Can relate this to Disinvestments and built environment from a public health course

20% to 40% of the poopulation's residents live in poverty MLK in only 17%

The quality of sleep is critical for work performance and other aspects of life as well

I actually hadn't thought about how my mac has the same keyboard shortcuts as other Apple products across the whole system. It makes sense and probably contributes to some people's choice to use mainly, or even exclusively, Apple products. Consistency seems like a very important heuristic when designing for a company that has multiple products, to ensure users have an easy experience with the interface.

I think this is a great point to consider. In the previous video, the record button had an icon and it would be interpretable by tech savvy people, but maybe not young children or older people who are not as familiar with newer technology. They could've done a walkthrough where they test the design against multiple personas, ensuring that their design is inclusive. Of course, there is the fact that designers want their product to work for their target audience. However, it is important to make designs more inclusive for every user base.

I found this section interesting because it shows how persona design can move beyond surface traits and actually reflect the way people think and behave. I agree that this approach makes evaluations more inclusive by considering these specific traits. It made me realize that realistic personas aren't just creative writing but they're grounded in real psychology and can reveal deeper issues pertaining to usability.

In this passage I know that In recent years, anxiety, depression, and damaged self-esteem have been on the rise among teenagers (especially girls). Meta's internal research shows a "perceived impact" rather than a clear "causal mechanism." For example, Meta's research asked teenagers, "How do you feel Instagram has affected your emotions?", rather than using a randomized controlled trial to measure it. Therefore, I think it's difficult to measure the specific effects and how they occur, because it's a very personal and emotional question. Therefore, one crucial detail remains unclear: whether "social media causes mental health problems" or "teenagers with mental health issues are more likely to use social media."

An interesting detail I found in a study they linked is that emotional contagions can be given in the absence of nonverbal cues. This may help us understand the significance of how social media having less ways of conveying body language.

I totally agree that social media influence on mental health. Because many people's actions are amplified online, and anyone can express their opinions to the poster, a lot of different ideas emerge. People may argue or post comments that are detrimental to their mental and physical health. This leads to people suffering from cyberbullying. Many people become depressed or choose to end their lives because of cyberbullying, so I think this is a terrifying thing. We should focus more on ourselves, instead of on the Internet.

I think that extreme cases, such as 4chan's incel community, should just be removed. If it's an obvious cycle of harm that only causes more problems, it's their responsibility to rectify that harm or at the very least prevent more of it.

This Wikipedia entry describes the evolutionary path of cetaceans, starting with the discovery of cetacean fossils on land, then moving to shallow waters, and finally becoming entirely marine. Modern cetaceans also fall into two main categories, such as Mysticeti and Odontoceti. Their evolution involves not only genetic and skeletal development but also cultural behaviors, such as the use of different tools for foraging. Environmental factors also influenced the divergence of cetaceans. One such detail is the radioactive events; I observed three large-scale radioactive events, the last one occurring 12-2 million years ago.

I looked at [l48] “We Need to Talk About Digital Blackface in GIFs” from Teen Vogue (2017). This article really stood out to me because it explains how using GIFs of Black people to express exaggerated emotions can unintentionally repeat old stereotypes — similar to how blackface mocked Black expression in the past. What I found powerful was how it connected something as casual as sending a reaction GIF to deeper issues of race and representation online.

This source made me think about how easy it is to participate in cultural appropriation without realizing it. It also connects to the chapter’s point about “copying” — that not all copying is harmless or funny; sometimes it carries history and meaning that needs to be respected. I think this article pushes readers to be more self-aware and ethical about what we share, even in small everyday actions on social media.

The Selfish Gene written by Richard Dawkins was mainly about how the evolution of genes was. The main goal was to change and survive. The book also mentioned “memes”, how people spread the picture from person to person, just like how genes work.

This article by Rick Paulas provides a visceral, first-hand feel for what it means to go “viral” in social media contexts—describing it as something like “doing cartwheels on the water-spout of a giant whale.” That kind of metaphor really brings home how thrilling yet unstable virality is: fun, exhilarating, but also out of control and potentially dangerous.

By the definition that a meme is a gene like organize that replicates and adapts, to a degree it can be argued that languages, or words in a language are also a form of memes. Certain words can adapt and change depending on circus stances in order to best suit the time's culture. This is also the case with words that become memes, as seen through the usually uninteresting number "67", which has since become a viral meme. Due to its meme status, this also changes how people perceive existing instances of the number "67".

By reading the wikipedia I learned the why memes are called memes, and understanding a viral word with the biological explanation is very interesting. Before reading the article, I thought the memes were just pictures we share online, but after learning the theory of memes, I realized that these online pictures are also a form of evolution.

This is an emoji reply from Monica Lewinski about the "worst thing she's ever done". It is a great example of how the internet adds onto each other still going with the thought of how it evolves.

I've seen this kind of video before and I thought this type of video could enormously improve the depth of communication among people online. Because people who filmed these videos with others need to spend a lot of time. Although their main purpose was to make their videos look good and receive more people's likes, the emotion these videos express followed the virtue ethics to make the platform community friendly and strongly emotionally bonded.

As much as I respect her efforts to create light out of her situation, part of me feels like this is an attempted grab at relevance. The Clinton scandal is so old now, and in my opinion, many of her joke tweets are unfunny and not meaningful in any way. Just feels like her bringing up something that many Internet users now weren't even alive for.

I find the origins of the term meme very interesting because I had never really considered how the term was coined. According to the article, meme is short for mimeme which means "imitated thing," and is also modeled after the word "gene." However, the explanation for the word meme makes sense because it's a piece of media that evolves and adapts through time and is shared by people. Furthermore, I found it surprising how the term was coined by Dawkins in 1976, long before the internet became widely available and accessible.

Ghyslain Raza became known as the "Star Wars Kid" after he recorded a video of himself pretending o fight in the Star Wars movie. A classmate posted the video online and it became viral. He was bullied severely and had to finish school is a psych ward. There are a lot of negative side effects of going viral and how that subjects you to unwanted and negative attention.

The "Star Wars Kid", aka Ghyslain Raza was a child featured in a viral video of a mock lightsaber battle. The video is estimated to have amassed over a billion views, and the internet was struck by the hilarity of the video. However, Raza was psychologically damaged and suffered emotionally from the video, even finishing school in a psych ward. This example demonstrates the dangers of the internet and how someone can suffer greatly from going viral.

This article caught my eye because I can remember listening to a podcast when I was younger about a pyramid scheme that someone didn't realize they were apart of. They just thought they were joining a good business to make some money in and then it all fell apart and they got in trouble for it. A pyramid scheme is a business model that asks its employees to pay a certain amount of money when they join and then are told that for every person they can recruit they will get a cut of their payment. This goes on to just make a ton of money for the higher ups in the pyramid and screws everyone else up and is nota legitimate business.

This source discussed the consequences that occur when you post your children without them being able to have a say in what they are posted in. One mother in particular suffered the consequences of this because she posted videos of her daughter dancing around the house, and most of the comments were on her daughter's appearance, which quickly prompted her to take the videos off the internet.

The reason that these types of videos are able to go viral is due to the experimental nature. Because no one is going to personally make something like that, they go online to see others do it for them. It’s interesting to see how people can find a niche like this and create a fan base and online persona.

This web page is dedicated to a chained letter campaign in the 80s and 90s. A chain letter is a letter that you send to someone with the intent for them to pass it on. This one specifically says please send a copy of this letter to 20 people. On the website you can open each iteration of the letter to see if it changes overtime.

As much as i dislike the song, it does bring me nostalgia. I'm surpised and sad to see that Rebecca got bullied for it. I though people liked the song.

A bean dad is about a person named John Roderick who tweeted a story of his 9-year-old daughter asking him to open a can of beans, but John Roderick thinks she should figure out how to open it herself. His daughter ended up spending hours just to open the can of beans. After he posted the tweet, people started accusing him, saying it is an abuse for him to treat his daughter like that. After this drama, people discovered more about John Roderick’s past controversies. Shortly, people turned this into a meme. Users edited this story to fit in a different scene. 

This article is a compilation of lots of Tweets made by Monica Lewinsky. The writer praises her humor and says she is a "light" in the cesspool of Twitter. I appreciate how she makes light of a bad situation. She knows that there are many eyes on her and she isn't fazed by it. I think this is a positive example of using social media for good. She uses it to make people laugh, instead of using it to tear people down.

This is content I often see on social media, it could be content related to books, shows or movies ("I read ___ so you don't have to"). However this kind of content related to food reminds me of tiktok trends in 2020 where people were trying weird food or watching people try weird foods out of pure boredom. This could be a kind of trolling, people trying bad or weird food combinations to get views, engagement or create discourses online.

This article really resonates with people. The author describes "going viral" as "doing a cartwheel in the blowhole of a giant whale" - it sounds funny, but just imagine how out of control it is. You post something on your social media, and suddenly it becomes the topic of discussion all over the world overnight. The pressure and absurdity hit you all at once. It reminds us that although going viral on the internet seems glamorous, it often comes with huge psychological burdens, exposure of privacy, and completely unpredictable consequences. In other words, "the light of going viral" has a lot of "burning heat" in it.

I'm surprised I haven't heard of this before. To be a little critical of the idea, I feel like the main character tends to be a few people exchanging the spotlight every time. Especially now that Musk has proven he is willing to rig the system

This article explains how social media rewards whatever grabs attention, even if it's shocking or negative. It connects really well with this chapter's idea of selection: the posts that get the most reactions are the ones that survive and spread. I thought it was interesting but also a bit depressing that success online often means being louder, not smarter. It made me realize how easily the attention economy can shape what we see and believe.

This article basically summarizes how posting children online could be putting them in jeopardy for unwanted attention. One example it talks about is a mother who is also a Tik Tok creator. After having a video of her child go viral, she noticed a lot of predatory comments towards her kid, and now wish she hadn't posted the video at all. The article also discusses family vlogging channels and child actors, and how these kids can't really consent to being featured publicly. Many of these children often have the deal with the consequences of being perceived so young, and as they grow up may feel the need to constantly be performing.

I found The Selfish Gene really thought-provoking because it completely changes how we think about evolution. Instead of seeing living things as the main focus, Dawkins makes us see genes—and even ideas—as the real “survivors.” Personally, I think this idea is still very relevant today, especially when we think about how memes and online trends spread. It’s amazing how something written in 1976 can explain how the internet works now.

I think Monica Lewinsky is a great example of virality. Before her scandal of being rumored to have had an affair with U.S. president Bill Clinton, no one really knew her. She was just a white house intern. But after news broke out and theories started to spread, her life changed drastically to constantly being ridiculed by the press and public. Although many years have passed, and she's now an activist for women's rights, people still reference the meme of her "being under the desk" to this day.

The Waterloo chain-letter example nails classic memetic tricks: authority (“around the world nine times”), urgency (“96 hours”), a fixed replication goal (“send 20 copies”), and fear/hope anecdotes. It’s basically early engagment bait—just offline and slower. The fixed “20” reads like an R0 target; if folks actually did it, growth would explode until friction kills it. Which element actually matter most in real-life replication—the deadline, the number target, or the vivid stories? My hunch is the deadline did most of the work (it fights procrastination and nudges action now), which is still how viraly prompts get us to click today, definitley.

