Just looking at how amazing it is to just sit with a group of racially diverse background people in DS I remind me how privileged I am to be able to sit in the classroom and join the conversation with people of such diverse racial and cultural background. It's such an asset that Canada is able to bring together.

As the Ministry of Education has turned Faculties of Education upside down and continues to give them all a shake: they are currently scrambling to reorganise, rewrite and attempt to have these new course programs fit the new mandate they have been given. Will they take the opportunity to bring in more space for inclusion and equity or will the status quo remain?

I have met a few exceptional teachers who were not giving permanent positions because their credentials were not from this country. This is a reality not only at the K-12 level teaching and administration hiring processes. Abawi pointed out how the obstacles to hiring and recruitment are linked to the same issues that cause poverty, unstable work, and lack of credential recognition in the BIPOC community. This connects to education in a way that opens the conversation on whether the institutions merely replicate social inequality rather than confront it. Of course, this leads me to think that maybe hiring schools cannot be improved separately from the systems that cause BIPOC people to be excluded before they even get into a job interview.

Although this article raises many valid issues that are still rampent in Ontario schools, I believe readers need to frame this entry as a place in time, which in some aspect, time has allowed for forward movement in addressing and changing practices. If you look at the Principle's Qualification program website, they offer AQ's in inclusivenes and mentor amongst others, with in a "culturally responsive" frame work. I would state the programs they offer are at least atttempting to be relevant and supporting of racialized groups in Ontario schools.

If you look at the Peel Region District School Board (PDSB), which is in an incredibly diverse region of the GTA, in 2025 they published a review of hiring practices ( hopefully carried through); was an attempt to establish a more transparent and equitable hiring practices within which included " Hiring Practices Procedure is to assist individual hiring managers" which has several sections including "1. Build a community of practice, action and accountability; 2. Institute practices that mitigate various biases (e.g., unconscious and implicit biases); 3. Expand the applicant pool to ensure that candidates with diverse identities and lived experiences reflect those of the students and community; This was after an extensive census report was taken on staffing and student ratios etc., (2021-2022) which in the conclusion was acknowled by the Board there were short comings in practices. This Board is probably not "perfect", but effect it seems is being made to advance inequities in hiring and practice.

Gloria Ladson-Billings' framework of Culturally Relevant and Responsive Pedagogy requires that we ensure students' cultural, linguistic and lived experiences are brought into classrooms and schools in meaningful ways. This has to include MORE than just visual representations and reading material (dare I call it 'low-hanging fruit'?). This comment, and our readings and discussions from the week have me reflecting on how educators can ensure that this happens in ways that have the potential to enact real change.

AND, the administrator gap cannot be closed without disrupting the upper administration gap. In the paragraph above this, they specify principals and vice principals when talking about administrators. If more than 90% of school administrators in Ontario are white - I wonder what that number looks like for Superintendents and Directors.I suspect it is even higher than 90%.

Linguistic differences are observed early in social interactions, much like characteristics like sex, skin color, or age, and as a result, they impact how people categorize others (Schulte et al., 2022). Recognizing linguistic differences can help us navigate conversations during both informal and formal exchanges. However, Rapid information processing based on stereotypes can result in discrimination if speakers are perceived as having unfavorable traits, potentially leading to biased treatment of them.

Schulte, N., Basch, J. M., Hay, H. S., & Melchers, K. G. (2024). Do ethnic, migration‐based, and regional language varieties put applicants at a disadvantage? A meta‐analysis of biases in personnel selection [Review of Do ethnic, migration‐based, and regional language varieties put applicants at a disadvantage? A meta‐analysis of biases in personnel selection]. Applied Psychology, 73(4). https://doi.org/10.1111/apps.12528

As someone who TA'd one of the mandatory courses in the Teacher Ed. program at Brock, you can't hire what doesn't exist. Gatekeeping structures that prevent access to BIPOC students becoming teachers must also be addressed. I have spoken with BIPOC students who would never consider teaching as a career because of the abuse they faced in the school boards as students. I wonder if that's part of the "plan"?

This is a beautiful example of how to push back against colonialist oppressions. "Divide and conquer" is the chief strategy. Quite often, a person alone cannot overcome a bully; when a group of friends, or their older sibling accompanies them, it's almost comical how quickly the bully caves. Bullies rely on isolation to enforce their terror. We must band together and resist.

Abawi contends that merit and fit often mask racialized decision-making, especially when administrators are overwhelmingly White. This is indicative of wider critiques of colour-blind racism and neoliberal policy language. It makes me think of how fairness is often claimed through procedure, while structural inequities persist underneath those procedures.

This is an unsettling statement to read. If Ontario's education system represents the best of Canada, then what do the other Provincial systems look like?

I was privileged to moderate the student and teacher panels at Future Black Female's "Breaking the Cycle" Conference this spring. It was very revelatory in the way that Administrators hold the decision-making power about what EDI practices/policies do/do not get enacted in individual schools. It also made me think about the Gaudry and Lorenz (2018) article, where "Inclusion" seems to be best concession that institutions are willing to make at this time. Perhaps moving towards Reconciliation Inclusion, and even Decolonization Inclusion would mean installing the types of bodies that would bring policy/systemic change, and that's the "fear"?

I think it is important to note that many racialized people are faced with systemic barriers that often limit their income. Having this flexibility provides them with the opportunity to pursue higher education without feeling the burden of thinking they have to leave their job

This is another form of colonialism where they are representing a unilateral way of learning. As we all know, across our various backgrounds, cultures, and other identities, there are multiple ways to learn and achieve the same/similar outcomes. It's like this system wants to produce robots that all act exactly the same way, with no difference.

I think that it is important to understand that limited accountability measures can also stem from the power dynamics that are present within the schooling system. The majority of administrators are predominantly white, as are teachers. When I was in high school, a white male accused me of bullying him when we had not spoken for 3 months. The vice-principal at the time did not ask me for my side of the story when she brought me into the office. She believed him and started telling me that I'll never go to university, that I'll be expelled, and that my future is over. It was not until my white best friend, who was with me during the time of this accusation, said that I was not involved, that she believed me.

In their article, Lauren Powell's article clearly states that "inclusion is not equity" (p. 3) when discussing the value of co-production in neurodivergent research. Gaudry & Lorenz call inclusion the 'low hanging fruit of Indigenization' (p.220). Across these three articles, we see authors calling out surface-level inclusion and calling for more meaningful change that moves past hiring quotas and addresses the foundational oppressive structures and practices in our institutions.

When I was graduating from high school, my school got their first BIPOC VP. Now reading this, I genuinely wonder how hard it was for them to get into that position. I remember being so happy to see someone who looked like me in a position of power, but I hated that it was in my last year of high school.

When I was in my M.Ed degree, I was a teaching assistant, and there were multiple professors who, once they saw me, questioned whether I was qualified to mark their students' work. One professor got to the point where they would log in to the LMS and monitor every grade I entered and the time I spent on the site. Yet, the university expresses that the TAs' positions are on a trust basis...

The author suggests that hiring practices are influenced by the social location of those in power and that diversity among leaders is as critical an issue as diversity among teachers—something that relates to the general discourse of educational gatekeeping practices, reproduction of institutional culture, and the necessity of those in power to question their assumptions and biases.

McQueen (2023) highlights the double-bind, which expresses that racialized women in higher education often experience two forms of discrimination at the same time, such as microaggressions against their race and gender. She expresses that the 'holistic' university experience is oftentimes limited or nonexistent to this demographic due to systemic discrimination

This comment brings to mind the "Fine Band of Misfits" discussion around the Gaudry & Lorenz article on Thursday, about the colonial strategy of divide and conquer. Saurabh commented about TikTok being used as a platform to connect different groups experiencing similar forms of oppression. This comment is an inspiring example of what is possible when groups can work together to affect change.

Settler-colonialism works alongside the colonized mind. As a person who was born in Canada and went through the Canadian public school system, many of the ideas and ways of learning are a result of colonialism. For example, I had a professor who used APA 7 formatting for papers due to university standards, but never enforced the formatting because it was a colonial and unilateral way of learning.

By 'removing' all biases, it creates space for tokenization of students and surface-level policies, because it is extremely difficult to eliminate unconscious biases. To create more inclusive policies that move beyond the surface-level requires having more BIPOC administrators and teachers present in these spaces and aiding in creating more inclusive policies that reflect the lived experiences, alongside white allies.

This comment surprised me and gave me pause. I wish that the author had cited where he found this 'label', champion of human rights, or at the very least provided more context. Perhaps because of our Charter of Rights and Freedoms? It leads me to wonder how exactly we are champions? Who are the champions? Indigenous groups, Japanese-Canadians during WWII, women and 2SLGBTQI+ individuals working in the Federal Government (armed forces and RCMP) and youth with special education needs navigating our public school system all come to mind as Canadians whose human rights are most definitely not being championed. Our readings from this week also suggest this label may be misleading.

Terms such as these are considered race-neutral. While people should be hired based on their qualifications, there should also be an acknowledgement that there has been a history of racial discrimination within the schooling systems. Taking accountability can create meaningful and long-lasting change. Rather than remaining neutral, it turns into active anti-racist practices.

This was my experience growing up as a racialized person, even through higher education. Most of my professors were white, and they were teaching racialized students. In my undergrad, the only professor who was a person of colour had more inclusive learning material compared to the white male professors. I think it shows that sometimes professors have a choice when it comes to creating inclusive learning environments

Abawi ( 2021) doesn't explain or illustrate the barriers these teachers face, but makes a broad statement and cites Pollock (2010) who's work is based around research mainly from the early 2000's. We have covered/ made up ground since then. If you go to the OCT website just for International Teachers, the steps are there on how to apply, as well as training and support and links to others sessions. Perfect, probably not, better since 2010, I would believe so.

This is only partially true. Each province and territory have Ministers of Education and they sit on a national council called the Council of Ministers. They meet to oversee trends in education, strategies and alignments with/about curriculum. Curriculum nationally is very much identical, fractions are still fractions in primary/junior. This system allows for regional priorities and knowledge to be implemented so as to meet the needs/view of that region. In the past because of teacher licencing in each regions; there was almost a protectivist attitude to teachers with licences out side of province, this is getting better.

