides a robust methodology. Comprehensive ChIP-seq experiments indicate PfMORC's interaction with sub-telomeric areas and genes tied to antigenic variation, suggesting its pivotal role in stage transition. The incorporation of Hi-C studies is noteworthy, enabling the visualization of changes in chromatin conformation in response to PfMORC knockdown.

Weaknesses:

Although disruption of PfMORC affects chromatin architecture and stage-specific gene expression, determining a direct cause-effect relationship requires further investigation. Furthermore, while numerous interacting partners have been identified, their validation is critical and understanding their role in directing MORC to its targets or in influencing the chromatin compaction activities of MORC is essential for further clarification. In addition, the authors should adjust their conclusions in the manuscript to more accurately represent the multifaceted functions of MORC in the parasite.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

Summary: The authors investigated the function of Microrchidia (MORC) proteins in the human malaria parasite Plasmodium falciparum. Recognizing MORC's implication in DNA compaction and gene silencing across diverse species, the study aimed to explore the influence of PfMORC on transcriptional regulation, life cycle progression and survival of the malaria parasite. Depletion of PfMORC leads to the collapse of heterochromatin and thus to the killing of the parasite. The potential regulatory role of PfMORC in the survival of the parasite suggests that it may be central to the development of new antimalarial strategies.

Strengths: The application of the cutting-edge CRISPR/Cas9 genome editing tool, combined with other molecular and genomic approaches, provides a robust methodology. Comprehensive ChIP-seq experiments indicate PfMORC's interaction with sub-telomeric areas and genes tied to antigenic variation, suggesting its pivotal role in stage transition. The incorporation of Hi-C studies is noteworthy, enabling the visualization of changes in chromatin conformation in response to PfMORC knockdown.

We greatly appreciate the overall positive feedback and cognisense of our efforts. Our application of CRISPR/Cas9 genome editing tools coupled with complementary cellular and functional approaches shed light on the importance of _Pf_MORC in maintaining chromatin structural integrity in the parasite and highlights this protein as a promising target for novel therapeutic intervention.

Weaknesses: Although disruption of PfMORC affects chromatin architecture and stage-specific gene expression, determining a direct cause-effect relationship requires further investigation.

Our conclusions were made on the basis of multiple, unbiased molecular and functional genomic assays that point to the relevance of the _Pf_MORC protein in maintaining the parasite’s chromatin landscape. Although we do not claim to have precise evidence on the step-by-step pathway to which _Pf_MORC is involved, we bring forth first-hand evidence of its role in heterochromatin binding, gene-regulation and its association with major TFs as well as chromatin remodeling and modifying enzymes. We however agree with the comment regarding the lack of direct effects of _Pf_MORC KD and have since provided additional evidence by performing ChIP-seq experiments against H3K9me3 and H3K9ac during KD. Our new results are presented in Fig. 5. We showed that the level of H3K9me3 decreased significantly during _Pf_MORC KD.

Furthermore, while numerous interacting partners have been identified, their validation is critical and understanding their role in directing MORC to its targets or in influencing the chromatin compaction activities of MORC is essential for further clarification. In addition, the authors should adjust their conclusions in the manuscript to more accurately represent the multifaceted functions of MORC in the parasite.

Validation of the identified interacting partners is indeed critical and essential to understanding their role in directing MORC to its targets. Our protein pull down experiments have been done using several biological replicates. Several of the interacting partners have also been identified and published by other labs and collaborators. To confirm our results, we completed a direct comparison of our work with previous published work. Results have now been incorporated into the revised manuscript to confirm the identified interacting partners and the accuracy of the data we obtained in our experiment. Molecular validation of novel proteins identified in our protein pull down requires generation of tagged lines and may take a few more years but will be submitted for publication in a follow up manuscript.

Reviewer #2 (Public Review):

Summary: This paper, titled "Regulation of Chromatin Accessibility and Transcriptional Repression by PfMORC Protein in Plasmodium falciparum," delves into the PfMORC protein's role during the intra-erythrocytic cycle of the malaria parasite, P. falciparum. Le Roch et al. examined PfMORC's interactions with proteins, its genomic distribution in different parasite life stages (rings, trophozoites, schizonts), and the transcriptome's response to PfMORC depletion. They conducted a chromatin conformation capture on PfMORC-depleted parasites and observed significant alterations. Furthermore, they demonstrated that PfMORC depletion is lethal to the parasite.