For my experience, nowadays meme is kind of trends. People will put the most hottest influencers on meme, or some funny graph or words on meme. A particular type of meme may become trendy for a period of time, but meme trends change very quickly. A new meme trend might emerge roughly every month. People frequently showcase currently popular memes on social media. For example, the Madagascar penguin is a very popular meme on Chinese social media right now; it's both funny and well-known.

Some of the memes are meaningless, so many of the people are actually seeing memes as downsides to our existing culture. For example, people think that if everyone is using these abstract words to express their feelings, especially for the young generation, they would lose their ability to express their thoughts formally, and not be willing to learn how to communicate with each other offline.

I think the consideration of the role of evolutionary forces in spreading true and false information is meaningful. Especially nowadays the internet allows the efficient spread of information, and it is hard for people to tell the reality of information online.

This reminds me of something quite silly but I think it's worth mentioning. While this term was later adapted to refer to what we today call a meme, it was still in use a this definition before and did circle through media, which made the media retroactively very comedic through the redefining of the word meme. My favorite example of this is the 2013 game Metal Gear Rising: Revengeance, which has a plot points revolving around how the only thing that truly matters to a persons self and decisions is memes and the ideas that their culture pass on to them. But with our modern definition, all the thoughtful speeches throughout the game become unintentionally very funny.

Before reading this chapter I had never tied meme with the biological evolution, and I didn't know that the term "meme" comes from gene. It is very interesting to me how memes are spreading just like evolution process. Memes are spread super easily, and people edit it and spread it in so many different ways but still keeps the main theme. It is very interesting how people find different ways to express themselves on the internet with different memes.

This passage made me think about how memes are almost like a game of telephone throughout online communities and across generations. A millennial may see the same meme in a completely different way than someone in generation z or alpha and visa versa.

Reading this section about The Selfish Gene really amazed me. I never realized how deeply connected biology and culture could be. The idea that memes evolve in the same way as genes made me think differently about how fast ideas spread on social media today. Personally, I find it both exciting and a little scary—exciting because creativity can spread so quickly, but scary because misinformation can too. It made me realize how powerful our sharing behavior is in shaping modern “evolution.”

The lack of knowledge held on these specific words can be weaponized as polarization to involve the aforementioned ingroup/outgroup thinking.

Main Takeaway: Bilingualism in class helps learning. teaching should be flexible, and not English only.

They are challenging English only teaching as they argue for balance.

Observed classes in Norway

Main idea: finding balance between target language and students first language.

Reading this section made me think about how normalized copying has become online. Platforms like TikTok, Twitter, and even meme pages thrive on remixing and reposting, but most people never think about who originally made something. Personally, I’ve shared memes and gifs without even realizing they came from artists who might want credit. I think Confucius’s idea of “li”—doing what’s proper and respectful—applies here: giving credit isn’t just a rule, it’s a way of showing respect for the creator and the community.

At the same time, I agree with Michael Wesch’s point that remixing can be a form of cultural expression and creativity, not just theft. It’s tricky, though, when remixing turns into cultural appropriation—like when certain slang or imagery from Black culture is taken and used for jokes by people outside the culture. I think the line between cultural exchange and appropriation comes down to intent and respect. If you’re sharing something to appreciate and understand, that’s exchange. But if it’s just for clout or laughs, it’s exploitation.

This section really made me rethink how I use memes and social media. I’m going to start paying more attention to where things come from—and maybe even give credit when I can, even if it’s just a tag or mention.

We should notify who made the content. For instance, if we repost a video or a picture, the author’s name or related tags should be mentioned in order to show respect. If the author mentioned that this is their private property, we should ask permission for its copyright and tagged all their information on it.

Memes are a great way to get an insight into the culture of people but what is important is that it is those people making memes about themselves. Looking at memes made by black people that are poking fun or making references in black culture can be a great way to better understand black culture. What is problematic is white people making memes about black culture, because that is not coming from a place of deep understanding and critique but from prejudice. Its kinda like doing accents, if you as a white person do a impression of a marginalized group it better be really damn good or its gonna be racist. And if you can do a really good impression its likely that you are coming from a place of deep understanding of the culture because you have spent a lot of time with its people or something else.

To be honest, this statement is not an exaggeration at all. Nowadays, memes, pictures, and jokes on the internet spread so fast that the original creators can hardly keep up. By the time you notice them, they have already been repackaged, filtered, and had their fonts changed by a dozen netizens. In the end, it's impossible to tell who the original creator is. Although the internet has a memory, sometimes it seems more like it remembers who spreads things faster. However, giving credit is not difficult. A simple "cr: original creator" can show respect and prevent you from becoming a "content pirate". Everyone will feel much more comfortable this way.

When your body builds a molecule (such as an amino acid or a sugar), its "left-handed" enzymes will only make the "left-handed" form. It doesn't create a 50:50 mix of both left and right gloves; it just makes a pure batch of left ones.

Compounds from living things (like sugar from a plant) are almost always pure "left-handed" or pure "right-handed."

The Result: Because life's enzymes are so specific, the compounds they produce are also specific. This purity (not being a 50:50 mix) is what makes them optically active.

How Racemates are Formed? If you make a "handed" (chiral) molecule using only symmetrical, "non-handed" (achiral) ingredients, the reaction has no preference. It's like flipping a coin—you'll end up making 50% "left-hand" molecules and 50% "right-hand" molecules. The result is always a racemate.

The only way to avoid getting a 50:50 mix is to use a "handed" ingredient (a chiral catalyst) in the reaction. This "handed" ingredient acts like a template, forcing the reaction to make more of one hand than the other.

What is a Racemate? A racemate (or racemic mixture) is a 50:50 mix of both "hands."

Because you have an equal amount of the "left-twister" and the "right-twister," their effects cancel each other out completely. The final mixture doesn't twist light at all—it has no optical activity.

Start with Special Light: The machine uses a special light (monochromatic) that is passed through a fixed filter (the polarizer). This filter acts like a vertical slot, forcing all the light to vibrate in only one direction (e.g., up-and-down).

The "Empty" Test: Before you add a sample, you have a second, movable filter (the analyzer) at the other end.

Max Light (0°): If you line up this second filter perfectly with the first one (both vertical), all the light passes through.

No Light (90°): If you turn the second filter sideways (to 90°, making a "+"), it completely blocks all the "up-and-down" light from the first filter. No light gets to the detector.

This "no light" position is the starting point. When you add a sample (like sugar water), if it's "optically active," it will twist the light. The "up-and-down" light might become "diagonal." This twisted light can now sneak past the 90° filter, and the detector will once again see light. You then have to turn the analyzer to find the new "no light" angle, and that angle tells you exactly how much the sample twisted the light.

I think the way the book compares all of these different examples of evolution and loops back around to how its related in social media is really well done. I believe it leaves me feeling engaged and intrigued to read more.

It is due to this phenomena of replication and reposting that created the common idea that anything on the interest will forever stay on the interest, and be impossible for any party to completely scrub of the interest. This is also the case for memes, and specifically the people who are features in the memes. Often times, due to different factors, the people in a given meme might face back clash from other users on the interest, resulting in the person being harassed and bullies, such was the case with the star wars kid back in the late 2000s. This also is the catalyst for interest cringe culture and the reasoning for the effectiveness of mutual surveillance by other users.

I think nowadays the algorithms will only provide information that people are interested in, which helps people have better access to the information they need. However, this also prohibits people to have a wider access to all type of information.

As someone who uses social platforms and watches how memes / posts spread, I’ve observed that sometimes the version that goes viral isn’t the original but a mutated one (someone adds a caption, remix, or cross-posts to another network). The chapter’s point that inheritance matters jumped out: once a variation exists and spreads with the change, future copies carry that change. That resonates with seeing e.g. a tweet being quote-retweeted, then everyone repeats the quote-tweet version, not the original tweet.

I think that this comment is very interesting and I would like to add my personal experience with the mutation of social media and the memes of social media that evolved into what memes are today. Memes have evolved from one another and continue to do so every day, an example of this being "brainrot," a form of memes that first began around 2020 but still evolved into being relevant today.

The ease at which things are able to duplicate and spread across the internet is what creates trends online. Being able to show your friends a funny video allows for more eyes to be on a video which leads to the algorithm picking it up and getting more people to see it.

I like how this section compares social media to evolution, and it actually makes a lot of sense. A post can reproduce when people share it, mutate when someone adds a caption or emoji, and then survive if it goes viral. It's funny but a little scary to realize how fast ideas can change and spread online. Sometimes the meaning of the original post completely disappears after being shared so many times. It made me think that maybe memes evolve faster than anything in nature!

I like how the chapter uses evolution to explain virality, but the “selection” part on social media feels more like artificial selection than natural. Platforms kinda breed certain traits on purpose (or at least by design): short, remix-able, high-arousal posts travel farther because the UI + metrics reward them. Remove visible like counts or add one extra click to repost and suddenly the “fitness” of outragey jokes drops—this isn’t nature, it’s a product decision, tbh. That ties back to algoritm ranking from last week: ranking isn’t a mirror, it’s a selector that shapes what even exists to be copied. So my question is: if platforms act as the main selector, how much responsiblity do they own for which memes win and which basically go extinct?

what if the speaker deems her dreams “little” bc she never got the chance to make it a reality so she downplays the dream as a result?

The Chemicaal trsucturs of MJ and heroin mimics how neurotransmitters work

It allows the drugs to attach and act on the neurons

I find this part very interesting. The concept of "cringe" in the 2010s was so prevalent, but now the lines are so blurred, as content many people enjoy can also be widely mocked as cringe. And on top of that, intentionally cringe content (which often goes viral and gets lots of success) makes this distinction even more difficult. But is this a good thing? It facilitates more diverse discourse and leads us away from homogenous thinking and opinions, as now there is disagreement. Which I think might actually be a good thing for internet culture.

I hated this song so much. I remember it was all my friends and siblings would sing and replay over and over. It got to the point were I just didn't find it funny or enjoyable.

This passage significantly advances the essay's core argument by incorporating the concepts of deep time and the Anthropocene into the analysis of Amitav Ghosh's The Hungry Tide. It argues that the geological timescale evoked by the Anthropocene, which the novel weaves into its historical narrative of the Sundarbans, adds a vital new dimension to the novel's central conflict.

Specifically, the author claims that framing the transcultural conflict between Western environmentalism and subaltern refugee agency in the Sundarbans within deep time suggests that these tensions are not merely historical (colonialism vs. decolonization), but are part of a broader, planetary crisis of agency unfolding across immense temporal scales, both past and future.

This perspective implies that concerns over biodiversity loss and social injustice are fundamentally linked at the level of planetary change. Consequently, addressing these complex tensions such as the historical trauma of the Morichjhapi massacre requires a "longer temporal perspective." This expanded view is necessary to fully grasp the history that conditioned past events and, critically, to anticipate the increasing agential challenges that geology and climate change will pose to cases of forced migration in South Asia in the future.

the thesis of an academic essay analyzing Amitav Ghosh’s novel, The Hungry Tide. The author frames their argument within recent scholarship on postcolonial ecocriticism, specifically addressing climate change and the Anthropocene, concerns previously underexplored in novel scholarship.