Connecting this to my experience within the Lebanese context, we actually have a centralized national framework (heavily corrupt), but the hiring in public schools is completely driven by religious and sectarian motivations instead. What surprises me is the difference in scale. Canada’s divide happens across massive provinces and territories, but Lebanon's rigid separation is within tiny neighborhoods, like hiring Eastern Orthodox teachers in certain parts of Beirut or Shia teachers in Dahiah. Ultimately, I think both setups show the same problem it doesn't matter if the split is by region or centralized by government, the people in power will always find a way to rig the hiring process to favor their own group.

When teacher training programs push this idea that a "good" teacher has to fit a specific white, middle-class mold, it can shape and alter teacher identity. I connect this to what Stein et al. say about the challenges in higher education, because it means the bias starts before anyone even gets a job. It sets up thiscycle where universities shape who feels like they belong in the profession, and then those same people graduate, move up into leadership, and become the gatekeepers. Since they’re the ones in charge of hiring, they just keep picking people who look and think like them.

This made me think of Lebanon’s parliament where seats are strictly divided by sect/religion, and whether Canadian schools should just allocate teaching roles based on a neighborhood’s demographics. Matching the community makes sense, but could definitely backfire by trapping BIPOC educators in specific areas instead of fixing systemic bias everywhere. We see this exact trap in Lebanese politics, where groups who were once considered a minority have outgrown the label but are still boxed within it by rigid structures.

This connects perfectly to why we need to explicitly teach these skills during university pre-service programs. Unearthing our biases isn't something we can just figure out alone; it’s a formal skill that has to be taught and practiced in communion with others. Ultimately, if we don't train future teachers and leaders how to engage in these heavy conversations from the start, can we expect administrators to magically unmask the biases that dictate who they let into the profession having not taught how?

The article lays out several great solutions to increase diversity, like alternative pathways to teaching and targeted mentorship for BIPOC leaders. While these ideas are valid and definitely effective, I don't see how we can sustain them without official mandates or legislated policy. The colonial mindset is just too deep in both our personal and professional lives to fade away on its own by some optional initiatives. Without real laws that force open cracks in the current system, it’s hard to imagine seeing any real, immediate change; especially at the decentralized level of Canada.

I found out about this website about a year ago, it helps me understand the true meanings of my favorite songs.

I too find it difficult to speak up in a whole class discussion. I hope I will be able to find social annotation as a great way to participate.

Every time I think about annotating, I don't take the time to think about what has been written. I am hoping to make my annotations more productive.

rendering issue

Human judgement can be messy. Inter-rater reliability is important because it separates an objective workplace from bias. The goal is to see if candidates are skilled rather then having to have chosen the correct answer. The generalizability theory helps calculate the candidates answers along with the toughness of the raters and the test conditions. What should companies do differently if they see low numbers consistently?

The lack of funding for I/O to expand has probably contributed to this. Corporations only pay for certain systems while NGOS and governments haven't realized the psychology needed in a workplace of a country that's growing. They should be allowed to apply their foundational information in order to grow their work and help the workforce. How would I/O psychology address global issues when shifting towards environmental sustainability?

y(a) = b0 is the y value @a y'(a) = b1 is the slope @a

Near the NM_000901.5:c.1868G>A variant with similar presentation

I picked this topic because I like how it expanded on simplification. I don't have to make my language less correct to help ELL students, instead I can use simple words, clear words and rephrase directions when needed so students can understand.

I agree with this sentence about students being able to show their understanding differently. Even without being able to understand the same ways as their peers, they can show understanding with different techniques.

This is something I liked because it emphasizes that ELL students should be included in grade-level learning from the start. They shouldn't have to wait until they are fluent in English to share the same content as their peers.

This stood out to me because silence doesn't always mean a student isn't paying attention. As a teacher, I want to give students time to become comfortable with things instead of expecting them to speak right away.

I picked this because it reminds me not to underestimate ELL students. Even if they can't fully understand or speak English yet, they are still capable of thinking critically and learning new concepts.

I like this because it reminds me that every student learns differently. Not every student can learn from one type of text. Using different genres and providing supports can help students better understand what they read.

I highlighted this because it reminds me that literacy isn't just about reading books. Students also need the opportunities to speak, listen and communicate because those are skills that help strengthen their reading and writing.

I picked this because it reminds me that teaching ELL students is not only the ELD teacher's job. As a future ELA teacher, I need to make sure my lessons support all students, including those who are learning English.

This stood out to me because some students understand the content but they struggle to show it because of language barriers. I think the assessment should focus on what students actually know instead of if they are good with the English language or not.

This sentence really stood out to me because I agree that students do learn better when they are comfortable and welcome. As a teacher, I want my classroom to be a place where every student feels safe to make mistakes and receive help when needed.

I highlighted this section because I really like how it reminds us that ELL students should be held to high expectations just like every other student. They may need different supports, but they are capable of learning challenging materials and accomplishing them.

你好啊

123345

Talking with teachers instead of to them, and working through a dialogue in which both adults can learn is what instructional coaching is all about!

Adult learning is absolutely intrinsically motivated! How can we improve adult education by learning from them what they want to learn. I teach SSN for elementary students and I'm not particularly interested in the district's high school AN focus, for example.

One of my pet peeves is when related service providers come to IEP meetings and sit on their computers the whole time. I don't even sit on my computer if I'm leading the meeting. I'll just edit a paper copy of the IEP throughout the meeting. Respect for parents is so important.

When we're teaching, we're teaching behaviors and modeling respect. When we punish behaviors like asking questions, we're showing kids that we don't care.

Why this matters to me: This is a really powerful historical assessment of repression on university campuses. Most people’s perception of university campuses today is that they are progressive spaces and “safe spaces” for students but this historical account really reveals the sites of university campuses as sites of marginalization and repression rather than not. This piece will allow for an assessment of the extent to which historical sites of trauma have yet to be unpacked on university campuses and how that campus climate shapes the perspectives of current and past faculty and administrators as well as current students in order to provide a really powerful lens through which to view current marginalization on university campuses.

Using ‘how is queer?’ as a lens through which to view research transforms the entire process of research from categorizing human beings into boxes and labeling them to examining the systems, the binaries and the language that create the oppression in the first place (heterosexual/homosexual, male/female etc). This way of viewing research leads to the use of critical, post-structuralist methodologies.

yes, because how can one person represent all queer experience? But often leaderships spots the "queer person in the room" and suddenly they're expected to be the voice of ALL queer people.

Coming out as an educational leader is dangerous for people in the public system but especially in the Catholic system where there are expectations of heteronormativity and expectations around patriarchal hierarchy that make high profile roles troublesome. Queer leadership is like a red flag for a bull to parents and members of the public who want to impose their stereotypes and biases on these people and the system at large. Having to deal with opposition from the public sphere can be taxing enough, but when people think they have the right to judge your capability solely based on your sexual identity, your soul gets worn down.

I have been involved in a national project interviewing educational leaders doing gender justice work. Many participants cited burnout as a leading pressure in doing this type of representation, because it's personal and exhausting work to always fight against barriers, limitations, and bias. Despite people coming to this work with passion and love, when you don't take care of yourself and get renewed with community and love for yourself, it can be very isolating.

Just a short comment on how students are often the ones who are enacting change and taking on leadership roles within their institution. Academic leaders rarely take on the responsibility to create these spaces unless there is a demand for it. This emotional, mental, and physical labour that student take on to better their college/university is added on top of their existing school, work, and personal life loads.

I found the format of this overview of literature difficult to digest. I understand that they wanted to separate the concepts and create a linear sort of evolution to make the information intelligible, but the tables and such were disorienting and interrupted the flow of the arguments for me.

As we have been discussing throughout this article, queer representation in leadership is needed, but as @TaliT mentioned earlier (their statistics post), there are a lot of barriers that queer people face in higher education where leaders are intentionally blocking them from taking on leadership roles or being open about their queerness.

We've expressed this common theme in our class discussion too where a lot of what we're reading make us say "yes, we need this" but they are not practiced in reality.

I wonder what we will do individually to action these "we need this" feelings in our everyday work life? Especially when the work days feel long.

I took a course in 1995 at York called Language, Gender and Power in which people from a variety of disciplines met to discuss these interests. Queer theory was very new, and in linguistic circles researchers began examining the reclamation of the word "queer" by the 2SLGBTQIA+. It was a time in which coming out was dangerous, and talking about queer issues, let alone talking about the word "queer" as a positive powerful label, was tough for lot of people. There was a lot of disagreement about using the term. Many people felt it evoked too much trauma for them; others saw it as a powerful reclamation of language. It is a demonstration of complexity and tension that is important to keep in mind. This kind of bravery in scholarship and in physical classrooms is a small example of the type of risks people took and take still (risk of real physical violence) to normalize and authorize queer scholarship.

This phrase as an introduction is an interesting choice: "given", as if they didn't fight for it, as if the dominant powers hadn't felt pressured to do it because of decades of action and protest and education. But it also has connotations of "bestowal." And it comes with pressure, being in a spotlight, because it opens people up to the bias and stereotypes and misplaced anger of bigots.

Great work here as well

Great job highlighting this distinction.

I wonder if you could add some additional context here. I cannot imagine some undergraduate students reading this and debating whether legitimate criminal acts, such as robberies or thefts, would be considered crimes if the perpetrator commits the act to buy food, pay for shelter, etc. In their minds, those excuses may amount to a defense.

I recommend adding "For example" at the beginning of this sentence to help students understand that this is an example of a crime of omission. Another option would be to place this sentence in a block quote in the middle of the paragraph to draw attention to it and isolate it from the rest of the text.

cách chạy bên trog câu lệnh đó

yo

I think this sentence should be split into two sentences to improve clarity.

In ở đây là kiểu đầu vô của m , như kiểu cho câu lệnh vào , thì In sẽ là nơi tiếp nhận nó -> chữ theo thứ tự xác định Out sẽ đưa ra kết quả, (không phải cái nào cũng có out như print, def(hàm trả về None), -> nhảy chữ lien tuc

Synthèse de la Classe Coopérative : Un Modèle de Pédagogie Active et de Responsabilisation

Résumé Exécutif

La classe coopérative, telle qu'expérimentée au collège Picasso sous l'impulsion d'Aurélie et de son équipe, repose sur la rupture de la verticalité traditionnelle entre enseignant et élèves.

Ce modèle s'articule autour de deux piliers centraux : le conseil coopératif, un espace de parole régulé où les élèves gèrent les conflits et la vie de classe en autonomie, et un projet collectif interdisciplinaire (ici sur le thème du cinéma) qui valorise les compétences transversales.

Les bénéfices observés incluent une amélioration significative du climat scolaire, une réduction du taux d'absentéisme et de retard, ainsi que le développement de l'aisance orale et de la confiance en soi, particulièrement chez les élèves en difficulté académique.

