Why is the focus on tilapia?

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.

Introducing 'bidialctism' as an approah to help students learn by accepting and affirming their nonformal dialects. some students do not know of a standard english and will grow up only speaking nonstardard english.

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.

Dialect prejudice is a real problem in many schools and instituitions, whereas if one does not speak 'properly', they are seen as lesser. who decides what is proper?

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 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.

Translation: “pulls out of his arse”

This is beyond jargon… this is farce.

This cannot be real.

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?

How can first-year students create theory if they feel inexperienced? Similar to how in robotics we hypothesize solutions before testing them.

(SAYS-DOES) He says students should form personal theories; this does empower learners as researchers.

People dont think about what we get from books.

take no advice.

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.

Ainsi posée, la question suggère déjà une réponse négative. Je suggère de décomposer la question : pensez-vous que cela puisse avoir un impact sur le lendemain ? quel est cet impact, etc ? Attention, certains contenus pourraient être positifs, d'autres négatifs.

la question n'est pas suffisamment liée aux contenus proposés précédemment. Je vous invite à faire une question tableau

vous pourriez proposer d'autres modalités de réponse, et proposer aussi un tableau de fréquence.

C'est un résultat de recherche déjà démontré.. Il n'est donc pas nécessaire d'enquêter sur cette question.

On connait assez bien les effets de l'hyperconnexion, mais on connait peut-être moins les motivations, les peurs, les justifications, les tentatives de se déconnecter, etc. Que pensez-vous d'explorer ces éléments auprès des jeunes/étudiants ?

Cela ressemble davantage à une hypothèse de recherche qu'à un objectif.

yes. Everyone has different preferences and opinions.

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

They have to be historically accurate and true.

If you can write good enticing books for both it is genuinley a talent.

Many people read these books and them read them to their children.

They have to make it futuristic, entertaining, and believable.

Must be able to differentiate and use both at once.

Making fun of a character who is someone you usually cant make these jokes about or to in real life.

Something that you get later.

Using humor to bring up important political topics.

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.

entertainment.

Made up stories.

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!

La question de la sensibilité est au cœur des intervention du mouvement

La dimension éthique est si centrale dans le mouvement que des questions de pertinence scientifique se posent immanquablement

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

Prospective memory (prospectief geheugen) is het vermogen om je te herinneren dat je in de toekomst nog iets moet doen.

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

Enantiomers are: they are molecules that are non-superimposable mirror images of each other.

contraindicative in opiod naive, so antitussive are general, in that case many are not?

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

Lebnsraum - takeover

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

counts引数の説明を追加して欲しい

こっちの方がよく使うので、先にあってもいいかなって思いました

下の図ですが、figureにタイトルを文字列として書いてください。可能なら、顔図としてもヒストグラムの画像そのものを入れて欲しい「図：~~~」の部分の画像が不要

https://pyhack.slack.com/archives/G01CKBZ7J8J/p1762173838549059

「表：」はトルでOK 他も同様

seed関数も表7.6で説明して欲しいな

下の画像に画像タイトルまで含まれているので、タイトルはちゃんと文字として入れて欲しい

https://pyhack.slack.com/archives/G01CKBZ7J8J/p1762173838549059

これなんだ? 脚注かなんかのミスかな

今回uvを追加するのでまるっと削除でもいいかも。

nits: PowerShellだとバックスラッシュの方がいいかも?

nits: PowerShellの方が最近は一般的だから上の方がいいかな

これ、.gitignoreの中が「*」なので、venv以下は全部対象外になる、という説明だけでいいと思います。

以下のgitの手順はなくていいかなと(情報が多くて逆に読みにくい) 以下だけでいいのでは感

$ python3.14 -m venv env $ cat env/.gitignore *

SHOULD: これよく考えたらpipの制限ではなくて、Python側の制限なんじゃないだろうか。

from /hyperpost/♖/indy/0/pad/next/0.0.2/index.hpmi

How do they know what skills the agent learns?

contextパラメーターも説明を書いて欲しい

ここに追加するなら、from_numberメソッドの説明は文章でもしてほしいかな

このあたりもコメントでどういう数値を作っているか書いて欲しいかな。

ここコメントでfloatの精度によってこうなっちゃうよって説明して欲しい

ここか7.2.1に組み込み関数のroundを紹介して、偶数丸めであることを書いて欲しいかなと思った

https://docs.python.org/ja/3.14/library/functions.html#round

これ、見出しレベルが違うかも?