The central claim is that the novel demonstrates the political value of a utopian approach to refugee agency in South Asia, particularly for populations facing climate-induced migration. This focus shifts critical attention to how the text models imaginative, hopeful solutions for empowerment and survival, moving beyond discussions solely focused on ecological degradation and conflict.

this part shows the methodology of postcolonial ecocriticism. It argues that analyzing complex conflicts between human and nonhuman interests requires a three-part lens: ecocriticism, environmental and postcolonialism. This confluence defines the field's mission: to critically assess representations of how mainstream environmentalism interacts with subaltern agency (marginalized groups). The field exists to analyze tensions and potential reconciliations, ensuring environmental action does not perpetuate historical injustices against the world’s most vulnerable populations. It is a necessary, synthesizing approach.

It's interesting to see this as I remember in the early days of the internet when digital chain mail would go around with a negative or scary incentive to repost the image or thread.

I think this is interesting because it reminds me of copypastas that can be found on the internet. Sometimes, there will be a TikTok in my feed that is of the same nature, urging people to repost and use the audio for good luck. I did not know chain letters were a thing and it's really interesting to see how they are carried over in the digital age.

This reminds me so much of the chain texts people sent in middle school. I remember receiving these texts and actually being scared that bad things would happen. I think it's interesting that this format has stayed the same and that it exploits people's superstitions through a carrot and stick method.

I like the analogy of sourdough for the Internet. It seemed silly to me at first, but it actually is quite accurate. The sourdough starter grows and develops over time. In the same way, a meme or an online joke starts with one user / one event, and morphs depending on who interacts with it. People can put viral topics in new contexts and give them a new light. The sourdough starter can be used into multiple different loaves.

It's a blessing in disguise, at least to the T&L world.

I had an interesting experience during covid when we were all locked indoors of going viral on tik tok and I will never forget it. I was always a bit obsessed with going viral during covid as any middle schooler in the time was. It was right when the video game among us was going viral itself and I decided to try and benefit off of that. I played the game a lot and really enjoyed playing, I decided to create a fresh tik tok account that would post funny among us content. Videos would be 60 seconds and of my game play along with funny sound effects over the gameplay and my videos went pretty viral. I worked up to 170 thousand followers and a total of around 5 million likes and even more views. It was a very fun but also stressful experience because once I reached that viral status, I was constantly worried about keeping it and not going down in views.

I agree that while A/B testing can help provide evidence of causality, there may be issues in verifying if the results can be trusted or not. This makes me think about concepts such as validity. How do we know that the results are because of the specified variable, and not other extraneous variables that may have influenced the results?

I like this section because it highlights how valuable failure can be in the process of designing something. I agree that usability testing is less about providing a design and more so about finding out where it breaks down. It reminded me that good design comes from observing and understanding in order to fix those breakdowns, until the interface works for people.

I think this highlights the most prominent weakness of these user studies. Despite that weakness, it seems like usability tests can be very valuable and worthwhile when designing an interface. It seems like all of the design methods we learn about are strong in one way and weak in another, so it's really starting to click for me why we should do so many types of tests and use different methods.

I like how the content from this chapter relates to the content from INFO 300 and all the ideas from randomized tests. I am also taking INFO 370 which approaches many concepts in parallel as well on how difference design choices for the participants have different pros and cons depending on which validities the study/research is aiming for.

See Ezra Klein's argument against using ChatGPT for writing even the first draft. https://podcasts.apple.com/us/podcast/how-i-write/id1700171470?i=1000710273359

Hoewel het voor mensen denk ik normaal is om vast te houden aan onze 'bijzondere' menselijkheid, ben ik het niet eens met deze stelling. Zodra wij mensen de AI-systemen zo kunnen instellen en ontwikkelen dat zij bijna ons als mens evenaren en onze waarden aanhouden, geloof ik dat AI ons ook op dit vlak enorm kan helpen. Wij moeten als mens de controle soms misschien ook iets meer loslaten en denken dat wij speciaal zijn. Dit kan overigens alleen als AI zich nog veel verder ontwikkelt naar onze kritische standaarden.

Hier ben ik het erg mee eens, vooral gezien het feit dat AI-gegenereerde 'creativiteit' tamelijk zielloos aandoet.

Vanwaar de focus op het waarborgen van diversiteit? Ik moest zelf meteen denken aan de gevaren van het baseren van succesvolle output op basis van klikgedrag en het toenemen van sensationele content.

Point 3 primary source for references

where is the ann

Elena Aguilar has so many great resources! We focused on her work as an academics team last year. It is so important to "pay attention" to our biases.

Given this is a membrane protein, did you have issues with solubility? Do you have data showing the protein is well folded after purification?

I am not sure that co-localization of those tags is the best way to confirm that the protein integrity contains both tags. It is unlikely that you could see single proteins based on your image resolution - I think the stronger claim is the western blot you have that shows the size change upon cleavage (fig 2E,D)- and also just the fact that your protein is of expected size with the two tags.

It is super helpful that you included so many tag designs already in the construct. It really adds to the usability right off the bat.

This sets up the reason epigenetics matters in schizophrenia: not just genetic inheritance but modifiable environmental impacts shape risk and progression. Epigenetic mechanisms such as DNA methylation, ncRNA regulation, histone modification serve as bridges between what you inherit and what you experience.

Key schizophrenia/autism genes with varying lifetime methylation patterns. Important for understanding neurodevelopment. This fits with article summary that epigenetic mechanism like methylation influence schizophrenia development. DLG4, DRD2, NOS1 and SOX10 are genes with varying methylations that influence schizophrenia.

delete this "of" to fix the grammatical flow of this sentence!

Interesting how politics can play such a role in the welfare of general public. Even stepping out of line to help has negative consequences, perhaps it's mirrored in our society in other ways. (Politics & Public Health)

I agree with this terminology a lot. Social media tends to make people feel fake already and the incentivization of fame only makes it worse. It's very annoying to go onto social media and need to differentiate ragebait, trolls and people being genuine.

Kimmerling's voice analyzes Benny Morris's shocking moral and political shift. Kimmerling critiques the notion that ethnic cleansing could be justified by the "overall, final good". This tackles the moral conundrum of nation-building (Q4). Morris’s quote, used here to frame the debate, provides an explicit, utilitarian justification for mass violence and expulsion, linking the successful establishment of the exclusive nation-state (rigidity) to necessary moral compromises such as breaking an egg for an omelette. The metaphor of breaking something for a better and more useful end result. My synthesis of this critique highlights the danger of using historical necessity to legitimize atrocities, fearing this intellectual legitimacy fuels current radical impulses for future ethnic cleansing, because I can't ever see myself thinking humans are mere eggs waiting to be broken in order to create a better territory.

This quote from Morris’s interview refers to the atrocities committed by Israeli forces during the 1948 war, some of which involved rape. The main point of this quote is that the number of 12 is unreliable because the reported cases are not even the entire story. Morris argues that because of the typical pattern of underreporting sexual assault, the number must be higher. He uses the metaphor of the iceberg to reveal the hidden realities of sexual violence. Morris’s quote shows that the Israeli “acts of massacre” and wrongdoings are more than the ones documented.

I think that a standard english is quite unreasonable for the average person to use consitently, and that even in professional situations, often when I have broken out of that mold, even for a joke or passing comment, it not only humanizes me but connects me to others, even when I say something standard english would not deem acceptable.

I think this does complicater someideas, as I think one can successfully code mesh but much of the success is deppendent on understanding ones audience and if they will understand the purpose of code meshing. I often speak to friends and family in multiple dialects, which I determine how and when to do such based of of my relationship with and knowledge of my audience. However, in education, such as grading, how does one differentiate bad writing vs code-meshing? I believe this can be determined from the usage and context, such asif code-meshing can add a stronger argument or foundation for ones work.

back to the students refered to earlier, when they are miscatorgized as unprofficeint reader, it harms their further education, their sense of self and confidence, and especially for younger students this can affect the rest of their education

This ssue is not about race, but about encouraging students and nuturing thier language skills

Minimizing the racial aspects of code switching can actually help people be more accepting to code-switching. When race is added to the equation, it becomes one of the only aspects people will focus on. Perhaps the racism that is equated with code-switching is actually peoples own biases, as they are not seeing the whole picture.

The racial aspects of code-switching cannot be denied, bit their are many other factors that would cause one to 'switch'

Even slight shifts in dialogues is normal across many different regions and dialects. This shift can depend on many different factors. How does one learn what shift they should use for different circumstances?

What I have been refering to as proffessional vs personal, many people will change their dialect depending on their situation, sometimes without meaning to. I have often used the so called 'customer service voice' at jobs and ets, but without even thinking about it conciously. I do make this switch to make others around me either more comfortable, or in some cases, to lessen aggression from others. By changing my tone and word choices, it is easier to navigate with certain people and their percetions of how a service worker should speak and act.

Language is used in many ways, and even subconciously can affect people's biases

Differences in dialects stem from more than just race, and I agree with the idea of a class difference. Along with this, those in higher classes are usually the ones to set the standards, even though they are not the majority of people.

When teachers learn how to differentiate dialects vs actual mistakes, they also feel more acclomplished as a teacher who helps their students compared to feelings as though the kids cannot learn.

When taught the differences in dialects, compared to only be taught a standard dialect, students are given the option to chose for themselves, showing their own compentence and opions. This shows many chose standard for more proffesinal cases, and their own dialect for creative freedom and expression. By embracing their unique dialects, students aare able to learn more comprehensively, and have shown improvement in their reading a writing capabilities, vs forcing students to use only one dialect. Along with use, stopping students from using their home dialect, it can also cause them to lose their sense of self and distance them from their communities.

prejudice and biases contributes to students not recieving a proper education. what world is putting a child in a corner all year vs actually teaching them? Melinda should've been fired and her treatment should be considered neglect.

The goal is not to stop students from using their preffered dialects, but to use it as a tool to continue to teach students grammar, spelling, and reading comprehension.

This form of standard english has changed as well, and other regions and languages have their own 'standard' or 'True and Real' form, as well has having other dialects that are regarded as non standard.

Code-switching/meshing has mixed opinions, especially in education. Is labeling this switch as 'code-meshing' racist? it is undenieable that people will speak different dialects depending on many factors, not just race,but also class, location, gender, etc

This line really stood out to me because it captures both empowerment and unity. Sojourner Truth reminds her audience that women’s strength has always been transformative, Eve “turned the world upside down,” and now women collectively have the power to restore justice and balance. I find this message timeless because it speaks not only to women’s resilience but also to the importance of solidarity in achieving equality. Truth’s words challenge the idea that women are passive or fragile; instead, she reframes them as powerful agents of change who can reshape society when they work together.

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Referee #2

Evidence, reproducibility and clarity

Summary:

The manuscript by Shukla et al described the "chromatin states" in the bryophyte Marchantia polymorpha and compared it with that in Arabidopsis thaliana. They described the generally common features of chromatin states between these evolutionally distant plant species, but they also find some differences. The authors also studied the connection between chromatin states and TF bindings, mostly in Arabidopsis due to the scarcity of the TF binding data in Marchantia. Their analyses lead to interesting finding that specific transcription families tend to associate with specific chromatin state, which tend to associate with specific genomic regions such as promoter, TSS, gene body, and fucultative heterochromatin. Overall, the authors provide novel piece of information regarding the evolutional conservation of chromatin states and the relationship between chromatin states and TFs.