Toutefois, cette approche exige un "lâcher-prise" de l'enseignant, une cohésion d'équipe forte et une vigilance constante pour éviter que la parole libre ne se transforme en un "tribunal" pour certains élèves.

I. Le Conseil Coopératif : Mécanique de l'Autonomie

Le conseil coopératif est une instance hebdomadaire d'une heure où les élèves se réunissent en cercle pour débattre, résoudre des problèmes et proposer des idées.

1. Structure et Rôles

Le conseil fonctionne de manière autonome grâce à une répartition précise des rôles entre les élèves, sans intervention directe de l'enseignant qui se positionne comme un participant parmi les autres.

| Rôle | Responsabilités | | --- | --- | | Le Président | Anime la séance et choisit les thèmes à aborder à partir d'une boîte à idées. | | Le Secrétaire | Prend des notes pour garder une trace écrite des discussions et décisions. | | Le Gardien de la parole | Répartit équitablement le temps de parole et donne la priorité à ceux qui ont le moins parlé. | | Le Gardien du temps | Limite chaque sujet à un maximum de 5 minutes pour garantir la fluidité. |

2. Règles de fonctionnement

• La boîte à sujets : Les thèmes sont déposés librement par les élèves avant la séance.

• La régulation : Le conseil suit des règles strictes : interdiction de se moquer, écoute active, demande de parole par main levée, et respect de la bienveillance.

• Le renforcement positif : Le conseil inclut des temps de félicitations et de remerciements entre pairs pour valoriser les efforts (ex: progrès sur le bavardage, prise de parole d'un élève timide).

II. Une Nouvelle Posture Enseignante

Le passage à une classe coopérative modifie radicalement la position de l'adulte.

• Rupture de la verticalité : L'enseignant s'assoit dans le cercle, lève la main pour parler et accepte de se mettre en retrait. Aurélie souligne que "le lâcher-prise est la clé".

• Autorité transformée : L'autorité n'est pas abolie mais s'appuie désormais sur le dialogue.

L'enseignant reste le garant de la sécurité et de l'ordre, conservant "le dernier mot" en cas de nécessité.

• L'enseignant comme "messager" : Dans ce système, l'enseignant peut servir d'émissaire entre les élèves et le reste du corps professoral pour résoudre des tensions pédagogiques ou comportementales.

III. Le Projet Coopératif : La Pédagogie par le Faire

Le projet de fin d'année, centré sur le cinéma, sert de catalyseur à la coopération.

Chaque jeudi, les élèves disposent de deux heures pour travailler en ateliers.

• Diversité des ateliers :

  • Couture/Upcycling : Création de costumes (ex: Aladin, Avatar) à partir de vêtements récupérés et transformés.

• Menuiserie : Construction de décors (rideau de scène, tapis rouge).

• Cuisine : Préparation du buffet pour la réception des parents (biscuits trompe-l'œil, rochers coco).

• CDI/Recherche : Création d'affiches, de jeux sur les métiers du cinéma et de recherches documentaires.

• Révélation des talents : Ces ateliers permettent aux élèves en difficulté dans les matières traditionnelles de briller.

L'absence du "stress de l'écrit" favorise l'épanouissement et la mise en avant de compétences techniques.

IV. Impacts et Résultats Observés

L'analyse de l'expérience au collège Picasso met en évidence plusieurs indicateurs de succès :

• Climat scolaire : Une ambiance de classe apaisée et une solidarité accrue, même entre élèves n'ayant pas d'affinités naturelles.

• Indicateurs administratifs : Selon la CPE, la classe coopérative affiche le plus faible taux d'absence et de retard de l'établissement, car les élèves sont "contents de venir".

• Compétences sociales : Développement de l'aisance à s'exprimer devant un public et face aux adultes.

Ces élèves se retrouvent fréquemment élus délégués ou membres du Conseil d'Administration (CA).

V. Défis et Limites du Modèle

Malgré les points positifs, les sources soulignent des points de vigilance :

• Le risque du "tribunal" : La parole libre peut conduire à une situation d'un contre tous lors du règlement de conflits, ce qui peut être ressenti comme "humiliant" par l'élève visé.

Un cadre strict et des garde-fous sont nécessaires.

• Pas une solution miracle : Le modèle n'empêche pas les situations de harcèlement ou les conflits majeurs.

Il doit être complété par les leviers traditionnels de l'établissement.

• L'hétérogénéité des élèves : Certains profils (tempérament compétitif ou rivalité forte) adhèrent moins au projet, et le retour dans une classe "classique" en 4ème peut être brutal pour certains élèves fragiles.

VI. Recommandations pour la mise en œuvre

Pour les enseignants souhaitant initier une telle démarche, les praticiens conseillent :

• Le travail d'équipe : La classe coopérative ne peut fonctionner sans une équipe pédagogique soudée.

Le binômage (deux professeurs principaux pour une classe) est fortement recommandé pour se rassurer et partager la charge.

• La progressivité : "Commencer petit" en introduisant d'abord le conseil coopératif avant de se lancer dans des projets complexes.

• La formation et l'accompagnement : Se faire aider par des référents ou des collègues expérimentés pour établir un cadre spécifique à l'établissement.

• L'acceptation de l'erreur : Tester des dispositifs, accepter qu'ils puissent échouer, et réajuster en fonction des réactions des élèves.

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as i said eariler i think i am sliglty more confident now

Till now what i understood is that - Put in raw data to map func it give structured key value of what you need - Give huge amount of structured key value generated from map to reduce func and it will summarize (for eg) and return thr output to you like total count of all the occurnces etc

这个注解的用途是检查是否真的有重复的方法。

https://www.merriam-webster.com/dictionary/vernissage

This comment is attached to something specific

I'm going to leave a comment here

what does this indicate? I don's see it as evidence of a lack of a welfare gain. Perhaps a friction (~transactions cost) or a stigma issue?

this is an annoying AI 'not this but that' again

applied economic theory

this is AI-speak ... the not this buut that juxtaposition

Maybe this missses the usual 'what's in it for the retailer'? and the answer is 'it helps them price discriminate by the (likely) single most indicative measure of willingness to pay -- the income (or adjusted income', helping them increase their profits

it seems a bit pretentious because 'the opportunity card' was never actually a thing, just an idea I had

I think the authors need to look into the criticisms of learning styles and MBTI as they seem at first glance to use both uncritically. They also seem to cite the theories and claim "tremendous opportunities" (p.1) for effectiveness without citing many of any actual empirical studies that would support such effectiveness.

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Reply to the reviewers

We thank the reviewers for their careful and constructive evaluation. We believe the requested revisions are feasible will substantially strengthen the manuscript.

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Referee #3

Evidence, reproducibility and clarity

In this manuscript, Halter and colleagues propose a novel approach to sample level analysis of single cell disease atlas by only considering cellular proportion. They perform an evaluation with several SOTA approaches including ones using both pseudo-bulk or combination of compositional and transcriptional changes. While bench-marking results are positive, the design of the study including selected data and evaluated statistics differs from previous studies possibly indicating some bias selections. The paper has also strong statements on the fact their method is not affected by batches, while this is only shown in a single data set and some of the evaluated data has batched samples removed. These results are very likely misleading and authors need to either remove such strong claims or show strong evidence supporting it.

Main points

Authors indicate only samples with 500 cells are included. How would the method work without such a filter? Evaluation should also be done on sparse data, as this might be frequent in single cell studies. Otherwise, tgus should be also discussed as a limitation of the study. Authors should discuss the potential risk of this variance-based thresholding inadvertently filtering out rare, low-variance cell populations that carry critical biological significance. Also, could it be that this filter simply selects condition specific cells (cancer cells in cancer samples?). Regarding the previous concern, it is unclear if this performance retention is unique to ECODA's log-ratio approach. For a fair comparison, the authors should benchmark the competing compositional methods using the exact same HVC subset to determine if the performance advantage stems from the algorithm itself or simply the feature selection. In this case, some methods, such as PILOT, need cell types. To comprehensively demonstrate ECODA's performance, the authors should compare their approach against recently introduced sample-level representation methods. QOT: https://academic.oup.com/bib/article/26/1/bbae713/7953914. Joodaki, M. (n.d.). PILOT-GM-VAE: patient-level analysis of single-cell disease atlas with optimal transport of Gaussian mixture variational autoencoders. Pang, K. (n.d.). PULSAR: a Foundation Model for Multi-scale and Multi-cellular Biology. Regarding the data selection, authors should include all the previous data as in previous studies. See PILOT, PILOT-GM-VEA or QOT for a larger selection of data sets. The current approach uses k-means but some of the evaluated methods are shown to work better with other clustering methods (Leiden) or similarity metrics (Cosine). Authors should improve the benchmarking to also include richer strategies on how to perform clustering and include metrics evaluating the distances directly (silhouette index). Data has very strong filters that remove confounding factors. For example, the authors handle demographic confounders (such as age) in the Gong & Sharma dataset by severely restricting the cohort (males <= 40 years), which limits real-world clinical applicability where cohorts have complex, overlapping covariates. Can ECODA deal with the data sets without any kind of filter on confounding factors?

In the Stephenson dataset, the authors manually restricted the data to a single clinical location ("Ncl site"). If ECODA is as robust to batch effects as claimed, manual exclusion of multi-center data should be unnecessary. The authors should explain why this pre-filtering was required and evaluate if ECODA can accurately stratify patients when all multi-center data is included. Another example of a batch rich data is the KPMP Kidney data, which include multicenter and multi protocols single nuc vs. single cell data. The claim that ECODA is robust to batch effects is currently supported only by ANOSIM scores on two datasets, which is insufficient by current single-cell benchmarking standards. To rigorously demonstrate that ECODA effectively resists batch effects while conserving biological variance, the authors should evaluate their sample-level embeddings by using more data and also utilizing metrics such as Silhouette batch, LISI, and so on (take a look at scvi-tools, https://docs.scvi-tools.org/en/1.3.3/tutorials/notebooks/scrna/harmonization.html). Besides, they need to drop all filters based on clinical variables and include additional data sets as previously discussed.

Referees cross-commenting

Reviewers do focus on distinct but non conflicting aspects, while some similar points regarding benchmarking are quite similar between us and reviewer 2. I would like however to stress the batch correction aspect, which is currently a big statement on the manuscript, but we could capture several design issues with this aspect.