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.

This article about Sampling a music talks about how musicians take parts of existing songs and reuse them to make something new. I thought this really connects to the idea of replication and variation from the chapter because music samples change and spread kind of like social media posts do. (From a small piece of a song can turn into something completely different when it’s used in another context.) It made me realize that creativity often comes from reusing and transforming what’s already there.

This type of content reminds me a lot of what we have learned about trolling. This type of non-sensical reaction-evoking content, often called rage bait, is made to prompt viewers to interact with the content and ultimately make the creator more money by getting them more views. I think it is interesting how emotionally invested people get in non-consequential, silly content that doesn't truly affect them.

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

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.”

self-curated wordCloudLines make everything in line and marked in line

When documents cease to be enclosures of discreste disconnected islands separated decontextualized and self standing of human externalization of human intellect but

but inclosures acting as portals

to complete transitive closure of bidirectionally meaningfully high-resolution linked associative complexes that connect people ideas and things into emerging co-evolving born bultiplayer co-laborative permanent evergreen coevolving spaces of associative conplexes

♖/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 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?

I thought it was really interesting how the chapter compared social media to evolution. It made me realize that posts kind of “evolve” too like when people add comments, quote tweets, or make new versions of memes. I see this a lot on TikTok, where one simple video turns into so many different versions as people keep adding their own twist. It’s interesting that how creative that can be, but also a little scary because no one really controls where it goes, and sometimes it ends up spreading negative stuff or misinformation.

Learn how to handle errors in Swift with do-try-catch, custom error types, and best practices. A complete step-by-step guide with examples to make your iOS apps more stable and reliable.

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.

Căn cứ Cam kết WTO

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.

https://www.reddit.com/r/BaseballScorecards/comments/14fkq8d/i_love_scoring_and_i_love_field_notes_so_this/

https://www.reddit.com/r/BaseballScorecards/comments/1df8ktt/heres_my_prototype_little_book_of_scorecards/

https://www.reddit.com/r/BaseballScorecards/comments/1omz0nt/la_dodgers_5_at_toronto_blue_jays_4_world_series/

the future of HyperText is HyperPlex

Hypertext Mark Up Language

HyperPlex Mark-in Notation HPMI

reimaginging HTML

local-first person-first self-husted autopoietic

Open commons based

Peer produced

Extendable

Integral Omnioptional omnipresent

emergent Open Stadards

not just open source

but Open Constructs

embodied Open de factor co-evolutionary Standards

liveing lively organicallly vo-evolving

field of emergent practices self-describing self-explicating

Meta-Plex is lexible extendable

self-hosted autipoietic Mark In Notation with its own atomic term/formula language

72

That’s true. Continuing to instill good habits into younger generations will help promote futility and prosperity. If we minus the hate in the world it’ll be even more fruitful.

Yeah and we wonder why they have shorter lifespans. It’s almost comical that they haven’t seem to figure it out for themselves, but I digress.

I wonder if this applies faster to those who decides to do drugs or even crazy things. Like for those people who end up doing harsh drugs I wonder if they’re bodies ages faster most than others.

I wonder truly if the life expectancy will continue to increase as humanity ages or will it end up declining. Maybe with better medicines the population will continue to grow and prosper, but if not we could see a potential decline in society. There’s no way to tell until we get there and see what the future holds.

I feel like this explains what our current generation in certain areas have been going through. There’s people out there who discriminate against older content creators because they may feel that it’s unrelated to their lives or situations or due to that they judge harshly. When in reality the content being produced wasn’t for them to begin with.

I feel like the concept of social aging is something that can be looked at from multiple perspectives. Like one person may have the idea that someone has to be in a younger audience category in order to by more recognizable and well known, however I feel like that even those who are older than us have gained plenty of respect and appreciation for their efforts that they’ve shown. Essentially this helps pave the way for the ones that come after them.