Major comments:

1. In the end of the abstract they state "The association with the +1 nucleosome defines a list of candidate pioneer factors we know little about in plants", which is one of their major points. This is based on the results Fig4F and 4G, described in P27 L16-17. Question is, is cluster 1 TFs really associated with the +1 nucleosome? From Fig. 1C, +1 nucleosome is characterized mostly by E1 state and also by E2, F3, F4. However, from Fig. 4F, cluster 1 TFs are not associated with E1/E2 and association is not particularly strong for F3/F4. Indeeed association with E1/E2 is much conspicuous for cluster 4 TFs. Therefore, authors should reconsider this point and consider rephrasing or showing further results of analyses.

2. P17 last line to P18, they state "The facultative heterochromatin states were primarily associated with the intergenic states I1 to I3, based on their enrichment in H3K27me3 and H2AK121ub, low accessibility, and low gene expression". I'm not sure about this statement. How can they say "primarily associated" from the data they cite? As far as the PTMs and variants patterns, I1 to I3 and facultative heterochromatin look different. The authors should explain more or rephrase.

3. P20 L15, the authors state "Contrary to Arabidopsis, the promoters of Marchantia defined by the region just upstream of the TSS showed enrichment of H2AUb and the elongation mark H3K36me3, along with other euchromatic marks. " I have a concern that the TSS annotation could be inaccurate in Marchantia compared to more rigorously tested annotation of Arabidopsis thaliana, so that the relationship between TSS and histone PTMs could be different between species. The authors should make sure this is not the case.

4. P21 last line to P22, they analyzed only H3K27me3 and H2Aub in the mutants of E(z) (Fig. 2E) and states that "we analyzed chromatin landscape in the Marchantia...". Is analyzing two histone marks enough to say "chromatin landscape"? In addition, they state "These findings suggest a strong independence of the two Polycomb repressive pathways in Marchantia. " However, they did not analyzed the effect of loss of PRC1 on H3K27me3; the opposite way. Actually, in Arabidopsis loss of PRC1 causes loss of H2Aub AND H3K27me3 (Zhou et al (2017) Genome Biol: DOI 10.1186/s13059-017-1197-z).

5. Related to the above comments, they states "To further compare the regulation by PRC2 in both species,". However, they did not describe the knowledge about regulation by PRC2 in Arabidopsis. They should consider describing.

6. P25 L14: "With this method to estimate TF activity, the scores of TF occupancy and activity converged. To look at different patterns of chromatin preferences among TFs, we kept ChIP-seq and DAP-seq data for ~300 TFs in Arabidopsis (after filtering out TFs with low scores of occupancy and activity)." This part is a little hard to follow. Perhaps better to explain in more detail.

7. In discussion section P30 L19-21: "This could be due to open chromatin, which is associated with highly expressed genes and permissive for TF binding, generating highly occupied target regions (HOT) with redundant or passive activity (19)." This part needs further explanation; espetially for the latter part, It's not clar what the authors claim.

Minor comments:

1. P17 L21: H2bUb should be H2Bub.

2. Legend of Fig. 4D: later should be latter.

3. Legend of Fig. 4G and H: "clusters defined in figure-H" should be "defined in Fig. 4F"?

Referee cross-commenting

Reviewer #1 raises thorough and important points that should be addressed before the manuscript is published. Particularly about the comparison of chromatin states between Arabidopsis and Marchantia, as this paper will make foundation for further research in the future and serve as a resource for community, the authors should thoroughly look into the points raised by reviewer #1 including annotation of transcriptional units.

Significance

Strength and limitation: Strength of this paper is the insights into chromatin-based transcriptional regulation by defining chromatin states using combination of many epigenome data and compare it with TF biding data. Limitation is lack of experimental support for their interesting claims by perturbing histone PTMs, for example. Also, a limitation is that comparing only two species can tell subjective "similar" or "different" between species.

Advance comparing past literature: One clear advance is studying chromatin states in a plant other than Arabidopsis thaliana. Another one is revealing that TFs can be classified into a number of groups according to the relationships with chromatin-based transcription regulation. However, experimental tests for these are awaited.

Audience: Epigenetics, chromatin, and transcription researchers, plant biologists interested in transcriptional regulation.

My expertise: Epigenome, genetics, histone PTMs, plants

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Referee #1

Evidence, reproducibility and clarity

Summary:

The authors characterize chromatin states in the flowering plant Arabidopsis thaliana and the bryophyte Marchantia polymorpha. Here, they draw from ChIP-seq data that was previously published, and from data generated as part of this study, in particular for Marchantia H2.A variants (H2A.X.1, H2A.X.1, H2A.Z, H2A.M.2). The authors compute chromatin states, which enables a comparison over more than 450 million years of land plant evolution. While comparisons of plant chromatin to other species highlighted conservation as well as differences, this study targets a knowledge gap of evaluating chromatin conservation during land plant evolution. The authors investigate a connection between Transcription Factors binding sites and chromatin states. They propose a list of candidate pioneer factors associating with the +1 nucleosome.

Major comments:

• For the Association of chromatin states with expression, the authors use the TAIR10 annotation for extracting TSSs and promoter sequences. When investigated, a comparison of data resolving TSS with this annotation (or Araport11) shows a pretty poor overlap between the TSS based on Tair10/Araport11 and experimentally derived TSSs. This information was captured in Arabidopsis genome annotation files where the experimental TSS matches the genome annotation. What is the advantage of using an annotation with the inaccurate TSSs in TAIR10? It seems to confound the study.

• The TSS annotation in Marchantia polymorpha (Tak1 v7.1) may also match poorly to the experimentally derived TSS. I suggest that the authors generate data to detect TSS in their tissue of choice and compare the positions to the genome annotation they use (f.x. PMID: 38831668).

• I am not convinced that it is a wise choice to utilize fewer ChIP-seq data in Marchantia than Arabidopsis. Can the missing Marchantia ChIP-seq experiments not be performed and included to complete the comparison?

• P. 26 onwards, the authors investigate different TF clusters and their association with chromatin states. They state "cluster 1 TFs primarily associated with the first nucleosome downstream of the TSS". However, if the gene is not really expressed in these "leave" tissues, then how can the authors be sure that the same TSS position would be used in "flower" tissue? It could be an artifact of a genome annotation file that misses flower-tissue TSS data. It is not an obvious to conclusion to name these factors "pioneer TFs". Experiments testing this are missing as far as I can gather.

Minor comments:

• Can the authors add files ( e.g. .bed) with their segmented chromatin states as part of their GEO submission? That could improve the impact and make the findings more accessible.

• Can the authors rule out issues with the Marchantia annotation, for example missing read-through transcription or alternative isoforms, that would essentially have the effect that the genomic segmentation they use contains elongating upstream transcripts in from of promoter TSS? This could be an alternative explanation for the enrichment of H2AUb/H3K36me3 just upstream of the TSSs as they describe on p.21. If it can´t be ruled, the limitations from genome annotations, and examples offering improvements could be highlighted in the discussion. This may also be supported by the long persistence of E4 after the TTS p.23.

• P.23 - This further suggests that in Marchantia, the orientation of genes defines

• distinct chromatin environment in their vicinity, through mechanisms yet to be uncovered. Does this correlate with the distance of the closest (annotated) transcript pairs?

• The E1 state highlighted on p.24 and in Fig.3A/d is not annotated in Fig.3A/D. It is also not clear in the legends which number it is.

• P.30 - The marks H3K4me1 and H3K36me3 reflecting transcriptional elongation and confined to the gene bodies in Arabidopsis, extend beyond the TTS in Marchantia, suggesting that signals for transcriptional termination differ between flowering plants and bryophytes. There are multiple alternative explanations. Likely a combination of missing transcripts in their genome annotation (e.g. lncRNAs), annotation errors (e.g. wrong ends) and the segmentation of these regions (e.g. the transcripts are closer than in Arabidopsis). The discussion could extended significantly to address these issues and include the efforts to improve the genome annotations.

Referee cross-commenting

Reviewer #2 raises fair and valuable questions.

Significance

Significance: The authors corroborate prior chromatin state analyses in Arabidopsis and provide a chromatin state analysis for Marchantia. These data represent a resource that will be used and appreciated by the plant and ChromEvoDevo communities. The quality of the analyses are high and the description is transparent. I am not aware of a similar study comparing bryophytes and a land plant, so this study addresses a gap in knowledge.

General assessment: The quality of the manuscript is high. The analyses are described well, and in sufficient detail to be understood. The effort going into documentation is high, I rate the study as reproducible. The linked github deposition looks good. The data generated as part of this study is available in the linked GEO deposition. An experimental design of 2 biological repeats is used, which is OK, but the lower limit. The GEO-deposited .bw files should be of interest to the ChromEvoDevo community, and researchers interested in Marchantia epigenetics and gene expression. The manuscript is written clearly and to the point. The figures condense a lot of data and match the text. The figures are rather complex and not easily accessible to someone browsing through a journal issue. However, that is fine for these types of papers. The manuscript is strong on data analysis. Other approaches, for example mutants to validate their hypothesis, are not utilized. The calculation of chromatin states offers a way to condense complex information into simpler terms. Nevertheless, it re-organizes information that largely existed before. To me, the biggest value of this study appears to be to regard it as a resource that calculated the chromatin states in a comparable fashion between organisms.

Advance: The manuscript provides several advances. It provides new ChIP-seq data for Marchantia, it generates a chromatin state map for Marchantia, it compares Chromatin state maps between distant evolutionary time, and it generates a new hypothesis regarding pioneer TFs in plants. Some of the points described in the article hold true for even larger evolutionary distances, for example comparing plants to yeast and metazoans. The manuscript fills a knowledge gap and has offers a comparison via the computation of comparable chromatin states.

Audience: The audience will be colleagues interested in chromatin and epigenetics, the Marchantia and plant communities as well as researchers interested in EvoDevo of chromatin organization. Even though the study uses plant models, it is highly relevant for non-plant models.

Ik denk dat hier een groot gevaar in schuilt. Alhoewel, afhankelijk van hoe dit wordt gebruikt. Als het voorspellen van gebeurtenissen op basis van historie wordt gebruikt om te kunnen anticiperen op en voor het voorbereiden van onderwerpen die waarschijnlijk nieuwswaardig gaan zijn, voegt het zeker toe. Dat geeft rust. Als dit wordt gebruikt om voorspellende nieuwsberichten naar buiten te brengen of feitelijke zinnen met wat de samenleving te wachten staat, kan dit juist een schadelijke 'self fulfilling prophecy' worden, dat de lezer er vanuit gaat dat iets zo zal lopen en daar mogelijk naar gaat gedragen. Kortom: misschien interessant voor vervolgonderzoek.

Why is the focus on tilapia?

This article is part of the special JITC series: SITC 40th Anniversary: I-O Progress and Potential

eLife Assessment

This important study examines the potential role of ARHGAP36 transcriptional regulation by FOXC1 in controlling sonic hedgehog signaling in human neuroblastoma. While there are many solid findings that strongly support this signaling pathway, there are some aspects of the study that are underdeveloped.

Reviewer #1 (Public review):

This thoughtful and thorough mechanistic and functional study reports ARHGAP36 as a direct transcriptional target of FOXC1, which regulates Hedgehog signaling (SUFU, SMO, and GLI family transcription factors) through modulation of PKAC. Clinical outcome data from patients with neuroblastoma, one of the most common extracranial solid malignancies in children, demonstrate that ARHGAP36 expression is associated with improved survival. Although this study largely represents a robust and near-comprehensive set of focused investigations on a novel target of FOXC1 activity, several significant omissions undercut the generalizability of the findings reported.