Significance

The discussion on how to analyze single cell data of patients cohorts is of strong significance. Authors make an interesting point that cell frequencies can work well, but the results are clearly biased by data selection and experimental design. The study will have a greater value if authors can tune down most of their claims and instead have a more balanced analysis on when compositional analysis is best and when transcriptional signatures are best. This is likelly to be very study/data dependent.

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Referee #2

Evidence, reproducibility and clarity

This manuscript benchmarks scRNA-seq sample representation methods for unsupervised patient stratification. The authors demonstrate that centered log-ratio (CLR)-transformed cell-type proportions (ECODA) match or outperform more complex state-of-the-art methods while requiring orders of magnitude fewer computational resources. The accompanying scECODA R package facilitates practical adoption.

Software availability. scECODA, integrated into Bioconductor and Seurat workflows as an open-source R package, substantially increases the practical impact of this work. scECODA comes with proper documentation and meaningful tutorials.

Major Concerns

1. The benchmark conflates methods with fundamentally different objectives. Several benchmarked methods were not designed primarily for unsupervised patient stratification by clustering. MrVI, for instance, explicitly targets the identification of local gene expression shifts that would be missed by a composition-based approach (scPoli likely behaves similarly though this manuscript lacks analysis). These methods are likely complementary to ECODA rather than competing with it. This is already partly visible in the benchmark: MrVI performs superior to ECODA on the Adams and Stephenson datasets while underperforming elsewhere. The authors should investigate and discuss why ECODA underperforms in these specific cases for example, whether transcriptional reprogramming rather than compositional shifts dominates the biological signal in those datasets. Such an analysis would provide practical guidance for method selection and reframe the benchmark as a characterization of complementary use cases rather than a simple ranking.
2. The Leiden clustering benchmark requires clarification and extension. This concern has several distinct components that should each be addressed:
   • Batch correction dependency: ECODA's robustness to batch effects may derive partly from cell types being called in batch-corrected embedding spaces (e.g., Harmony or scVI), rather than from ECODA itself. The authors should clarify whether cell-typing in batch-corrected embeddings are a prerequisite for ECODA's batch robustness, and if so, make this explicit in the workflow recommendations.
   • Performance in challenging compositional scenarios: The Gong Sharma dataset, which requires fine-grained immune cell-type resolution to separate CMV-positive from CMV-negative individuals, is arguably the most demanding test case for Leiden-based annotation. Would unsupervised Leiden clustering achieve comparable performance to expert labels in this scenario? This is not addressed in the current analysis.
   • Resolution parameter guidance: The manuscript implies that higher Leiden resolutions are generally better for ECODA, but there must be practical limits to this - overclustering introduces noisy, sparsely populated clusters that destabilize CLR estimates. The authors should provide clearer guidance on how to select the resolution parameter in the absence of expert labels.
   • Stability of the identified marker cell types: Figure 3B shows that the top contributing cell types identified by ECODA differ substantially between HiTME and authors_HR annotations for the Adams dataset. How stable are these "marker" cell types across annotation strategies, and what are the implications for biological interpretation?
3. The range of zero-handling values tested is too narrow. The benchmarked pseudocount values are in a relatively small range, and performance differences among them are minimal. To establish that the recommended default (pseudocount of 0.5) is genuinely optimal rather than arbitrarily chosen, the authors should extend the analysis to much smaller values (e.g., 1×10⁻³) and much larger values (e.g., 100). This would demonstrate that performance degrades at the extremes and that the current default sits in a robust optimum.
4. The HVC analysis would benefit from a more rigorous feature selection framework. The current approach selects highly variable cell types based on unsupervised variance ranking. Since the goal is patient stratification, it would be informative to compare this approach against supervised feature selection methods (e.g., LASSO-penalized classification). This comparison would clarify whether the variance-based approach approximates supervised selection, or whether meaningful discriminative signal is being left on the table. Separately, the claim that HVC-based ratios are directly translatable to clinical platforms such as flow cytometry should be made more carefully: several of the identified marker cell types (e.g., KLRF1⁻ GZMB⁺ CD27⁻ memory CD4 T cells) require multi-parameter panels that are not routinely employed in clinical practice.
5. Cell-type annotation subjectivity introduces a potential source of bias not discussed. The authors evaluate annotation robustness across strategies but do not discuss a related concern: future studies may define cell types in a manner that, intentionally or not, introduces a desired sample stratification, while cell populations associated with unwanted variation (e.g., technical confounders) may be merged or excluded. This subjectivity could inflate ECODA's apparent performance in practice and should be acknowledged explicitly in the Discussion as a caveat of annotation-dependent methods.
6. The title and framing overstate a causal claim. "Cell type composition drives patient stratification" implies that compositional differences causally determine clinical phenotypes. The benchmark establishes that compositional representations perform well for stratification, not that composition causes the underlying biology. The title and several passages in the manuscript should be revised to reflect this distinction for example, replacing "drives" with "predicts" or "reflects."
7. Foundation model-based methods should be discussed and ideally benchmarked. PULSAR, which uses the Universal Cell Embeddings (UCE) foundation model as a basis for sample-level representations, was recently introduced and represents an emerging class of methods not covered in this benchmark. Including PULSAR would future-proof the comparison. If the computational burden of full benchmarking is prohibitive, it would be a valuable addition to the Discussion.
8. The benchmark should address large-scale and multi-study scenarios. Studies with thousands of samples (e.g., OneK1K) and multi-study atlases (e.g., the Human Lung Cell Atlas) represent the trajectory of the field. Including at least one such dataset would substantially strengthen the claim that ECODA is scalable and robust to batch effects. This is especially critical as deep-learning based methods might scale favorably with the number of samples.

Minor Points

• The number of nearest neighbors used for modularity computation is three, which is small and may introduce instability. A sensitivity analysis across a range of neighbor values is warranted.
• A comparison to the cLISI (cell-type Local Inverse Simpson's Index) metric, widely used in single-cell integration benchmarks, would be appreciated, though cLISI operates at the cell level rather than the sample level and adaptation for this setting might be necessary.

Significance

Given rapidly increasing scRNA-seq cohort sizes, rigorous evaluation of sample-level representation strategies is pressing. The field has invested heavily in complex methods, making this comparative analysis valuable.

This is a well-executed benchmarking study that delivers a clear and practically useful message: properly handled cell-type compositional data is a powerful and underappreciated representation for scRNA-seq cohort analysis.

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Referee #1

Evidence, reproducibility and clarity

This paper presents the ECODA method and scECODA package, which uses simple sample-level representations based on transformed cell-type abundances to recover biological stratification of the samples. This compares favourably to more complex embedding-based methods across 11 datasets.

I would like to congratulate the authors on a well written and interesting manuscript. My most important concern is that there is potentially some circularity in the labels and disease classifications used for evaluation. Some of this is mitigated by the cluster-derived cell labels, but not all of it, since part of the concern relates to how the sample-level disease labels were defined. A negative-control benchmark using labels not expected to be composition-driven would help clarify this.

The manuscript appears to motivate ECODA partly as a tool for de novo exploratory patient stratification, but the benchmark mainly evaluates recovery of known biological labels. This is a reasonable validation strategy, but it does not directly assess whether the method can discover previously unknown patient subgroups, or determine the number and clinical relevance of such groups without label guidance. This distinction should be made clearer early in the paper.

p3:

'across 11 patient cohorts' - are there common aspects to these? It would be good to indicate why these were chosen and what the strategy is.

p7, Fig 2 A:

The runtime for ECODA and GloProp seem to be significantly less than the shuffled baseline, but the implication is that they do more calculations, which seems surprising. Is this an artifact?

p8/9:

A possible limitation is that the benchmark may be enriched for studies where compositional shifts are already expected to be strong.

In a similar sense, for disease states that are conventionally defined in a specific cell type, the analysis could be recovering label-associated annotation structure rather than independent disease biology. This is especially relevant for high-resolution manual annotations, where disease-associated cell states may already encode part of the biological contrast being evaluated.

A negative-control or stratified benchmark on labels not expected a priori to be composition-driven, or on composition-matched sample groups, would be a useful cross-check here.

p10:

It would also be interesting to see the robustness to small sample sizes, as this isn't mentioned elsewhere, but potentially could be a confounder in many patient derived samples.

p11:

The distinction between ECODA and scECODA could be a little clearer here. As far as I understand scECODA is the R tool and ECODA is the general method?

p13:

The claim that inter-sample biological variation is largely explained by cell-type abundance may need some qualification, because some of the disease or disease-state labels may themselves be partly compositionally defined. If disease state was assigned or refined using histopathology, cellular composition, or the presence of particular cell populations, then a composition-based method is partly being evaluated against labels that already contain composition-like information. This might weaken the interpretation that composition is an independent driver of disease biology, rather than a correlate of how the disease category was operationally defined.

It would therefore be useful to give more detail on how these disease labels were derived. This concern is not removed by using cluster-derived cell labels, because the possible circularity is in the disease-state definition rather than the cell annotation procedure.

'For example, ratios...immunotherapy response.' - A citation here would be good.

'Cost effective diagnostic assays...' - Perhaps a bit of an overstatement. This would likely require validation across larger cohorts, clinical sample-processing conditions, sequencing depth/cell recovery difference, etc. This claim could be softened or framed as a future direction.

p14:

'Expert author annotations...' - this could be clarified. Were these all manually curated annotations from the original studies, or did some original studies use automated/reference-based annotation followed by curation?

p15:

'We further controlled...' - was the analysis also evaluated over these separate subsets (other than just males <= 40) as a cross check?

p19:

HVCs: Could you comment on this procedure, versus something more akin to highly variable gene selection?

Significance

This paper presents a fast and interpretable method for sample-level stratification of single-cell RNA-seq cohorts, based primarily on compositional differences in cell-type abundance. This makes it of broad interest to researchers performing cohort-level scRNA-seq analysis, since its speed and simplicity make it an attractive baseline even when more involved downstream analyses are planned. However, as presented, its performance is less well established for datasets without reliable high-resolution annotations, for settings where the relevant structure is not primarily compositional, and for genuine de novo discovery of patient subgroups rather than recovery of known labels.

My background is in computational biology and biophysics.

this can impact school success,

Synthèse : Enjeux de santé mentale et parcours de soins du public LGBT+

Ce document de breffage synthétise les interventions et les données issues de la journée d'échanges organisée par l'association ENIPSE, avec le soutien de l'ARS Occitanie, consacrée aux problématiques de santé mentale spécifiques aux personnes LGBT+.