Strengths: This study significantly advances our understanding of PfMORC's role in establishing heterochromatin. The direct consequences of the PfMORC depletion are addressed using chromatin conformation capture.

We appreciate the Reviewer’s comments and reflection on the importance of our work.

Weaknesses: The study only partially addressed the direct effects of PfMORC depletion on other heterochromatin markers.

Here again, we agree with the reviewer’s comment and have performed additional experiments to delve deeper into the multifaceted roles of _Pf_MORC. We have performed additional ChIP-sequencing analysis on _Pf_MORC depleted conditions focusing on known heterochromatin and euchromatin markers H3K9me3 and H3K9ac respectively. We hope our new results presented in figure 5 will shed light on the more direct implications of _Pf_MORC on heterochromatin and gene silencing.

Reviewer #1 (Recommendations For The Authors):

Suggestions for improved or additional experiments, data or analyses.

• Why does MORC, which was used in the pull-down, seem to be only minimally enriched in the volcano plot, while a series of proteins (marked in red) and AP2 (highlighted in green) are enriched with log2 fold changes exceeding 15?

We apologize for the confusion. MORC was detected with the highest number of peptides (97 and 113) and spectra (1041 and 1177) confirming the efficiency of our pull-down. However, considering the relatively large size of the MORC protein (295kDa) and it weak detection in the control (5 and 7 peptides; 16 and 43 spectra), the Log2 FoldChange and Z-statistic after normalization are minimal compared to smaller proteins that were not identified in the control samples.

Additionally, can you explain why these proteins appear to be enriched at the same fold? 

We can postulate that these proteins form a complex with a ratio of 1:1. Two of these three proteins are described to interact with MORC in several publications, supporting a strong interaction between them.

Variations in the interactome could result from the washing buffer's stringency.

We agree that the IP conditions could affect the detection of the interactome as well as the parasite stage used. As indicated below, the overlap with previous publications and the presence of AP2 TFs and chromatin remodelers strongly support our results.

It would be highly appropriate for the authors, similar to the co-submitted article (Maneesh Kumar Singh et al.), to present their mass spectrometry data in relation to previous purifications in Plasmodium (Bryant et al. 2020; Subudhi et al. 2023; Hillier et al. 2019) and also in Toxoplasma (Farhat et al. 2020). It would be good if authors could also put their results into perspective in light of the following pre-prints:

We agree with the reviewer’s comment. In this revised manuscript, we compared our IP-MS data to previous published manuscripts. Key proteins including the AP2-P (PF3D7_1107800) and HDAC1 were indeed identified in several experiments validating our initial findings of the formation of large complexes with MORC. However, it’s important to highlight that the MORC protein was not used as the bait protein in previously published papers, and thus some discrepancies can be observed.

Given the tendency of MORCs to form multiple complexes with AP2 factors, have you explored whether specific AP2s are conserved between Plasmodium and Toxoplasma, within the phylum?

P. falciparum encodes for 27 putative AP2s, while T. gondii has over 60 AP2s, making direct comparison challenging. Some Plasmodium AP2s have multiple counterparts in T. gondii and typically conservation is limited to the AP2 binding domains. Attempts to identify sequence homology among AP2s and the regions of conservation have been performed (PMID: 30959972, PMID: 30959972, PMID: 16040597). Although this information would provide interesting insight, we believe exploring this topic at this time would diverge from our primary objectives. It would be more appropriate to address this in future studies.

Could this conservation be identified either through phylogenetic means or by using tools such as AlphaFold, especially considering not just the AP2 domains but also any existing ACDC domains?

Although this may reveal important information regarding the association between MORC proteins and AP2 domains, we believe investigating the conservation between AP2 across apicomplexan parasites may prove too challenging and is beyond the scope of this work.

Most of the genes are depicted without their immediate surroundings (Fig. 2d and Fig S2c, d). For instance, the promoter region of AP2g is not shown (Fig. 2d). It is therefore very challenging to determine the presence or absence of MORC upstream or downstream; considering that this factor, which can create DNA loop protrusions, might bind at a distance from the genes in question.