(1) It is notable that the volcano plot in Figure 1a does now show evidence of canonical Hedgehog gene regulation, even though the subsequent studies in this paper clearly demonstrate that ARHGAP36 regulates Hedgehog signal transduction. Is this because canonical Hedgehog target genes (GLI1, PTCH1, SUFU) simply weren't labeled? Or is there a technical limitation that needs to be clarified? A note about Hedgehog target genes is made in conjunction with Table S1, but the justification or basis of defining these genes as Hedgehog targets is unclear. More broadly, it would be useful to see ontology analyses from these gene expression data to understand FOXC1 target genes more broadly. Ontology analyses are included in a supplementary table, but network visualizations would be much preferred.

(2) Likewise, the ChIP-seq data in Figure 2 are under-analyzed, focusing only on the ARHGAP36 locus and not more broadly on the FOXC1 gene expression program. This is a missed opportunity that should be remedied with unbiased analyses intersecting differentially expressed FOXC1 peaks with differentially expressed genes from RNA-sequencing data displayed in Figure 1.

(3) RNA-seq and ChIP-seq data strongly suggest that FOXC1 regulates ARHGAP36 expression, and the authors convincingly identify genomic segments at the ARHGAP36 locus where FOXC1 binds, but they do not test if FOXC1 specifically activates this locus through the creation of a luciferase or similar promoter reporter. Such a reagent and associated experiments would not only strengthen the primary argument of this investigation but could serve as a valuable resource for the community of scientists investigating FOXC1, ARHGAP36, the Hedgehog pathway, and related biological processes. CRISPRi targeting of the identified regions of the ARHGAP locus is a useful step in the right direction, but these experiments are not done in a way to demonstrate FOXC1 dependency.

(4) It would be useful to see individual fluorescence channels in association with images in Figure 3b.

(5) Perhaps the most significant limitation of this study is the omission of in vivo data, a shortcoming the authors partly mitigate through the incorporation of clinical outcome data from pediatric neuroblastoma patients in the context of ARHGAP36 expression. The authors also mention that high levels of ARHGAP36 expression were also detected in "specific CNS, breast, lung, and neuroendocrine tumors," but do not provide clinical outcome data for these cohorts. Such analyses would be useful to understand the generalizability of their findings across different cancer types. More broadly, how were high, medium, and low levels of ARHGAP36 expression identified? "Terciles" are mentioned, but such an approach is not experimentally rigorous, and RPA or related approaches (nested rank statistics, etc) are recommended to find optimal cutpoints for ARHGAP36 expression in the context of neuroblastoma, "specific CNS, breast, lung, and neuroendocrine" tumor outcomes.

Reviewer #2 (Public review):

FOXC1 is a transcription factor essential for the development of neural crest-derived tissues and has been identified as a key biomarker in various cancers. However, the molecular mechanisms underlying its function remain poorly understood. In this study, the authors used RNA-seq, ChIP-seq, and FOXC1-overexpressing cell models to show that FOXC1 directly activates transcription of ARHGAP36 by binding to specific cis-regulatory elements. Elevated expression of FOXC1 or ARHGAP36 was found to enhance Hedgehog (Hh) signaling and suppress PKA activity. Notably, overexpression of either gene also conferred resistance to Smoothened (SMO) inhibitors, indicating ligand-independent activation of Hh signaling. Analysis of public gene expression datasets further revealed that ARHGAP36 expression correlates with improved 5-year overall survival in neuroblastoma patients. Together, these findings uncover a novel FOXC1-ARHGAP36 regulatory axis that modulates Hh and PKA signaling, offering new insights into both normal development and cancer progression.

The main strengths of the study are:

(1) Identification of a novel signaling pathway involving FOXC1 and ARHGAP36, which may play a critical role in both normal development and cancer biology.

(2) Mechanistic investigation using RNA-seq, ChIP-seq, and functional assays to elucidate how FOXC1 regulates ARHGAP36 and how this axis modulates Hh signaling.

(3) Clinical relevance demonstrated through analysis of neuroblastoma patient datasets, linking ARHGAP36 expression to improved 5-year overall survival.

The main weaknesses of the study are:

(1) Lack of validation in neuroblastoma models - the study does not directly test its findings in neuroblastoma cell models, limiting translational relevance.

(2) Incomplete mechanistic insight into PKA regulation - the study does not fully elucidate how FOXC1-ARHGAP36 regulates PKAC activity at the molecular level.

(3) Insufficient discussion of clinical outcome data - while ARHGAP36 expression correlates with improved survival in neuroblastoma, the manuscript lacks a clear interpretation of this unexpected finding, especially given the known oncogenic roles of FOXC1, ARHGAP36, and Hh signaling.

Reviewer #3 (Public review):

Summary:

The focus of the research is to understand how transcription factors with high expression in neural crest cell-derived cancers (e.g., neuroblastoma) and roles in neural crest cell development function to promote malignancy. The focus is on the transcription factor FOXC1 and using murine cell culture, gain- and loss-of-function approaches, and ChIP profiling, among other techniques, to place PKAC inhibitor ARHGAP36 mechanistically between FOXC1 and another pathway associated with malignancy, Sonic Hedgehog (SHH).

Strengths:

Major strengths are the mechanistic approaches to identify FOXC1 direct targets, definitively showing that FOXC1 transcriptional regulation of ARHGAP36 leads to dysregulation of SHH signaling downstream of ARHGAP36 inhibition of PKC. Starting from a screen of Foxc1 OE to get to ARHGAP36 and then using genetic and pharmacological manipulation to work through the mechanism is very well done. There is data that will be of use to others studying FOXC1 in mesenchymal cell types, in particular, the FOXC1 ChIP-seq.

Weaknesses:

Work is almost all performed in NIH3T3 or similar cells (mouse cells, not patient or mouse-derived cancer cells), so the link to neuroblastoma that forms the major motivation of the work is not clear. The authors look at ARHGAP36 levels in association with the neuroblastoma patient survival; however, the finding, though interesting and quite compelling, is misaligned with what the literature shows about FOXC1 and SHH, their high expression is associated with increased malignancy (also maybe worse outcomes?). Therefore, ARHGAP36 expression may be more complicated in a tumor cell or may be unrelated to FOXC1 or SHH, leaving one to wonder what the work in NIH3T3 cells, though well done, is telling us about the mechanisms of FOXC1 as an oncogene in neuroblastoma cells or in any type of cancer cell. Does it really function as an SHH activator to drive tumor growth? The 'oncogenic relevance' and 'contribution to malignancy' claimed in the last paragraph of the introduction are currently weakly supported by the data as presented. This could be improved by studying some of these mechanisms in patient-derived neuroblastoma cells with high FOXC1 expression. Does inhibiting FOXC1 change SHH and ARHGAP36 and have any effect on cell proliferation or migration? Alternatively, does OE of FOXC1 in NIH3T3 cells increase their migration or stimulate proliferation in some way, and is this dependent on ARHGAP36 or SHH? Application of their mechanistic approaches in cancer cells or looking for hallmarks of cancer phenotypes with FOXC1 OE (and dependent on SHH or ARHGAP36) could help to make a link with cellular phenotypes of malignant cells.

Author Response:

Thank you for forwarding these helpful and thoughtful reviews - at a time when the review process in some journals can be a bit of a 'bloodsport', it is refreshing to receive such constructive and excellent comments.  We essentially agree with the key points the reviewers have made, and as an interim response provide clarification of two areas:

1) As the reviewers highlighted, genome-wide analysis of ChIP-seq data from Foxc1 over-expression is indeed very worthwhile, and may offer insights for diverse malignancies where FOXC1 is over-expressed.  We have a manuscript in preparation integrating this data set with ATAC-and RNA-seq data to identify genes transcriptionally regulated by elevated levels of Foxc1.  In the interim, our full ChIP-seq data are available via the GEO accession number listed in the manuscript.

2) Analysis in neuroblastoma cell lines and then xenografts is equally important. Experiments manipulating ARHGAP36 levels in human neuroblastoma cell lines are underway, however a detailed mechanistic understanding of how ARHGAP36 influences neuroblastoma prognosis will take time, and lies beyond the scope of the current manuscript.

decay, entropy, the relentless passing of time; what's the point?

presents Marlowe's shepherd as an unreliable narrator; dishonesty of men, distrustfulness of women

introduces passage of time as a central theme

Internal is personal. External is others.

They make them likable and relatable.

are they warm and bubbly? dry and mean? cold and bitter?

The main message. less about what is being said. more about what it really means.

Usually what the plot revolves around.

the narrator is not always the main character.

What they story is about and how and why the story unfolds.

The time can and will effect characters and how they are treated or what they are dealing with.

is it the desert? is it cold? rainy? is it night?

finding the setting, plot, characters, theme.

You must have an understanding as to what you are reading about.

Even minimal preparation can spark meaningful classroom engagement. I like that he turns an unprepared moment into a learning opportunity.

Charlton argues for experimentation instead of over-focusing on rigid form. I like his honesty that "focus" can limit invention; reminds me to explore ideas before narrowing. In my personal projects, I also "drift" before finding structure, it's the same creative process. He claims too much focus harms learning, which is true but I believe some structure helps.

He ends by warning against over-valuing focus and routine. Challenging the "focus = good writing" myth makes sense, originally needs uncertainty. I like that he closes by inviting curiosity and collaboration, it turns writing into exploration. Feels similar to innovation in engineering, embracing trial and error as part of design.

Relates to the public communication, like adapting a game demo for investors. Could invention projects like this replace traditional essays? This reaffirms that audience awareness develops through experimentation, not memorization.

(SAYS-DOES) Charlton says Brittany’s creative testing aligns audiences, and this does illustrate authentic transfer of learning.

Brittany tests the link between reading and writing improvement, later critiques standardized testing. Her evolution from essay to exam design shows creative transfer. Her mock ACT logically aligns testing with actual writing tasks. What grading criteria did she use for her mock exam, was it ever tested?

They were dealing with trauma after trauma.

Everyone will feel differently based on their own lives and experiences.

You have to put in effort to reading and enjoying a story.

This is beyond jargon… this is farce.

This cannot be real.

yes. Everyone has different preferences and opinions.

Timing and historical facts are true. Only the characters are not.

Must be able to differentiate and use both at once.

Novels are longer like typical books.

They keep it short and simple.

During a journey a merchant could only transport a limited amount of good while still having enough money to trade. The paper, credit, trust based systems helped fix these problems.

Yes we read for pleasure or entertainment.

So much of the information we know about Latin literature came from here and places like Baghdad.

Its incredible to me how they adapted to the rainfall dropping and changed their lifestyle to suit it without having to migrate!

Im shocked at how quick they were to cut ties with King Olaf after he threatened them.

Its shocking to see how the Huns migration ultimately lead to the looting of Rome.

It was such an important position being close to their enemies and being on trade routes.

This seems like a point of conflict thats going to grow later.

The definitions were of their "mob rule" based on who perceived it.

The Huns through their migration had caused a conflict between the Goths and Rome.

casuals

Stereoisomers have the exact same "blueprint" or "wiring" (connectivity), but they are just different 3D shapes (cis/trans)

May be an issue for us as we want to only watch for high-level items in a directory. Watching recursively is: 1. potential performance hit 2. unnecessary items in a database (storage hit)

I like this revision to the OER-Enabled Pedagogy

As educators, this question should be the core driving force for the adaptation of a new or modification of existing instructional pedagogy.