Synthèse de la direction

Les personnes LGBT+ ne présentent pas de fragilité intrinsèque, mais sont exposées de manière structurelle à une violence polymorphe (rejet familial, harcèlement, discriminations systémiques) qui dégrade leurs indicateurs de santé.

Le concept central pour comprendre cette réalité est le stress minoritaire, un stress chronique lié à l'appartenance à un groupe stigmatisé, qui entraîne des modifications neurobiologiques durables et peut mener au trouble du stress post-traumatique complexe.

L'accès à la santé est intrinsèquement lié à l'accès aux droits.

L'insécurité administrative (non-conformité de l'état civil) et le manque de formation des professionnels de santé constituent les principaux freins, menant à un renoncement aux soins massif (jusqu'à 67 % chez les personnes trans).

L'approche recommandée repose sur la santé communautaire, l'autodétermination et une prise en charge pluridisciplinaire (psychologique, médicale et administrative) visant l'empowerment des individus.

I. Analyse des vulnérabilités : Le stress minoritaire et le psychotraumatisme

1. Le modèle du stress minoritaire (Meyer, 2003)

Le stress minoritaire explique la prévalence supérieure des troubles psychiques (dépression, anxiété, conduites addictives, comportements suicidaires) par une exposition à un environnement hostile.

Il se décompose en deux niveaux :

• Stress distal : Événements objectifs (insultes, agressions, rejet familial, discriminations au travail ou à l'école).

• Stress proximal : Processus internes résultant de l'exposition chronique (anticipation du rejet, hypervigilance, dissimulation de l'identité, honte intériorisée).

2. Le Trouble du Stress Post-Traumatique (TSPT) Complexe

Contrairement au TSPT simple lié à un événement isolé, le TSPT complexe (reconnu par l'OMS mais pas encore dans le DSM-5) est le résultat de traumatismes répétés, précoces et souvent relationnels.

Ses symptômes incluent :

• Dérégulation émotionnelle : Épuisement du système de régulation dû au port prolongé d'un "masque social".

• Altération de la perception de soi : La honte intériorisée est le cœur du tableau clinique.

• Perturbations relationnelles : Sentiment d'illégitimité et troubles de l'attachement.

3. Mécanismes neurobiologiques et Théorie Polyvagale

Le stress chronique modifie le fonctionnement cérébral :

• L'amygdale : Agit comme une alarme incendie constante, court-circuitant le cortex préfrontal (réflexion).

• L'hippocampe : Perturbé, il ne parvient plus à classer les souvenirs, provoquant des reviviscences physiologiques.

• Théorie Polyvagale (Steven Porges) : Le système nerveux dispose de trois modes hiérarchiques :

  • Sécurité/Engagement social : Communication et connexion possibles.

• Mobilisation (Sympathique) : Combat ou fuite (rythme cardiaque élevé, muscles tendus).

• Effondrement/Dissociation (Vagal dorsal) : Stratégie de survie archaïque où le corps ralentit faute d'issue perçue.

II. Le lien entre accès aux droits et accès aux soins

L'accompagnement administratif est un préalable indispensable au soin.

Sans "identité administrative" conforme (nom, prénoms, pronoms, numéro de sécurité sociale), le parcours de santé devient une source de traumatismes répétés.

1. Conséquences de la non-conformité administrative

• Mégenrage et "Deadnaming" : Utilisation de l'ancien prénom à l'hôpital, à la pharmacie ou auprès de l'assurance maladie.

• Outing forcé : Obligation de révéler son parcours trans pour justifier des documents d'identité discordants.

• Phobie administrative : Réponse défensive à l'anticipation des discriminations et de l'humiliation.

• Renoncement aux soins : Entraîne des errances diagnostiques graves (parfois supérieures à 3 ans) pour des pathologies non liées à la transition (gastriques, urologiques, etc.).

2. L'approche pluridisciplinaire du dispositif Sésame

Le dispositif Sésame propose une synergie entre trois types d'entretiens :

• Psychologiques : Stabilisation émotionnelle et traitement des mémoires traumatiques (EMDR, TCC).

• Accès aux droits : Aide à la constitution de dossiers (changement d'état civil, MDPH, aide médicale d'État).

• Accompagnement médical : Écoute, conseil et orientation vers des spécialistes formés ("safe").

III. Recommandations de la Haute Autorité de Santé (HAS)

Les nouvelles recommandations de la HAS (publiées pour une application pleine en 2025) visent à simplifier les parcours des personnes trans et rompre avec les approches normatives :

• Dépsychiatrisation : Fin de l'obligation d'évaluation psychiatrique systématique pour accéder aux soins ou aux chirurgies.

• Rôle du Médecin Généraliste : Positionné comme le coordinateur central du parcours de santé.

• Autodétermination : Respect du choix de la personne sans jugement de légitimité par le soignant.

• Accompagnement global : Intégration des dimensions psychologiques, sociales et administratives.

IV. Les défis spécifiques de la ruralité

La "métronormativité" laisse croire que la vie LGBT+ n'est possible qu'en ville.

Or, 39 % de la population d'Occitanie vit en zone rurale, où les inégalités de santé sont exacerbées.

1. Inégalités territoriales de santé

| Indicateur | Zone Urbaine | Zone Rurale | | --- | --- | --- | | Couverture médicale | 1 médecin pour 5 km² | 1 médecin pour 30 km² | | Densité de psychologues | 6 fois plus élevée (ex: IDF) | Très faible (ex: Bourgogne-F-C) | | Accès à des pros formés | Plus facile | 85 % n'y ont pas accès à proximité |

2. Risques accrus en milieu rural

• Hypervisibilité : Dans les petites communes, l'identité peut être connue de tous, augmentant la vigilance identitaire.

• Isolement social : 72 % des personnes LGBT+ rurales se sentent isolées.

• Renoncement géographique : L'absence de transports publics et les coûts de déplacement vers les agglomérations freinent le soin.

3. Solutions innovantes : Le format numérique

Le "Café Psycho" en visio, mis en place par le Sésame, permet de :

• Maximiser l'accessibilité pour les zones les plus isolées.

• Protéger la confidentialité (moins de risques qu'un lieu physique local).

• Favoriser les identifications positives entre pairs sans contrainte géographique.

V. Outils et ressources pour les professionnels

Pour améliorer l'accueil et la prise en charge, plusieurs outils communautaires ont été développés :

| Outil | Description | | --- | --- | | Feel Safe Box | Outil de stabilisation émotionnelle (respiration, ancrage sensoriel, écriture) pour gérer les crises de stress minoritaire. | | Guide d'autodéfense verbale | Conseils pour une communication assertive face aux agressions dans l'espace public, familial ou professionnel. | | Cartographie ENIPSE | Recensement des lieux ressources et structures d'aide juridique/sociale (Haute-Garonne et Hérault). | | Bans d'accueil Sésame | Badge utilisé en permanence pour signaler son prénom, ses pronoms et son état émotionnel (gommettes vert à rouge). |

Postures cliniques recommandées :

• Approche Trauma-Informed : Prioriser la sécurité physique et émotionnelle du lieu d'accueil.

• Transparence : Expliquer chaque étape du processus pour restaurer la confiance envers les institutions.

• Horizontalité : Reconnaître l'usager comme expert de son propre vécu.

• Validation : Ne jamais contester le ressenti de douleur ou de discrimination exprimé par la personne.

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Reply to the reviewers

The authors do not wish to provide a response at this time.

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Referee #3

Evidence, reproducibility and clarity

Summary

During mitosis transcription is silenced (except at centromeres) and the majority of chromatin-bound RNA is removed from chromosomes. Previous work has shown that retention of elongating RNAPII on mitotic chromosomes through a WAPL degron leads to transcription-dependent chromosome segregation errors. Additionally, retention of chromatin bound RNAs through mutation of HNRNPU/SAF-A also leads to chromosome segregation errors. However, the field lacks a complete understanding of the mechanisms that remove RNA from chromatin in mitosis. Previous work from the Oliveira lab (and others) demonstrated that depletion of Lds/TTF2 also leads to chromosome segregation errors, but it was not possible to directly link chromosome segregation defects to persistent mitotic transcription. In this current work the authors examine the role of TTF2 in mitosis in human cells. They nicely show that TTF2 is required to release transcripts from chromatin during mitosis and that retention of transcripts on chromatin leads to chromosome segregation errors. Interestingly the authors find that depletion of TTF2 leads to an increase in the number of R-loops present on mitotic chromosomes and that R-loop-containing DNA is a major component of anaphase bridges. The results presented in the manuscript are high quality and the data support the conclusions. This work is important because it provides further evidence that the removal of RNA and transcription complexes from mitotic chromosomes is important for accurate chromosome segregation. There are a couple of points that the authors should consider prior to publication listed below and some minor issues with data presentation that should be corrected.

Major points

1. The authors use RNAi to deplete TTF2 and examine mitosis following depletion. The authors state that TTF2 depletion requires 48 hours, or approximately 2 cell cycles. Since TTF2 is depleted for the entire cell cycle it is possible that transcription termination defects caused by TTF2 depletion during interphase causes defects in mitosis and that the observed phenotypes are not a result of mitotic function of TTF2. This concern is somewhat addressed by the observation where the authors inhibit transcription using triptolide and show that this treatment rescues chromosome segregation defects observed following TTF2 depletion. However, the TRP treatment is for 4 hours, which includes a substantial portion of interphase. The length of TTF2 depletion is a significant concern and I think there are two ways that this could be addressed:

a. Create a TTF2-AID (or dTAG) cell line and analyze chromosome segregation defects following TTF2 depletion only in mitosis. This is a difficult and time-consuming experiment but is also the most direct test of the role of TTF2 in mitosis. This experimental system would be required for publication in a high-impact journal.

b. Include a section in their discussion acknowledging that indirect effects could be a cause of the chromosome segregation errors observed following TTF2 depletion. 2. The authors nicely show that TTF2 depletion leads to a significant retention of EU-labeled RNA on mitotic chromosomes but do not address the nature of these transcripts. Additionally, the authors do not show that TTF2 depletion leads to changes in transcription in interphase cells. This work could be improved by the addition of EU-RNA sequencing data showing that TTF2 depletion leads to transcriptional changes in interphase (e.g. transcription past the normal termination site) and EU-RNA sequencing to identify that transcripts that are retained on mitotic chromosomes. Neither of these experiments are absolutely necessary for publication but would significantly improve the general interest of this work. 3. The authors show that TTF2 depletion leads to an increase of R-loops on anaphase bridges. Previous work has shown that R-loops are present at mitotic centromeres (29170278) and that activation of ATR through these R-loops is necessary for accurate chromosome segregation. This work is clearly relevant to the authors results and has not been cited or discussed. This previous work should be included in the discussion and interpretation of the authors' work.