All gene coverage plots, including AP2-G, show 500 bp up- and downstream of the displayed gene. We have modified our figure legends to make sure that this information is provided.

Upon examining Figure S3, it is evident that the authors have indicated a decline in PfMORC expression, represented as percentages over two unique time frames. The methodology behind this quantification remains ambiguous. It's essential for the authors to specify whether normalization was done using a loading control. As a benchmark, Singh et al. (2021) in their Figure 4 transparently used GAPDH as a loading control and included an untreated sample in their western blot analysis.

We thank the Reviewer for bringing this to our attention. Our initial quantification was performed using ImageJ. To address the Reviewer’s comment, we have reperformed the experiment. Our quantitative analysis was performed through Bio-Rad ImageLab software using aldolase expression as a loading control (50% of the MORC loading). This information has now been incorporated into the supplementary figures (Figure S3).

There's a striking observation that, despite significant degradation of PfMORC (as depicted in Figures S1 and S3), only the upper band in the western blot diminishes. This inconsistency needs addressing, as it can raise questions about the interpretation of the results.

We agree with the reviewer's comment. We experienced some challenges upon performing a Western Blot on such a large protein (295kDa). Our initial attempts required long exposure that may have highlighted non-specific signals of smaller proteins. To address the reviewer’s comment, we have performed the experiment one more time and made necessary changes to our WB protocol. Our new result better reflects the expected down regulation of _Pf_MORC. These changes have been incorporated to our manuscript and Fig S3.

Recommendations for improving the writing and presentation.

MORC KD quantification and consistency with previous findings (Figure S3): When comparing their results with those from another study (Singh et al. 2021), it's critical to ensure that the experimental conditions, especially the methodology for KD and the quantification of protein levels, are similar. If not, a direct comparison might be misleading.

We greatly appreciate the suggestions and have made efforts to redesign the MORC KD quantifications according to the reviewer’s recommendations.

While the manuscript mentions the level of KD, it does not delve into the functional consequences of such a decrease in protein levels. It would be of interest to understand how this level of KD affects the parasite's biology, especially in the context of the paper's main findings.

We have addressed this question by looking at the changes in chromatin structure in WT versus KD parasites upon atc removal. We have also validated this initial result by designing an additional ChIP-seq experiment against histone marks in WT versus KD parasites upon atc removal. Our findings showed a significant downregulation in H3K9me coverage in heterochromatin regions, specifically in genes associated with antigenic variation and invasion genes. These findings suggest that PfMORC regulates at least partially gene silencing and chromatin arrangements. The manuscript has been edited accordingly. 

Concluding page 5, the authors present an interpretation of their findings that suggests a multi-faceted role of PfMORC in regulating stage-specific gene families, particularly the gametocyte-related genes and merozoite surface proteins. While the narrative they present is intriguing, several concerns arise:

Over-reliance on correlation: The authors draw a direct line between the levels of PfMORC binding and the function of these genes in the parasite's life cycle. However, a mere correlation between PfMORC binding and stage-specific gene activity does not necessarily imply causation. They would need to provide experimental evidence showing that manipulation of PfMORC levels directly impacts these genes' expression.

We agree with the reviewer's comment. We have however partially addressed this issue by comparing our ChIP-seq, RNA-seq and Hi-C experiments. We concluded that several of the transcriptional changes observed were due to an indirect effect of PfMORC KD and were most likely induced by a cell cycle arrest and partial collapse of the chromatin structure. The collapse of the heterochromatin structure was validated using our Hi-C experiment. To further address additional concerns the review’s had, we have included additional ChIP-seq experiments targeting histone marks to confirm our initial hypothesis. Result of this additional experiment has been incorporated in the revised version of the manuscript.

Ambiguity surrounding "low levels" and "high levels": The terms "low levels" and "high levels" of PfMORC binding are qualitative and could be subject to interpretation. Without quantification or a clear benchmark, these descriptions remain vague.