Identifying the challenges your students are facing will serve as an evidential data, which will prompt the appropriate pedagogical mitigation to address these students' challenges for optimal and deeper learning to occur.

Oh......I think I am doing this right. When the truth comes out about anything the loudest people usually get very quiet rather than admitting they are wrong.

Stated in their 1920 '25-point programme' (quoted in United States Holocaust Mermorial Museum, 2025), the far-right 'NSDAP' in Germany sought from their creation to abolish liberal democracy in it's entirety. Point 25 demanded 'the creation of a strong central state power for the reich', the Nazis having 'unconditional authority...over the entire Reich'. While the program was made early in the far-right extremist groups history, the threat from the far-right in 1920 seemingly minimal in comparision to the hard-left who's sparticist uprising the previous year had sparked terror, once the Nazi party gained a larger following after the crisi, the programme, still forming the heart of Nazi aims, provides evidence of the threat the Nazis posed () to the German people.

Here, no other parties ig?

aim to get the middle-class on their side

Possible good quotation

The right (under the Nazis) were a particular threat in Germany due to their understanding of party politics. They often changed their focus on ideology based on where they were (reference, please)

Shows how the point-plan were their aimswhich they wanted to fulfil

The predictions held up well. Hansen’s 1988 paper projected warming trends under three emission scenarios:

• A (high emissions) – ran hotter than observed.
• B (moderate, realistic) – tracked observed global warming closely.
• C (low emissions) – ran cooler than observed.

Later comparisons (e.g., Hausfather et al., 2019, Geophysical Research Letters) found that Hansen’s model produced nearly the correct warming rate once actual greenhouse gas concentrations were used as input.

The main difference came from emission assumptions, not from the physics of the model.

BOOM

How do they know what skills the agent learns?

Comments from the community are welcome! You'll need a https://hypothes.is/ account.

Please sign your comments and be respectful in your contributions to the initiative.

For more details on expectations on contributors to CCP-AHC, please read https://github.com/ccpahc/ccpahc.github.io/blob/main/CONTRIBUTING.md#code-of-conduct

♖/indy/0/pad/index.html

Gyuri Lajos, [03/11/2025 09:53]

Hyper Plex Mark In Editor - indy Pad Plex - transitional logs

reimagining html as hpmi

Universal Hyper Plex Marked In named networks of intentionally deeply interconnected documents people and capabilities

It's a new month, a new week

Over the weekend I started to work on turning indy 0 Pad to be the next level indy 0 Pad Plex HyperPlex Mark In document editor

Dpoing it by making Peer gos Custom App development slef-verioning and self-documenting. So I am in a double transition, trying to get two mutually interdependednt things right.

Hence I resort to document the work, again in Telegram. This one point to the urgent need to create indy 0 gram exapting indy 0 Plex which in turn is and exaption thorugh mixins of Indy 0 Pad which is already an exaption of CK Editor Peergos Custom App

Gyuri Lajos, [03/11/2025 10:01] started out documenting the work in a change log document which itself is not quite the right way to name/organize this onformation reified in named folder structure

Gyuri Lajos, [03/11/2025 10:03] I installed the indy 0 pad / next version and added a sandbox link called next to oen that hpmi document in the next version of indy 0 pad under development

Gyuri Lajos, [03/11/2025 10:04] The curren tlayout is for a Peergos Custom App created for the Indy web is to have the currentlyactive vesion at the root

eLife Assessment

This study is important as it demonstrates that 4-aminoquinoline antimalarials antagonize artemisinin activity under physiologically relevant conditions. Using isogenic parasite lines and a chemical probe, the authors provide mechanistic insight and compelling evidence implicating PfCRT in this antagonism. However, some weaknesses have been identified that limit full interpretation of the findings, which are based solely on in vitro assays, though the results have implications that will be of importance in optimizing future antimalarial combination strategies.

Reviewer #1 (Public review):

Summary:

This study set out to investigate potential pharmacological drug-drug interactions between the two most common antimalarial classes, the artemisinins and quinolines. There is a strong rationale for this aim, because drugs from these classes are already widely used in Artemisinin Combination Therapies (ACTs) in the clinic, and drug combinations are an important consideration in the development of new medicines. Furthermore, whilst there is ample literature proposing many diverse mechanisms of action and resistance for the artemisinins and quinolines, it is generally accepted that the mechanisms for both classes involve heme metabolism in the parasite, and that artemisinin activity is dependent on activation by reduced heme. The study was designed to measure drug-drug interactions associated with a short pulse exposure (4 h) that is reminiscent of the short duration of artemisinin exposure obtained after in vivo dosing. Clear antagonism was observed between dihydroartemisinin (DHA) and chloroquine, which became even more extensive in chloroquine-resistant parasites. Antagonism was also observed in this assay for the more clinically-relevant ACT partner drugs piperaquine and amodiaquine, but not for other ACT partners mefloquine and lumefantrine, which don't share the 4-aminoquinoline structure or mode of action. Interestingly, chloroquine induced an artemisinin resistance phenotype in the standard in vitro Ring-stage Survival Assay, whereas this effect was not apparent for piperaquine.

The authors also utilised a heme-reactive probe to demonstrate that the 4-aminoquinolines can inhibit heme-mediated activation of the probe within parasites, which suggests that the mechanism of antagonism involves the inactivation of heme, rendering it unable to activate the artemisinins. Measurement of protein ubiquitination showed reduced DHA-induced protein damage in the presence of chloroquine, which is also consistent with decreased heme-mediated activation, and/or with decreased DHA activity more generally.

Overall, the study clearly demonstrates a mechanistic antagonism between DHA and 4-aminoquinoline antimalarials in vitro. It is interesting that this combination is successfully used to treat millions of malaria cases every year, which may raise questions about the clinical relevance of this finding. However, the conclusions in this paper are supported by multiple lines of evidence, and the data are clearly and transparently presented, leaving no doubt that DHA activity is compromised by the presence of chloroquine in vitro. It is perhaps fortunate that the clinical dosing regimens of 4-aminoquinoline-based ACTs have been sufficient to maintain clinical efficacy despite the non-optimal combination. Nevertheless, optimisation of antimalarial combinations and dosing regimens is becoming more important in the current era of increasing resistance to artemisinins and 4-aminoquinolines. Therefore, these findings should be considered when proposing new treatment regimens (including Tripe-ACTs) and the assays described in this study should be performed on new drug combinations that are proposed for new or existing antimalarial medicines.

Strengths:

This manuscript is clearly written, and the data presented are clear and complete. The key conclusions are supported by multiple lines of evidence, and most findings are replicated with multiple drugs within a class, and across multiple parasite strains, thus providing more confidence in the generalisability of these findings across the 4-aminoquinoline and peroxide drug classes.

A key strength of this study was the focus on short pulse exposures to DHA (4 h in trophs and 3 h in rings), which is relevant to the in vivo exposure of artemisinins. Artemisinin resistance has had a significant impact on treatment outcomes in South-East Asia, and is now emerging in Africa, but is not detected using a 'standard' 48 or 72 h in vitro growth inhibition assay. It is only in the RSA (a short pulse of 3-6 h treatment of early ring stage parasites) that the resistance phenotype can be detected in vitro. Therefore, assays based on this short pulse exposure provide the most relevant approach to determine whether drug-drug interactions are likely to have a clinically relevant impact on DHA activity. These assays clearly showed antagonism between DHA and 4-aminoquinolines (chloroquine, piperaquine, amodiaquine, and ferroquine) in trophozoite stages. Interestingly, whilst chloroquine clearly induced an artemisinin-resistant phenotype in the RSA, piperaquine did not appear to impact the early ring stage activity of DHA, which may be fortunate considering that piperaquine is a currently recommended DHA partner drug in ACTs, whereas chloroquine is not!

The evaluation of additional drug combinations at the end of this paper is a valuable addition, which increases the potential impact of this work. The finding of antagonism between piperaquine and OZ439 in trophozoites is consistent with the general interactions observed between peroxides and 4-aminoquinolines, and it would be interesting to see whether piperaquine impacts the ring-stage activity of OZ439.

The evaluation of reactive heme in parasites using a fluorescent sensor, combined with the measurement of K48-linked ubiquitin, further supports the findings of this study, providing independent read-outs for the chloroquine-induced antagonism.

The in-depth discussion of the interpretation and implications of the results is an additional strength of this manuscript. Whilst the discussion section is rather lengthy, there are important caveats to the interpretation of some of these results, and clear relevance to the future management of malaria that require these detailed explanations.

Overall, this is a high-quality manuscript describing an important study that has implications for the selection of antimalarial combinations for new and existing malaria medicines.

Weaknesses:

This study is an in vitro study of parasite cultures, and therefore, caution should be taken when applying these findings to decisions about clinical combinations. The drug concentrations and exposure durations in these assays are intended to represent clinically relevant exposures, although it is recognised that the in vitro system is somewhat simplified and there may be additional factors that influence in vivo activity. I think this is reasonably well acknowledged in the manuscript.

It is also important to recognise that the majority of the key findings regarding antagonism are based on trophozoite-stage parasites, and one must show caution when generalising these findings to other stages or scenarios. For example, piperaquine showed clear antagonism in trophozoite stages, but not in ring stages under these assay conditions.

The key weakness in this manuscript is the over-interpretation of the mechanistic studies that implicate heme-mediated artemisinin activation as the mechanism underpinning antagonism by chloroquine. In particular, the manuscript title focuses on heme-mediated activation of artemisinins, but this study did not directly measure the activation of artemisinins. The data obtained from the activation of the fluorescent probe are generally supportive of chloroquine suppressing the heme-mediated activation of artemisinins, and I think this is the most likely explanation, but there are significant caveats that undermine this conclusion. Primarily, the inconsistency between the fluorescence profile in the chemical reactions and the cell-based assay raises questions about the accuracy of this readout. In the chemical reaction, mefloquine and chloroquine showed identical inhibition of fluorescence, whereas piperaquine had minimal impact. On the contrary, in the cell, chloroquine and piperaquine had similar impacts on fluorescence, but mefloquine had minimal impact. This inconsistency indicates that the cellular fluorescence based on this sensor does not give a simple direct readout of the reactivity of ferrous heme, and therefore, these results should be interpreted with caution. Indeed, the correlation between fluorescence and antagonism for the tested drugs is a correlation, not causation. There could be several reasons for the disconnect between the chemical and biological results, either via additional mechanisms that quench fluorescence, or the presence of biomolecules that alter the oxidation state or coordination chemistry of heme or other potential catalysts of this sensor. It is possible that another factor that influences the H-FluNox fluorescence in cells also influences the DHA activity in cells, leading to the correlation with activity. It should be noted that H-FluNox is not a chemical analogue of artemisinins. Its activation relies on Fenton-like chemistry, but with an N-O rather than O-O bond, and it possesses very different steric and electronic substituents around the reactive centre, which are known to alter reactivity to different iron sources. Despite these limitations, the authors have provided reasonable justification for the use of this probe to directly visualise heme reactivity in cells, and the results are still informative, but additional caution should be provided in the interpretation, and the results are not conclusive enough to justify the current title of the paper.