I came back and when I reread it, I realized I didn't fully read this paragraph. Is code-meshing and code-switching different?

読みましたが、特にコメントはありません。LGTM。

1.1.0

1.1.0

MUST: 表タイトル入れてください

もうちょっと説明がほしいかな。 ハッシュがあるから改ざんに対応できるとか、さっきの課題にどう対応しているかの説明があるとうれしい。

でもそこまで書くとコラムとして書きすぎかなぁ。

の?

PEP 751だけだとわかりにくいし脚注もリンクだけなので、どういう内容のPEPかを最初に書いて欲しいです。

◯◯に関するPEP 751が、みたいな

数字の箇条書きであることに意味があるのかが気になった。 優先順位とかがないのであれば、数字なしの箇条書きがいいかなと思います

nits 最新版の4.0. でもいいかなと思った

ここの説明だけ「ですます」調になっているので、他を合わせた方がよさそうです。

test

後ろに余分なスペースが1個入っています。

後ろに余分なスペースが1個入っています。

後ろに余分なスペースが1個入っています。

Artificiality is used in the film to emphasize the visual design of the film and how they used cinematic tools to create a world in the story. The film did not have the intention of being realistic to the time period or setting, but to create emotion and suspense in the storyline of Macbeth. With its use of shadows and high contrast black and white shadows, it gives attention to the amount of detail and high contrast efforts that were implemented in the film.

The bare rooms and attention to placement in the film allowed for the audience to pay attention to what really mattered in each scene, instead of letting the atmosphere take away from the plot.

The heightened self-awareness was something I haven't seen in many films but it adds to the suspenseful and cold/uncomfortable feeling of the story leading to some big event--which ended up being when he was killed.

I agree with this statement. It felt very cold and dark, with a lot of the shots seeming to lack fluff around them. Most of the rooms that scenes were shot in were empty and had one center object for the audience to pay attention to.

Elaborating on the term "movie magic", I think that this film uses a lot of emotion and design to give meaning to the storyline and additionally make it almost satisfying to watch. There was no clear storyline, which made you not know what would come next.

「お勧めです。」の方ががよさそうです。

公式ドキュメントを読むと、excludeという引数はないようです。 https://docs.python.org/ja/3/library/tarfile.html#tarfile.TarFile.add

laramはparamのtypo？

this is an interesting comment.and helps explain why efficient code is less common

「お勧めです。」のほうがよさそうです

The text ends by emphasizing capacity building, sustainable policy depends on educators who can research, reflect, and adapt their language practices to meet diverse needs.

Leadership and administrative involvement are crucial. This shows how policy success depends on collaboration and the integration of language concerns into leadership training.

This pragmatic statement recognizes schools as implementation hubs for national goals. It emphasizes bottom-up application rather than top-down control.

The scope of language policy has expanded to embrace diversity and equity, reflecting changing demographics and the need to address linguistic pluralism in education.

This indicates a shift from macro to micro perspectives; recognizing schools as key agents in enacting and adapting national language visions to local realities.

The Swann Report stresses the efficiency and coherence that a clear language policy brings. Without coordination, schools risk fragmented practices and unequal support for multilingual learners.

This highlights a research gap; while national policies exist, there’s less focus on how language evolves inside schools, where real interactions occur daily. It argues that schools are laboratories of linguistic change.

The Australian example serves as a model of comprehensive national planning, contrasting with school-level efforts. It shows how governmental policies can inspire and align local initiatives.

This broad definition underlines language’s pervasive influence in society, from national identity to classroom interaction. It sets up a bridge between macro (national) and micro (school) policies.

Here, school language policies are framed as micro-level reforms capable of improving daily teaching and learning practices, showing that systemic change can start locally.

余分な文字が入っています。

typo→集めた

makes a copy of the previous state to avoid directly changing the previous state

Research is very often used when trying to figure stuff out or trying to solve questions.

I know there is a lot of content posted online that is created by people stealing others' work without crediting them. I don't think this is a just way to create content. When people's work is being reposted, I believe it should be credited as such. I have noticed some content creators adding watermarks over their videos to ensure people know where their content came from, despite the ways it is distributed. I think this is a simple way to ensure attribution.