Minor points

1. In Figure 1 the authors show a comparison of the levels of EU-labeled RNA in control and TTF2-depleted cells at each stage of mitosis. The graphs in B and D would be much easier to compare if these were combined into a single graph.
2. There are a number of quantitative plots that are lacking statistical comparisons between key groups. These are: Figure 1B and D, Figure 1F, S2B, Figure 4DE, S6C,

Significance

This advance is incremental but adds to accumulating evidence that transcription termination is important for normal chromosome segregation.

The strengths of this work are high quality data, careful analysis, and the fact that conclusions follow directly from the data presented.

The weaknesses are that the model system in not optimal to address the question being posed and that the authors have not completely characterized their model system.

This work will primarily be of interest to groups working on mitotic chromatin:RNA and transcriptional regulation.

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Referee #2

Evidence, reproducibility and clarity

Summary:

In this original article, authors attempt to define the molecular and cellular consequences of retaining spurious transcriptional activity in mitotic chromosomes. The study uses the depletion of TTF2 (transcription termination factor 4) in tissue culture of HeLa cells as a model. Results show the accumulation of nascent RNAs and R-loops as a product of such aberrant transcription which, strikingly, increases the incidence of chromosome segregation errors. Importantly, authors elegantly show that these errors are corrected by inhibition of RNA Polymerase2 activity with previously reported drugs. This reviewer finds the study very interesting and compelling, very well written and structured. However, I consider that the adjustment of several aspects might improve the manuscript:

Major comments.

1. The main limitation of the study relies on the approach followed to deplete TTF2. Authors used siRNAs in order to decrease the levels of this factor, which implies an incubation time of 48h. This might represent a limitation. Regarding this aspect, this referee request to show the proof of TTF2 depletion in the main figure with accurate quantification when possible.
2. Most of the main conclusions are very well supported by quantification of imaging data. However, this referee suggests the generation of superplots (ref) where values and average for each replicate can be clearly visualized. Statistical analyses comparing the median from each different conditions by t-student (unpaired test) will better support the outcome.
3. Authors show a beautiful correlation between the presence of R-loops and chromosome segregation errors (Figure 6). I request the authors to replicate the experiment with cn or TTF2 siRNAs in combination with the triptolide treatment. Additionally, and to provide further evidence about the functional consequences of retaining R-loops, I wonder if authors can drive specific R-loop depletion by using RNase H activity. This will definitely reveal whether transcription activity or/and its product underlies the chromosome segregation defect.

Minor comments.

1. I highly recommend to include the value of all the statistic tests in each plot.
2. I would suggest the incorporation of a final paragraph at the end of the introduction summarizing the most important results of the study.
3. In the introduction, and based on wide evidences showing transcriptional activity at centromere regions (Chan 2012, Liu 2016, Perea-Resa 2024), and to a much lower extend, at the chromosome arms of mitotic chromosomes (Palozola, 2017), I would rephrase to make clear that transcription is generally repressed rather than globally silenced in mitosis.

Referees cross-commenting

After reading the comments from the other two referees, I maintain my view about the quality/interest of the manuscript. I endorse the potential publication of this work, after addressing the recommendations, in a reasonable period of time.

Significance

Authors provide a compelling study addressing the functional relevance of repressing transcription early in mitosis to properly segregate chromatids to daughter cells. In addition, the study also pursuits to illuminate the molecular and cellular consequences of inactivating TTF2 function and to provide insight into the chromosome segregation defects found under TTF2 misfunction. The study considers and discusses the most important aspects of the literature relevant for the proposed questions. The results are very encouraging and mostly confirmed by several orthogonal approaches.

The major strength is the direct correlation found between R-loop retention and chromosome segregation defects. The major limitation is, however, the usage of slow siRNA-based strategies to deplete TTF2. The use of degron-protac alternatives would be very beneficial although I do not consider this an essential aspect.

The study is sound and address very interesting still open questions. The publication of the results will influence and benefit a wide audience specially researches working on the mitosis and transcription fields. Research on R-loops, a field full of open questions, would also acknowledge the insights from this study.

Overall, I consider the study very interesting and compelling. I support the evaluation and envision that its publication, once addressed the above described comments, will be feasible within roughly 3-6 months of revision.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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Referee #1

Evidence, reproducibility and clarity

In this manuscript, Tovini and colleagues investigate the role of transcriptional silencing during mitosis using depletion of TTF2, a factor implicated in mitotic transcription termination. The authors show that TTF2 depletion leads to retention of elongating transcripts on mitotic chromatin, defects in chromosome organization and compaction, delayed mitotic progression, and increased chromosome segregation errors, particularly DNA bridges and UFBs. They further report accumulation of R-loops and demonstrate that transcription inhibition largely suppresses the observed segregation defects.

Overall, this manuscript is interesting and potentially important, and the study is technically sound. In particular, I liked the experiments showing continued transcript elongation during mitosis in TTF2-depleted cells. The rescue experiments with triptolide also support the conclusion that transcription contributes significantly to the observed phenotypes. The methods and statistical analyses appear generally appropriate and sufficient for reproducibility.

At the same time, I think that several aspects of the mechanistic interpretation are somewhat overstated, and some important alternative explanations remain insufficiently addressed. For these reasons, I believe the manuscript would benefit from substantial revision before publication.

Major comments

1. The authors favor a model where persistent transcription and R-loop accumulation interfere with Top2A-mediated sister chromatid resolution. While this is certainly possible, I do not think the presented data fully support such a specific interpretation. The authors demonstrate a pronounced chromosome organization phenotype. TTF2 depletion causes broadened metaphase plates and approximately 1.5-fold increased chromosome volume. These observations suggest a substantial defect in mitotic chromosome compaction. An important alternative possibility should be considered, namely that persistent mitotic transcription interferes with Condensin I and/or Condensin II loading and/or retention. Given the current understanding that chromosome condensation and loop organization are highly dynamic processes, it is conceivable that ongoing transcription could impair Condensin-mediated chromosome assembly. At minimum, the authors should assess chromosomal localization of Condensin I and II in mitotic cells after TTF2 depletion (for example CAP-H/CAP-D2 and CAP-H2/CAP-D3). Such analysis, ideally also including triptolide rescue, would substantially strengthen the mechanistic interpretation and help distinguish between a primarily transcription/R-loop-mediated defect and a more global chromosome assembly defect.
2. The authors conclude that kinetochore-microtubule attachments are largely normal, based on Mad2 localization, inter-centromere distance, and the absence of strongly uncongressed chromosomes. In my opinion, these measurements are indirect and do not sufficiently demonstrate robust mature end-on attachments. Given the observed metaphase spreading, mild congression defects, and SAC-dependent delay, it remains possible that TTF2 depletion causes more subtle defects in k-fiber stability or attachment robustness. I think a cold-stability assay of spindle microtubules would be important here. Such experiment would directly address whether cold-stable k-fibers are normally formed and maintained in TTF2-depleted cells. Ideally, this should be combined with kinetochore markers and quantification of k-fiber intensity. Without such analysis, the statement that KT-MT attachments are unaffected should be toned down.
3. The manuscript generally frames mitotic transcription as detrimental to chromosome segregation. However, several previous studies, including work from Hongtao Yu's lab, reported that localized centromeric/kinetochore transcription during mitosis contributes positively to chromosome segregation, including correct Sgo1 localization and centromeric function (this literature is not discussed, despite appearing conceptually relevant to the present study). This is not necessarily a contradiction; it seems possible that a limited and spatially restricted mitotic transcription program at centromeres may be beneficial, whereas persistent chromosome-wide elongation caused by TTF2 depletion becomes pathological. However, this distinction should be discussed explicitly. As currently written, the manuscript risks giving the impression that mitotic transcription is generally deleterious, which would not be fully consistent with the literature. The authors should check whether Sgo1 is localized correctly after TTF2 depletion in both Noc-arrested and metaphase (MG132- or ProTAME-arrested) cells.
4. I was somewhat confused by the interpretation of RPA70-positive fibers. The authors state that they "did not detect a major increase" in RPA70-coated UFBs, arguing against an important contribution of replication-associated intermediates. However, in the next sentence, they report that approximately 20% of TTF2-depleted anaphases display an RPA70-positive fiber and, importantly, this phenotype is largely reverted by triptolide. In my opinion, this is not a trivial observation and appears difficult to fully reconcile with the conclusion that replication-associated events contribute minimally to the phenotype. In principle, RPA70 positivity should not necessarily be interpreted exclusively as evidence of replication stress. Persistent transcription, R-loops, or transcription-associated topological stress could generate ssDNA intermediates that can recruit RPA. Do RPA-positive fibers co-localize with R-loops? Therefore, the Discussion would benefit from a more balanced interpretation of these findings.
5. The rescue experiments with triptolide are convincing and support a transcription-dependent contribution to the phenotype. However, because triptolide treatment was performed over several hours, these experiments do not formally distinguish between ongoing transcription elongation during mitosis and perturbations arising earlier in the cell cycle, including possible transcription-replication conflicts during late S/G2. This limitation should be acknowledged more clearly in the Discussion, especially given the recently described role of TTF2 in replisome eviction. It would be informative to perform a time-course analysis of triptolide rescue. Demonstration that the phenotype can be substantially reverted within ~30 minutes of treatment would strongly support the interpretation that persistent transcription during mitosis, as opposed to earlier cell-cycle perturbations, is the major contributor to the observed defects.

Minor comment:

It would be useful to check whether RHINO-positive/R-loop-associated structures preferentially localize to centromeric versus chromosome arm regions. Such information may help distinguish physiological mitotic transcription from pathological transcript retention.

Referees cross-commenting

I have carefully read the other reviews and they do not change my overall assessment of the manuscript or my recommendations.

Significance

General assessment:

This is an interesting and potentially important study addressing how transcriptional silencing contributes to mitotic fidelity. The strongest aspect of the work is the convincing demonstration that transcription elongation persists during mitosis after TTF2 depletion and contributes to chromosome segregation defects. The main limitation is that the mechanistic interpretation currently appears somewhat narrower than supported by the data.