We agree with the reviewers that the terms "low levels" and "high levels" of PfMORC binding are qualitative and could be subject to interpretation. We have however quantified our change in DNA binding using normalized reads (RPKM). In trophozoite and schizont stages, most of the genes contain a mean of <0.5 RPKM normalized reads per nucleotide of Pf_MORC binding within their promoter region, whereas antigenic gene families such as _var and rifin contain ~1.5 and 0.5 normalized reads, respectively (Fig. 2b). Similar results are also obtained for the gametocyte-specific transcription factor AP2-G  that contains levels of Pf_MORC binding similar to what is observed in _var genes (Fig. 2c and S2c, d).

Shift in Binding Sites: The observed minor switch in PfMORC binding sites from gene bodies to intergenic and promoter regions is mentioned, but without context on how these shifts impact gene expression or any comparative analysis with other proteins showing similar shifts. The claim that this shift implicates PfMORC as an "insulator" is a leap without direct evidence.

We apologize for the confusion. We  have compared our ChIP-seq with RNA seq results at different time points of the cell cycle and demonstrated that the shift observed has an effect in gene expression. We have edit the manuscript to clarify these results.

Overextension of PfMORC's Role: The authors suggest that PfMORC moves to the regulatory regions around the TSS to guide RNA Polymerase and transcription factors. This is a substantial claim and would require additional experiments to validate. Simply observing binding in a region is insufficient to assign a specific functional role, especially one as critical as guiding RNA Polymerase. Historically, the MORC family has been primarily linked with gene silencing across Apicomplexan, plants, and metazoans. On page 7, the authors noted a minimal overlap between the ChIP-seq and RNA-seq signals (Fig. 4e). They also acknowledged that the pronounced gene expression shifts at schizont stages result from a combination of direct and indirect impacts of PfMORC degradation, which could cause cell cycle arrest and potential heterochromatin disintegration, rather than just decreased PfMORC binding. Therefore, the authors should adjust their conclusions in the manuscript to more accurately represent the multifaceted functions of MORC in the parasite.

We agree with the reviewer's comment and have edited the manuscript accordingly.  

DISCUSSION:

The authors concluded that "Using a combination of ChIP-seq, protein knock down, RNA-seq and Hi-C experiments, we have demonstrated that the MORC protein is essential for the tight regulation of gene expression through chromatin compaction, preventing access to gene promoters from TFs and the general transcriptional machinery in a stage specific manner."

Again, the assertion that MORC protein is essential for tight regulation of gene expression, based purely on correlational data (e.g., ChIP-seq showing binding doesn't prove functionality), assumes causality which might not be fully substantiated. The phrase "preventing access to gene promoters from TFs and the general transcriptional machinery in a stage-specific manner" needs also validation. Asserting that MORC is essential for this function might oversimplify the process and overlook other critical contributors.

We agree with the reviewer’s comments and the conclusion has since been edited accordingly.

The discussion is quite poor. It would be pertinent to put MORC in perspective within the broader picture of regulatory mechanisms of chromatin state at telomeres and var genes. For instance, how do SIR2 and HDAC1 (associated with MORC) divide the task of deacetylation? Or the contribution of HP1 and other non-coding RNAs.

We agree with the reviewer’s suggestion. However, in order to put MORC in perspective within a broader picture, we would need to measure changes in localization of several molecular components regulating heterochromatin in WT versus KD condition. This will require access to several molecular tools and specific antibodies that we do not currently have. We have addressed these issues in our discussion.  

Minor corrections to the text and figures.

Figure 1d: Could you provide the ID for each AP2 directly on the volcano plot? While some IDs are referenced in the manuscript, visual representation in the plot would facilitate a clearer understanding of their enrichment levels.

ID for unknown AP2 proteins have been added on the volcano plot.

I recommend presenting Figure S2b as a panel within a primary figure. This change would offer readers a more quantitative understanding of the distinct differences between developmental stages. Notably, there seems to be a limited number of genes in common when considering the total, and there is an apparent lack of enrichment in the ring stage.

This has been done.

The captions are very minimally detailed. An effort must be made to better describe the panels as well as which statistical tests were used. 

We have improved the figure legends and add the number of biological replicates as well as the statistic used in each figure legend.

Figure 1A: The protein diagram with its domains does not take scale into account.

The figure has been modified.

Reviewer #2 (Recommendations For The Authors):

(1) The study lacks a direct link between PfMORC's inferred function and the state of heterochromatin in the genome post-depletion.