Another interesting finding that was not elaborated by the authors is the impact of chloroquine on the DHA dose-response curves from the ring stage assays. Detection of artemisinin resistance in the RSA generally focuses on the % survival at high DHA concentrations (700 nM) as there is minimal shift in the IC50 (see Figure 2), however, chloroquine clearly induces a shift in the IC50 (~5-fold), where the whole curve is shifted to the right, whereas the increase in % survival is relatively small. This different profile suggests that the mechanism of chloroquine-induced antagonism is different from the mechanism of artemisinin resistance. Current evidence regarding the mechanism of artemisinin resistance generally points towards decreased heme-mediated drug activation due to a decrease in hemoglobin uptake, which should be analogous to the decrease in heme-mediated drug activation caused by chloroquine. However, these different dose-response curves suggest different mechanisms are primarily responsible. Additional mechanisms have been proposed for artemisinin resistance, involving redox or heat stress responses, proteostatic responses, mitochondrial function, dormancy, and PI3K signaling, among others. Whilst the H-FluNox probe generally supports the idea that chloroquine suppresses heme-mediated DHA activation, it remains plausible that chloroquine could induce these, or other, cellular responses that suppress DHA activity.

The other potential weakness in the current manuscript is the interpretation of the OZ439 clinical data. Whilst the observed interaction with piperaquine and ferroquine may have been a contributing factor, it should also be recognised that the low pharmacokinetic exposure in these studies was the primary reason for treatment failure (Macintyre 2017).

Impact:

This study has important implications for the selection of drugs to form combinations for the treatment of malaria. The overall findings of antagonism between peroxide antimalarials and 4-aminoquinolines in the trophozoite stage are robust, and this carries across to the ring stage for chloroquine (but not piperaquine).

The manuscript also provides a plausible mechanism to explain the antagonism, although future work will be required to further explore the details of this mechanism and to rule out alternative factors that may contribute.

Overall, this is an important contribution to the field and provides a clear justification for the evaluation of potential drug combinations in relevant in vitro assays before clinical testing.

Reviewer #2 (Public review):

Summary:

This manuscript by Rosenthal and Goldberg investigates interactions between artemisinins and their quinoline partner drugs currently used for treating uncomplicated Plasmodium falciparum malaria. The authors show that chloroquine (CQ), piperaquine, and amodiaquine antagonize dihydroartemisinin (DHA) activity, and in CQ-resistant parasites, the interaction is described as "superantagonism," linked to the pfcrt genotype. Mechanistically, application of the heme-reactive probe H-FluNox indicates that quinolines render cytosolic heme chemically inert, thereby reducing peroxide activation. The work is further extended to triple ACTs and ozonide-quinoline combinations, with implications for artemisinin-based combination therapy (ACT) design, including triple ACTs.

Strengths:

The manuscript is clearly written, methodologically careful, and addresses a clinically relevant question. The pulsing assay format more accurately models in vivo artemisinin exposure than conventional 72-hour assays, and the use of H-FluNox and Ac-H-FluNox probes provides mechanistic depth by distinguishing chemically active versus inert heme. These elements represent important refinements beyond prior studies, adding nuance to our understanding of artemisinin-quinoline interactions.

Weaknesses:

Several points warrant consideration. The novelty of the work is somewhat incremental, as antagonism between artemisinins and quinolines is well established. Multiple prior studies using standard fixed-ratio isobologram assays have shown that DHA exhibits indifferent or antagonistic interactions with chloroquine, piperaquine, and amodiaquine (e.g., Davis et al., 2006; Fivelman et al., 2007; Muangnoicharoen et al., 2009), with recent work highlighting the role of parasite genetic background, including pfcrt and pfmdr1, in modulating these interactions (Eastman et al., 2016). High-throughput drug screens likewise identify quinoline-artemisinin combinations as mostly antagonistic. The present manuscript adds refinement by applying pulsed-exposure assays and heme probes rather than establishing antagonism de novo.

The dataset focuses on several parasite lines assayed in vitro, so claims about broad clinical implications should be tempered, and the discussion could more clearly address how in vitro antagonism may or may not translate to clinical outcomes. The conclusion that artemisinins are predominantly activated in the cytoplasm is intriguing but relies heavily on Ac-H-FluNox data, which may have limitations in accessing the digestive vacuole and should be acknowledged explicitly. The term "superantagonism" is striking but may appear rhetorical; clarifying its reproducibility across replicates and providing a mechanistic definition would strengthen the framing. Finally, some discussion points, such as questioning the clinical utility of DHA-PPQ, should be moderated to better align conclusions with the presented data while acknowledging the complexity of in vivo pharmacology and clinical outcomes.

Despite these mild reservations, the data are interesting and of high quality and provide important new information for the field.

Reviewer #3 (Public review):

Summary:

The authors present an in vitro evaluation of drug-drug interactions between artemisinins and quinoline antimalarials, as an important aspect for screening the current artemisinin-based combination therapies for Plasmodium falciparum. Using a revised pulsing assay, they report antagonism between dihydroartemisinin (DHA) and several quinolines, including chloroquine, piperaquine (PPQ), and amodiaquine. This antagonism is increased in CQ-resistant strains in isobologram analyses. Moreover, CQ co-treatment was found to induce artemisinin resistance even in parasites lacking K13 mutations during the ring-stage survival assay. This implies that drug-drug interactions, not just genetic mutations, can influence resistance phenotypes. By using a chemical probe for reactive heme, the authors demonstrate that quinolines inhibit artemisinin activation by rendering cytosolic heme chemically inert, thereby impairing the cytotoxic effects of DHA. The study also observed negative interactions in triple-drug regimens (e.g., DHA-PPQ-Mefloquine) and in combinations involving OZ439, a next-generation peroxide antimalarial. Taken together, these findings raise significant concerns regarding the compatibility of artemisinin and quinoline combinations, which may promote resistance or reduce efficacy.

Throughout the manuscript, no combinations were synergistic, which necessitates comparing the claims to a synergistic combination as a control. The lack of this positive control makes it difficult to contextualize the observed antagonism. Including a known synergistic pair (e.g., artemisinin + lumefantrine) throughout the study would have provided a useful benchmark to assess the relative impact of the drug interactions described.

Strengths:

This study demonstrates the following strengths:

(1) The use of a pulsed in vitro assay that is more physiologically relevant than the traditional 48h or 72h assays.

(2) Small molecule probes, H-FluNox, and Ac-H-FluNox to detect reactive cytosolic heme, demonstrating that quinolines render heme inert and thereby block DHA activation.

(3) Evaluates not only traditional combinations but also triple-drug combinations and next-generation artemisinins like OZ439. This broad scope increases the study's relevance to current treatment strategies and future drug development.

(4) By using the K13 wild-type parasites, the study suggests that resistance phenotypes can emerge from drug-drug interactions alone, without requiring genetic resistance markers.

Weaknesses:

(1) No combinations are shown as synergistic: it could be valuable to have a combination that shows synergy as a positive control (e.g, artemisinin + lumefantrine) throughout the manuscript. The absence of a synergistic control combination in the experimental design makes it more challenging to evaluate the relative impact of the described drug interactions.

(2) Evaluation of the choice of drug-drug interactions: How generalizable are the findings across a broad range of combinations, especially those with varied modes of action?

(3) The study would also benefit from a characterization of the molecular basis for the observed heme inactivation by quinolines to support this hypothesis - while the probe experiments are valuable, they do not fully elucidate how quinolines specifically alter heme chemistry at the molecular level.

(4) Suggestion of alternative combinations that show synergy could have improved the significance of the work.

(5) All data are derived from in vitro experiments, without accompanying an in vivo validation. While the pulsing assay improves physiological relevance, it still cannot fully capture the complexity of drug pharmacokinetics, host-parasite interactions, or immune responses present in living organisms.

(6) The absence of pharmacokinetic/pharmacodynamic modeling leaves questions about how the observed antagonism would manifest under real-world dosing conditions.

Author response:

Reviewer #1:

We thank the reviewer for their thoughtful summary of this manuscript. It is important to note that DHA-PPQ did show antagonism in RSAs. In this modified RSA, 200 nM PPQ alone inhibited growth of PPQ-sensitive parasites approximately 20%. If DHA and PPQ were additive, then we would expect that addition of 200 nM PPQ would shift the DHA dose response curve to the left and result in a lower DHA IC50. Please refer to Figure 4a and b as examples of additive relationships in dose-response assays. We observed no significant shift in IC50 values between DHA alone and DHA + PPQ. This suggests antagonism, albeit not to the extent seen with CQ. We will modify the manuscript to emphasize this point. As the reviewer pointed out, it is fortunate that despite being antagonistic, clinically used artemisinin-4-aminoquinoline combinations are effective, provided that parasites are sensitive to the 4-aminoquinoline. It is possible that superantagonism is required to observe a noticeable effect on treatment efficacy (Sutherland et al. 2003 and Kofoed et al. 2003), but that classical antagonism may still have silent consequences. For example, if PPQ blocks some DHA activation, this might result in DHA-PPQ acting more like a pseudo-monotherapy. However, as the reviewer pointed out, while our data suggest that DHA-PPQ and AS-ADQ are “non-optimal” combinations, the clinical consequences of these interactions are unclear. We will modify the manuscript to emphasize the later point.

While the Ac-H-FluNox and ubiquitin data point to a likely mechanism for DHA-quinoline antagonism, we agree that there are other possible mechanisms to explain this interaction.  We will temper the title and manuscript to reflect these limitations. Though we tried to measure DHA activation in parasites directly, these attempts were unsuccessful. We acknowledge that the chemistry of DHA and Ac-H-FluNox activation is not identical and that caution should be taken when interpreting these data. Nevertheless, we believe that Ac-H-FluNox is the best currently available tool to measure “active heme” in live parasites and is the best available proxy to assess DHA activation in live parasites. Both in vitro and in parasite studies point to a roll for CQ in modulating heme, though an exact mechanism will require further examination. Similar to the reviewer, we were perplexed by the differences observed between in vitro and in parasite assays with PPQ and MFQ. We proposed possible hypotheses to explain these discrepancies in the discussion section. Interestingly, our data corelate well with hemozoin inhibition assays in which all three antimalarials inhibit hemozoin formation in solution, but only CQ and PPQ inhibit hemozoin formation in parasites. In both assays, in-parasite experiments are likely to be more informative for mechanistic assessment.

It remains unclear why K13 genotype influences RSA values, but not early ring DHA IC50 values. In K13^WT parasites, both RSA values and DHA IC50 values were increased 3-5 fold upon addition of CQ. This suggests that CQ-mediated resistance is more robust than that conferred by K13 genotype. However, this does not necessarily suggest a different resistance mechanism. We acknowledge that in addition to modulating heme, it is possible that CQ may enhance DHA survival by promoting parasite stress responses. Future studies will be needed to test this alternative hypothesis. This limitation will be acknowledged in the manuscript. We will also address the reviewer’s point that other factors, including poor pharmacokinetic exposure, contributed to OZ439-PPQ treatment failure.

Reviewer #2:

We appreciate the positive feedback. We agree that there have been previous studies, many of which we cited, assessing interactions of these antimalarials. We also acknowledge that previous work, including our own, has shown that parasite genetics can alter drug-drug interactions. We will include the author’s recommended citations to the list of references that we cited. Importantly, our work was unique not only for utilizing a pulsing format, but also for revealing a superantagonistic phenotype, assessing interactions in an RSA format, and investigating a mechanism to explain these interactions. We agree with the reviewer that implications from this in vitro work should be cautious, but hope that this work contributes another dimension to critical thinking about drug-drug interactions for future combination therapies. We will modify the manuscript to temper any unintended recommendations or implications.