Advance:

The study extends previous work on TTF2 by linking defective mitotic transcriptional silencing to chromosome organization and chromosome segregation defects. The demonstration of transcription elongation during mitosis after TTF2 depletion is particularly interesting.

Audience

The work will likely be of interest to researchers studying mitosis, chromosome biology, transcription, genome stability, and chromosome organization.

Expertise:

mitosis, chromosome organization, nuclear organization, cell biology, biochemistry. I do not consider myself an expert in R-loop biology or transcription-coupled repair.

This website no longer exists. Domain's for sale if anyone has $1,396.

Nice summary

hi

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Yes

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nice way of putting it

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eLife Assessment

This valuable study investigates the mechanisms underlying inter-item biases in visual working memory. By experimentally manipulating the relative noise levels of target and non-target items, the authors report bias patterns that are broadly consistent with predictions of their previously proposed normative demixing theory. However, the supporting evidence remains incomplete, as the manuscript lacks a sufficient description of the underlying theory, key assumptions, and a quantitative link between the model and behavioral data. The manuscript would be substantially strengthened by clearer exposition and stronger tests, including analyses of the full error distributions and comparisons with alternative models, which would increase its potential interest to the cognitive neuroscience and computational cognitive science communities.

Reviewer #1 (Public review):

Summary:

Many previous studies have reported inter-item biases in visual working memory tasks. These biases can be either attractive or repulsive, depending on the particular experiments. It has been difficult to explain these biases in a unifying theoretical framework. Recently, Chetverikov (the first author of the current manuscript) proposed a demixing model for explaining these biases in Ref 22. That paper shows that both attractive and repulsive biases could emerge in the demixing framework depending on the noise properties. The current manuscript seeks to test the predictions of the demixing model experimentally in a series of new experiments and find evidence supporting the demixing model.

Because previous modeling results described in reference 22 (which is a preprint) are essential in interpreting the results reported in the current manuscript, I also studied that preprint and used the results reported in that paper to help interpret the results in this paper. My comments below will also contain discussions of that modeling paper.

Strengths:

Overall, the computational model tested in the paper is novel and interesting.

The demixing framework represents an appealing hypothesis that deserves further investigation.

The current paper provides new empirical data showing that the target stimuli with the same absolute noise level can be either repelled from or attracted to non-target items, depending on the relative noise levels. The observation that biases depend on the relative noise levels is by itself an interesting one, and is consistent with the prediction of the demixing model.

Weaknesses:

While this manuscript contains interesting new experimental observations and theoretical ideas, it has several substantial problems in its current form, which limit the conclusions that can be drawn. The description of the computational model is too brief. The key modeling assumptions need to be better motivated and explained. As the computational models generate different predictions in different regimes, it is a bit difficult to evaluate how well the experimental data support the model at a more quantitative level. Also, the results focused on studying the biases in the behavior; it is unclear whether the model can fully explain the behavior data (such as error distributions or behavioral precision).

Major concerns:

(1) Concerns/suggestions regarding the computational modeling

The current paper seeks to test the predictions of the demixing-based computational model proposed in reference 22. There are several problems with the modeling component in the current paper.

(1a) The description of the model is too brief and difficult to understand. Although the model was proposed in reference 22, it would still be beneficial to provide more details of the model so that readers can understand and appreciate the strengths/limitations of the model.

The generative model and the inference procedure could be better explained to better link the model to the behavior. In particular, how was the observer's behavioral report in each trial modeled? This requires more explanation because currently the demixing procedure estimates four parameters for a given trial, yet for a given trial, only one behavioral report was produced (e.g., current Experiment 1), or two reports were produced sequentially (e.g., current Experiment 2).

(1b) Key modeling assumptions need better justification.

One such key assumption is that on a given trial, each stimulus triggers many samples (or approximately, an entire response distribution), rather than a single sample. This assumption deviates substantially from prior work on ideal observer models. It was not clear whether this assumption is realistic. For the type of stimuli used in the current experiments, perhaps one can argue that each pixel corresponds to one sample of brain activity, thus collectively each stimulus should trigger many samples of activity in the brain. If this were to be the case, it would have two implications. First, the noise parameter in the model should be directly related to the magnitude of the stimulus noise. Thus, one should be able to plug these experimentally-controlled parameter values into the model to directly generate predictions about the biases. Second, when using stimuli with no stimulus variability (e.g., simple grating stimuli), the predicted biases should change. However, it wasn't clear whether this would hold experimentally, i.e., using gratings would lead to different biases or no biases.

If the variability of the samples for a given stimulus involves neural noise, it would be useful to justify why it is reasonable to consider that many samples were generated per stimulus.

(1c) As mentioned in (1b), the model assumes that on each trial, a large number of samples was generated. It would be useful to study and report how the prediction would change when the number of samples generated per stimulus is small. In particular, what happens when each stimulus only generates one measurement? This might be useful for interpreting previous experiment results with grating stimuli.

(1d) Reference 22 studies how the predicted biases depend on the d-prime of the identifying dimension and found that the pattern of the biases varies substantially depending on the information available for the identifying dimension. However, the current paper didn't really discuss this important point. It is also unclear what parameters the authors used for the d-prime of the identifying dimension. Was it fitted directly to the data? The Methods section has some description on the "identifiability dimension", but it was a bit obscure.

Intuitively, when the d-prime of the identifying dimension is very large, the demixing problem becomes irrelevant. In this case, there should not be any biases induced by demixing. In the case of the d-prime for the identifying dimension is 0, the problem should reduce to the simplified 1-d problem studied in reference 22. If my reading of reference 22 was correct, they reported different conclusions. It would be useful to clarify these points.

In any case, the d-prime of the identifying dimension appears to be a key parameter. It would be great to constrain this parameter using the empirical data. When the d-prime of the identifying parameter is small, the observer would easily confuse the probed stimulus with the other stimulus in a given trial. This should lead to poor task performance. Thus, it may be possible to directly estimate the value of the d-prime of the identifying dimension based on the observer's performance, and then use this parameter to generate model predictions accordingly.

(1e) The current model assumes that a large number of samples are generated per stimulus and the brain can manipulate this information to perform the demixing task. It was well documented that visual working memory has a capacity limit (i.e., it can only hold information about a few items); this discrepancy needs to be clarified or addressed.

(2) How well the computational model can explain the experimental data remains not entirely clear

The authors show that there exists a parameter regime that can qualitatively explain the experimental finding. They also show that it is possible to fit the model to the data to explain the bias patterns. However, given that the model is flexible, it would be stronger if the authors could show that the same parameters that explain the biases could also explain other aspects of the behavior, for example, the magnitude of the errors.

In other words, the model is not well constrained in the way it was tested in the paper. But it should be possible to improve it. First, if the noise parameter in the model is determined by the stimulus variability, one can determine it directly based on the external noise in the stimuli (discussed also in 1b) and see what prediction it leads to. Second, from the behavioral data, it may be possible to estimate the noise for the identifying dimension. Doing so will help better constrain the model.

It would also help if the authors could report the best-fitted parameters from the experimental data. From these parameters, one can simulate synthetic data and apply the demixing model to see if the error distribution of the simulated observers is indeed similar to the experimentally measured error distribution. That way, one can check whether the fitted parameter explains the observer's behavioral performance beyond the biases.

Other comments:

(1) How does the model account for the swap errors? I am not sure I understood the way how the swap errors were treated in the paper. To me, substantial swap errors seem to be a consequence of having low d-prime values for the identifying dimension; that is, if there is only little information to discriminate the identity of the two stimuli, swap errors would be large. However, this possibility didn't seem to be mentioned in the paper.

(2) Since the solution of the demixing problem was obtained using a numerical procedure based on EM. It would be useful to check whether the initialization has affected the biases obtained.

Reviewer #2 (Public review):

Summary:

This manuscript investigates the origins of inter-item biases in visual working memory. The authors proposed a computational model where overlapping memory signals are disentangled, inducing memory biases that depend on relative noise levels across items. The key theoretical advance is the prediction that bias direction depends not only on absolute memory noise but on the relative noise levels of target and non-target representations. Using four experiments with color mosaics whose color variability manipulates memory precision, the authors report that biases reverse as a function of relative noise in a manner predicted by the model.

Strengths:

The manuscript is clearly written and theoretically motivated. The experiments are well designed and provide converging evidence for a distinctive and non-intuitive prediction of the proposed model. I found the central result compelling: independently manipulating target and non-target noise leads to qualitatively different bias patterns, consistent with the model's prediction that relative noise is a key determinant of bias direction.

Weaknesses:

The main limitation is that the evidence establishes consistency of the data with the proposed Demixing Model, but does not demonstrate that the model provides a unique explanation of the data. Although the manuscript argues that dominant theories struggle to account for the observed reversals, no formal comparison with alternative computational frameworks is presented. In addition, model fitting results are reported only briefly, making it difficult to evaluate fit quality at the level of individual observers.

Author response:

Reviewer #1 (Public review):

Strengths:

Overall, the computational model tested in the paper is novel and interesting.

The demixing framework represents an appealing hypothesis that deserves further investigation.

The current paper provides new empirical data showing that the target stimuli with the same absolute noise level can be either repelled from or attracted to non-target items, depending on the relative noise levels. The observation that biases depend on the relative noise levels is by itself an interesting one, and is consistent with the prediction of the demixing model.

We are grateful for the positive evaluation of the model and the empirical observations.

Weaknesses:

While this manuscript contains interesting new experimental observations and theoretical ideas, it has several substantial problems in its current form, which limit the conclusions that can be drawn. The description of the computational model is too brief. The key modeling assumptions need to be better motivated and explained. As the computational models generate different predictions in different regimes, it is a bit difficult to evaluate how well the experimental data support the model at a more quantitative level. Also, the results focused on studying the biases in the behavior; it is unclear whether the model can fully explain the behavior data (such as error distributions or behavioral precision).

We agree that the model description should be expanded and that quantitative agreement with the data should be assessed more thoroughly, and we plan to address this during the revision. In the initial version of the manuscript, we aimed to highlight the qualitative agreement of the data with the novel and counterintuitive predictions by the model. While the reviewer is correct that the model "generates different predictions in different regimes," the particular predictions we test (the interaction between the target noise level and the parity of the target and non-target noise levels in Experiments 1-3, and the effects of non-target noise when the target noise is held constant in Experiment 4) hold across regimes (Figure S1 shows this for the former prediction). We aim to further expand on this point in the revision.

Major concerns:

(1) Concerns/suggestions regarding the computational modeling

The current paper seeks to test the predictions of the demixing-based computational model proposed in reference 22. There are several problems with the modeling component in the current paper.