We agree with the reviewer's comment and have included additional ChIP-seq experiments to measure changes in histone marks in PfMORC depleted parasite line. We show a significant decrease in histone H3K9me3 marks in PfMORC KD condition.

Conducting ChIP-seq on well-known heterochromatin markers such as H3K9me3, HP1, or H3K36me2/3 could shed light on the consequences of PfMORC depletion on global heterochromatin and its boundaries.

With no access to an anti-HP1 antibody with reasonable affinity, we have not been able to study the impact of MORC KD on HP1 but have successfully observed the impact on H3K9me3 marks. These results have been added to the revised manuscript in (Fig. 5).

(2) The authors should conduct a more comprehensive analysis of PfMORC's genomic localization, comparing it to ApiAP2 binding (interacting proteins) and histone modifications. This would provide valuable insights.

We have performed a more comprehensive genome wide analysis of MORC binding through ChIP-seq on WT and MORC-KD conditions. Our results show that Pf_MORC localizes to heterochromatin with significant overlap with H3K9-trimethylation (H3K9me3) marks, at or near _var gene regions. When downregulated, level of H3K9me3 was detected at a lower level, validating a possible role of _Pf_MORC in gene repression. Regarding the comparison with AP2 binding, our proteomics datasets have shown extensive MORC binding with several AP2 proteins.

(3) RNA-seq data reveals that only a few genes are affected after 24 hours of PfMORC depletion, with an equivalent number of up-regulated and down-regulated genes. The reasons behind down-regulation resulting from a heterochromatin marker depletion are not clearly established.

We agree with the reviewer’s comment. At this stage (24 hours), _Pf_MORC depletion is limited and the effects at the transcriptional level are quite restricted. Furthermore, it is highly probable that down-regulated genes are most likely due to an indirect effect of a cell cycle arrest. We have edited the manuscript to address this comment. 

The relationship between this data and the partial depletion of PfMORC needs further discussion.

We agree with the reviewers and have improved our discussion in the revised version of the manuscript.

(4) The authors did not compare their ChIP-seq data with the genes found downregulated in the RNA-seq data. Examining the correlation between these datasets would enhance the study.

We apologize for the confusion. We have compared ChIP-seq and RNA-seq data and identified a very limited number of overlapping genes indicating that most of the changes observed in gene expression are in fact most likely indirect due to a cell cycle arrest and a collapse of the chromatin. We have edited the manuscript to clarify this issue.

(5) The discussion section is relatively concise and does not fully address the complexity of the data, warranting further exploration.

We have improved the discussion section in the revised version of the manuscript.

pages 1-15

• Hay un aumento en personas jóvenes con al menos un producto financiero
• Menos del 50% de las personas adultas tienen una cuenta bancaria

This was a really cool study/experiment I enjoyed reading it. I've never really heard about heterogeneity before so this was new for me to learn about!

I had no idea what senescent meant but when I looked it up it means something that has aged or a state of being old.

peergos mirror

self-certifying data

TLS

Amanda Mireles is using hypothesis for students to get their own video clips and to annotate them for other students.

Students took scenes from THE BIG BANG and used literature and academic references to relate to the scene

Rachel Derr showing how hypothesis can be used to deal with ethical and private issues in nursing care

I am watcing hypothesis annial awards and using hypothesis to gather our collective wisdom.

I have just placed this text into that seminar. Hypothesis is a modrn digital example of how ancient Druids were recorded by Celtic Monks to keep wisdom. A monk wrote down what a Druid said. They wrote the material in large letters with large gaps between the lines. When the monk died, a new monk woiuld take over the wisdom keeping. The new wisdom keeper wrote IN BETWEEN THE LINES and ON THE SIDE LINES. These were called glossaries becase they wrote in GREEN INK. Glas is the Irish word for Green.

militaristic discourse can connect countries across national borders

link to gender and military

military masculinity

othering connected to militarism, enacted through it and created by it

militarism is entangled with race

This shows that the cat might have been cared for and it was not just a wild cat that lingers around.

By using this choice of words the author is making the connection that the cat isn't just a thing that's there. It's a thing that goes where the person goes.

This shows the challenges and hardships the cat Pangur Ban went through his life journeys.

More than loud acclaim its bigger than you expect

one person's attempt just so he can be alive