The reviewer notes that we conclude “artemisinins are predominantly activated in the cytoplasm”. We recognize that the site of artemisinin activation is contentious. We were very clear to state that our data combined with others suggest that artemisinins can be activated in the parasite cytoplasm. We did not state that this is the primary site of activation. We were clear to point out that technical limitations may prevent Ac-H-FluNox signal in the digestive vacuole, but determined that low pH alone could not explain the absence of a digestive vacuole signal.

With regard to the “reproducibility” and “mechanistic definition” of superantagonism, we observed what we defined as a one-sided superantagonistic relationship for three different parasites (Dd2, Dd2 PfCRT^Dd2, and Dd2 K13^R539T) for a total of nine independent replicates. In the text, we define that these isoboles are unique in that they had mean ΣFIC50 values > 2.4 and peak ΣFIC50 values >4 with points extending upward instead of curving back to the axis. As further evidence of the reproducibility of this relationship, we show that CQ has a significant rescuing effect on parasite survival to DHA as assessed by RSAs and IC50 values in early rings.

Reviewer #3:

We thank the reviewer for their positive feedback. We acknowledge that no combinations tested in this manuscript were synergistic. However, two combinations, DHA-MFQ and DHA-LM, were additive, which provides context for contextualizing antagonistic relationships. We have previously reported synergistic and additive isobolograms for peroxide-proteasome inhibitor combinations using this same pulsing format (Rosenthal and Ng 2021). These published results will be cited in the manuscript.

We believe that these findings are specific to 4-aminoquinoline-peroxide combinations, and that these findings cannot be generalized to antimalarials with different mechanisms of action. Note that the aryl amino alcohols, MFQ and LM, were additive with DHA. Since the mechanism of action of MFQ and LM are poorly understood, it is difficult to speculate on a mechanism underlying these interactions.

We agree with the reviewer that while the heme probe may provide some mechanistic insight to explain DHA-quinoline interactions, there is much more to learn about CQ-heme chemistry, particularly within parasites.

The focus of this manuscript was to add a new dimension to considerations about pairings for combination therapies. It is outside the scope of this manuscript to suggest alternative combinations. However, we agree that synergistic combinations would likely be more strategic clinically.

An in vitro setup allows us to eliminate many confounding variables in order to directly assess the impact of partner drugs on DHA activity. However, we agree that in vivo conditions are incredibly more complex, and explicitly state this.

We agree that in the future, modeling studies could provide insight into how antagonism may contribute to real-world efficacy. This is outside the scope of our studies.

eLife Assessment

This study presents vassi, a Python package that streamlines the preparation of training data for machine-learning-based classification of social behaviors in animal groups. This package is a valuable resource for researchers with computational expertise, implementing a framework for the detection of directed social interactions within a group and an interactive tool for reviewing and correcting behavior detections. However, the strength of evidence that the method is widely applicable remains incomplete, performance on benchmark dyadic datasets is comparable to existing approaches, and performance scores on collective behavioral datasets are low. While the package can analyze behavior in large groups of animals, it only outputs dyadic interactions within these groups and does not account for behaviors where more than two animals may be interacting.

Reviewer #1 (Public review):

Summary:

In this manuscript, Nührenberg et al., describe vassi, a Python package for mutually exclusive behavioral classification of social behaviors. This package imports and organizes trajectory data and manual behavior labels, and then computes feature representations for use with available Python machine learning-based classification tools. These representations include all possible dyadic interactions within an animal group, enabling classification of social behaviors between pairs of animals at a distance. The authors validate this package by reproducing the behavior classification performance on a previously published dyadic mouse dataset, and demonstrate its use on a novel cichlid group dataset. The authors have created a package that is agnostic to the mechanism of tracking and will reduce the barrier of data preparation for machine learning, which can be a stumbling block for non-experts. The package also evaluates the classification performance with helpful visualizations and provides a tool for inspection of behavior classification results.

Strengths:

(1) A major contribution of this paper was creating a framework to extend social behavior classification to groups of animals such that the actor and receiver can be any member of the group, regardless of distance. To implement this framework, the authors created a Python package and an extensive documentation site, which is greatly appreciated. This package should be useful to researchers with a knowledge of Python, virtual environments, and machine learning, as it relies on scripts rather than a GUI interface and may facilitate the development of new machine learning algorithms for behavior classification.

(2) The authors include modules for correctly creating train and test sets, and evaluation of classifier performance. This is extremely useful. Beyond evaluation, they have created a tool for manual review and correction of annotations. And they demonstrate the utility of this validation tool in the case of rare behaviors where correct classification is difficult, but the number of examples to review is reasonable.

(3) The authors provide well-commented step-by-step instructions for the use of the package in the documentation.

Weaknesses:

(1) While the classification algorithm was not the subject of the paper, as the authors used off-the-shelf methods and were only able to reproduce the performance of the CALMS21 dyadic dataset, they did not improve upon previously published results. Furthermore, the results from the novel cichlid fish dataset, including a macro F1 score of 0.45, did not compellingly show that the workflow described in the paper produces useful behavioral classifications for groups of interacting animals performing rare social behaviors. I commend the authors for transparently reporting the results both with the macro F1 scores and the confusion matrices for the classifiers. The mutually exclusive, all-vs-all data annotation scheme of rare behaviors results in extremely unbalanced datasets such that categorical classification becomes a difficult problem. To try to address the performance limitation, the authors built a validation tool that allows the user to manually review the behavior predictions.

(2) The pipeline makes a few strong assumptions that should be made more explicit in the paper.

First, the behavioral classifiers are mutually exclusive and one-to-one. An individual animal can only be performing one behavior at any given time, and that behavior has only one recipient. These assumptions are implicit in how the package creates the data structure, and should be made clearer to the reader. Additionally, the authors emphasize that they have extended behavior classification to animal groups, but more accurately, they have extended behavioral classification to all possible pairs within a group.

Second, the package expects comprehensive behavior labeling of the tracking data as input. Any frames not manually labeled are assumed to be the background category. Additionally, the package will interpolate through any missing segments of tracking data and assign the background behavioral category to those trajectory segments as well. The effects of these assumptions are not explored in the paper, which may limit the utility of this workflow for naturalistic environments.

(3) Finally, the authors described the package as a tool for biologists and ethologists, but the level of Python and machine learning expertise required to use the package to develop a novel behavior classification workflow may be beyond the ability of many biologists. More accessible example notebooks would help address this problem.

Reviewer #2 (Public review):

Summary:

The authors present a novel supervised behavioral analysis pipeline (vassi), which extends beyond previously available packages with its innate support of groups of any number of organisms. Importantly, this program also allows for iterative improvement upon models through revised behavioral annotation.

Strengths:

vassi's support of groups of any number of animals is a major advancement for those studying collective social behavior. Additionally, the built-in ability to choose different base models and iteratively train them is an important advancement beyond current pipelines. vassi is also producing behavioral classifiers with similar precision/recall metrics for dyadic behavior as currently published packages using similar algorithms.

Weaknesses:

vassi's performance on group behaviors is potentially too low to proceed with (F1 roughly 0.2 to 0.6). Different sources have slightly different definitions, but an F1 score of 0.7 or 0.8 is often considered good, while anything lower than 0.5 can typically be considered bad. There has been no published consensus within behavioral neuroscience (that I know of) on a minimum F1 score for use. Collective behavioral research is extremely challenging to perform due to hand annotation times, and there needs to be a discussion in the field as to the trade-off between throughput and accuracy before these scores can be either used or thrown out the door. It would also be useful to see the authors perform a few rounds of iterative corrections on these classifiers to see if performance is improved.

While the interaction networks in Figure 2b-c look visually similar based on interaction pairs, the weights of the interactions appear to be quite different between hand and automated annotations. This could lead to incorrect social network metrics, which are increasingly popular in collective social behavior analysis. It would be very helpful to see calculated SNA metrics for hand versus machine scoring to see whether or not vassi is reliable for these datasets.

Author response:

We thank the reviewers and editors for their assessment and for identifying the main issues of our framework for automated classification of social interactions in animal groups. Based on the reviewers’ feedback, we would like to briefly summarize three areas in which we aim to improve both our manuscript and the software package.

Firstly, we will revise our manuscript to better define the scope of our classification pipeline. As reviewer #1 correctly points out, our framework is built around the scoring and analysis of dyadic interactions within groups, rather than emergent group-level or collective behavior. This structure more faithfully reflects the way that researchers score social behaviors within groups, following focal individuals while logging all directed interactions of interest (e.g., grooming, aggression or courtship), and with whom these interactions are performed. Indeed, animal groups are often described as social networks of interconnected nodes (individuals), in which the connections between these nodes are derived from pairwise metrics, for example proximity or interaction frequency. For this reason, vassi does not aim to classify higher-level group behavior (i.e., the emergent, collective state of all group members) but rather the pair-wise interactions typically measured. Our classification pipeline replicates this structure, and therefore produces raw data that is familiar to researchers that study social animal groups with a focus on pairwise interactions. Since this may be seen as a limitation when studying group-level behavior (with more than two individuals involved, usually undirected), we will make this distinction between different forms of social interaction more clear in the introduction.

Secondly, we acknowledge the low performance of our classification pipeline on the cichlid group dataset. We included analyses in the first version of our manuscript that, in our opinion, can justify the use of our pipeline in such cases (comparison to proximity networks), but we understand the reviewers' concerns. Based on their comments, we will perform additional analyses to further assess whether the use of vassi on this dataset results in valid behavioral metrics. This may, for example, include a comparison of per-individual SNA metrics between pipeline results and ground truth, or equivalent comparisons on the level of group structure (e.g., hierarchy derived from aggression counts). We thank reviewer #2 for these suggestions. As the reviewers further point out, there is no consensus yet on when the performance of behavioral classifiers is sufficient for reliable downstream analyses, and although this manuscript does not have the scope to discuss this for the field, it may help to substantiate discussion in future research.

Finally, we appreciate the reviewers feedback on vassi as a methodological framework and will address the remaining software-related issues by improving the documentation and accessibility of our example scripts. This will reduce the technical hurdle to use vassi in further research. Additionally, we aim to incorporate a third dataset to demonstrate how our framework can be used for iterative training on a sparsely annotated dataset of groups, while broadening the taxonomic scope of our manuscript.

Modern sports are usually jobs. Such a pipeline is inherently speculative, not because you shouldn't make a living with art or fitness, but because in doing so, you are the product, and people will rate you.

I think going viral can honestly be a double end sword. There are many cases where people have gained huge financial success and reputation from going viral weather intentionally or unintendedly. For example, many Youtubers and Tik Tokers have created huge brands for themselves from simply going viral off their respected app. But going viral can also bring harassment, and even ruin people's lives. For example, I remember watching a interview where a woman talks about how an ad she modeled for became a massive meme online, causing people to mock, harass, and even send death threats over her looks. This just goes to show although something may sound good in retrospect, there are many downsides that may also come with it.

CPC 86130 Legal documentation and certification services Preparation, drawing up and certification services of legal documents. The services generally comprise the provision of a number of related legal services including the provision of advice and the execution of various tasks necessary for the drawing up or certification of documents. Included are the drawing up of wills, marriage contracts, commercial contracts, business charters, etc.