(1a) The description of the model is too brief and difficult to understand. Although the model was proposed in reference 22, it would still be beneficial to provide more details of the model so that readers can understand and appreciate the strengths/limitations of the model.

The generative model and the inference procedure could be better explained to better link the model to the behavior. In particular, how was the observer's behavioral report in each trial modeled? This requires more explanation because currently the demixing procedure estimates four parameters for a given trial, yet for a given trial, only one behavioral report was produced (e.g., current Experiment 1), or two reports were produced sequentially (e.g., current Experiment 2).

We will provide more details about the model and how it was fitted to the data. Please note that the model parameters were fitted per subject and condition, not per trial: 2 hue noise parameters,  and , corresponding to the noise of the target and non-target item across 4 noise combination conditions, plus a shared identifiability noise, , across conditions, determining the discriminability of the items along the identifying dimension. This strongly limits model flexibility as only 3 parameters (including the shared  across conditions) are used to create the bias curve for each subject in each condition.

(1b) Key modeling assumptions need better justification.

One such key assumption is that on a given trial, each stimulus triggers many samples (or approximately, an entire response distribution), rather than a single sample. This assumption deviates substantially from prior work on ideal observer models. It was not clear whether this assumption is realistic. For the type of stimuli used in the current experiments, perhaps one can argue that each pixel corresponds to one sample of brain activity, thus collectively each stimulus should trigger many samples of activity in the brain. If this were to be the case, it would have two implications. First, the noise parameter in the model should be directly related to the magnitude of the stimulus noise. Thus, one should be able to plug these experimentally-controlled parameter values into the model to directly generate predictions about the biases. Second, when using stimuli with no stimulus variability (e.g., simple grating stimuli), the predicted biases should change. However, it wasn't clear whether this would hold experimentally, i.e., using gratings would lead to different biases or no biases.

If the variability of the samples for a given stimulus involves neural noise, it would be useful to justify why it is reasonable to consider that many samples were generated per stimulus.

We are grateful to the reviewer for raising this point, and we will provide more details on it in the revision. In brief, we believe that it is the standard ideal observer assumption of one sample per trial that is unrealistic and works only in cases when there is a single signal source, so that the samples can be simplified to a single average. Consider that determining a stimulus value is a similar problem for an ideal observer to the one that a researcher who aims to decode neural data from populations of neurons (or fMRI voxels) has to solve. Different populations of neurons would provide responses that match different stimuli – in essence, creating different samples in an ideal observer framework. Thus, even without external noise, the demixing problem would be present when there is more than one stimulus, but internal noise is much more difficult to control, so in our experiments, we used multi-colored stimuli.

(1c) As mentioned in (1b), the model assumes that on each trial, a large number of samples was generated. It would be useful to study and report how the prediction would change when the number of samples generated per stimulus is small. In particular, what happens when each stimulus only generates one measurement? This might be useful for interpreting previous experiment results with grating stimuli.

This is an interesting point that we aim to address in the revision.

(1d) Reference 22 studies how the predicted biases depend on the d-prime of the identifying dimension and found that the pattern of the biases varies substantially depending on the information available for the identifying dimension. However, the current paper didn't really discuss this important point. It is also unclear what parameters the authors used for the d-prime of the identifying dimension. Was it fitted directly to the data? The Methods section has some description on the "identifiability dimension", but it was a bit obscure.

Intuitively, when the d-prime of the identifying dimension is very large, the demixing problem becomes irrelevant. In this case, there should not be any biases induced by demixing. In the case of the d-prime for the identifying dimension is 0, the problem should reduce to the simplified 1-d problem studied in reference 22. If my reading of reference 22 was correct, they reported different conclusions. It would be useful to clarify these points.

We are grateful for the suggestion to expand the discussion of this point and will do so in the revision. The reviewer is correct that for very large d-prime in the identifying dimension, the demixing problem solution is trivial. However, the 2D case does not resolve to the 1D case when d-prime reaches zero. This is because the identifying dimension is still used to identify which item to report—unlike in the 1D case, when the reported dimension is the same as the identifying one. Consider what happens if the observer in our task does not remember at all which stimulus was left and which was right. It would report the other item in 50% of cases, leading to a strong attractive bias.

In any case, the d-prime of the identifying dimension appears to be a key parameter. It would be great to constrain this parameter using the empirical data. When the d-prime of the identifying parameter is small, the observer would easily confuse the probed stimulus with the other stimulus in a given trial. This should lead to poor task performance. Thus, it may be possible to directly estimate the value of the d-prime of the identifying dimension based on the observer's performance, and then use this parameter to generate model predictions accordingly.

We apologize for the confusion. We constrain the discriminability of items in the "identifying" dimension using the  parameter that determines the noise in that dimension for both items. The means in this dimension are fixed at an arbitrary value, as means and noise are interchangeable when considering discriminability. We will revise the description of the fitting procedure accordingly. Regarding the use of the same values in predictions, while possible, we prefer to keep predictions separate from fitting to avoid them becoming postdictions. The curves for the fitted model in Figure 2 already illustrate what the model predicts under the fitted parameter values.

(1e) The current model assumes that a large number of samples are generated per stimulus and the brain can manipulate this information to perform the demixing task. It was well documented that visual working memory has a capacity limit (i.e., it can only hold information about a few items); this discrepancy needs to be clarified or addressed.

We are grateful to the reviewer for raising this point, which we will address in the revised discussion. Briefly, we believe that the number of samples in the ideal observer model does not correspond directly to the working memory “slots”.

(2) How well the computational model can explain the experimental data remains not entirely clear

The authors show that there exists a parameter regime that can qualitatively explain the experimental finding. They also show that it is possible to fit the model to the data to explain the bias patterns. However, given that the model is flexible, it would be stronger if the authors could show that the same parameters that explain the biases could also explain other aspects of the behavior, for example, the magnitude of the errors.

It would also help if the authors could report the best-fitted parameters from the experimental data. From these parameters, one can simulate synthetic data and apply the demixing model to see if the error distribution of the simulated observers is indeed similar to the experimentally measured error distribution. That way, one can check whether the fitted parameter explains the observer's behavioral performance beyond the biases.

We are grateful to the reviewer for raising this point. We both agree and disagree with the reviewer here. The predictions reported come from an earlier paper describing the model (ref. 22). In our opinion, this represents a pure hypothesis-driven approach, where a prediction is formulated first and then tested with subsequently collected data. The model we test is normative, not descriptive; its goal is not to fit the data as closely as possible, but rather to make predictions about internal brain mechanisms. We do not suggest, for example, that demixing is the sole source of biases, so the resulting bias pattern might differ significantly from the predictions. That the model fits the data is, therefore, an additional bonus. At the same time, we agree that it is interesting to test whether the model can explain other parameters of the data. Note that our current fitting procedure was not geared toward this; we optimized the model to explain only the bias curve. In the revision, we aim to test whether the model can also explain the error variability.

In other words, the model is not well constrained in the way it was tested in the paper. But it should be possible to improve it. First, if the noise parameter in the model is determined by the stimulus variability, one can determine it directly based on the external noise in the stimuli (discussed also in 1b) and see what prediction it leads to. Second, from the behavioral data, it may be possible to estimate the noise for the identifying dimension. Doing so will help better constrain the model.

External noise accounts for only a portion of the total noise, as evidenced by behavioral errors. Even for a single item, the total noise consists of the amount of information the observer samples from the stimulus, the variability of these samples (external noise), and early (applied to each sample) and late (applied after integration) internal noise. Therefore, external noise alone might not constrain the model in the right regime. Regarding the identifying-dimension noise, as noted above, we do constrain it with the data. However, we aim to explore these points further in the revision.

Other comments:

(1) How does the model account for the swap errors? I am not sure I understood the way how the swap errors were treated in the paper. To me, substantial swap errors seem to be a consequence of having low d-prime values for the identifying dimension; that is, if there is only little information to discriminate the identity of the two stimuli, swap errors would be large. However, this possibility didn't seem to be mentioned in the paper.

We apologize for the confusion. We will further clarify and perhaps reassess the treatment of swap errors in the revision. The model itself produces swap errors when the stimuli sources are misidentified.

(2) Since the solution of the demixing problem was obtained using a numerical procedure based on EM. It would be useful to check whether the initialization has affected the biases obtained.

Indeed, this is a valid point, and it's why we use a multi-initialization strategy. For each simulation of a single trial sample set (e.g., 100 random samples), we use a large number of initial points (50 in the initial submitted manuscript) to ensure the obtained EM solution is truly optimal. Additionally, we conduct a large number of simulated trials (10,000 for each parameter combination) to ensure the accuracy of the bias distribution we obtain.

Reviewer #2 (Public review):

Summary:

This manuscript investigates the origins of inter-item biases in visual working memory. The authors proposed a computational model where overlapping memory signals are disentangled, inducing memory biases that depend on relative noise levels across items. The key theoretical advance is the prediction that bias direction depends not only on absolute memory noise but on the relative noise levels of target and non-target representations. Using four experiments with color mosaics whose color variability manipulates memory precision, the authors report that biases reverse as a function of relative noise in a manner predicted by the model.

Strengths:

The manuscript is clearly written and theoretically motivated. The experiments are well designed and provide converging evidence for a distinctive and non-intuitive prediction of the proposed model. I found the central result compelling: independently manipulating target and non-target noise leads to qualitatively different bias patterns, consistent with the model's prediction that relative noise is a key determinant of bias direction.

We are grateful for the positive evaluation of the model and the empirical observations.

Weaknesses:

The main limitation is that the evidence establishes consistency of the data with the proposed Demixing Model, but does not demonstrate that the model provides a unique explanation of the data. Although the manuscript argues that dominant theories struggle to account for the observed reversals, no formal comparison with alternative computational frameworks is presented. In addition, model fitting results are reported only briefly, making it difficult to evaluate fit quality at the level of individual observers.

We agree and we aim to provide a comparison with alternative models and an expanded description of the fitting results in the revision. Note, however, that the majority of existing models are descriptive, while we believe that as a normative model, the Demixing Model should be compared with other normative models, thus limiting the selection of competitors significantly.

export and add it to ghost

This is good!

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eLife Assessment

This useful study reports on lifespan extension in C. elegans males that carry a mutation in a gene for an insulin receptor; while the observations are striking, the strength of evidence is currently incomplete. The central claim of male-specificity is undermined by the absence of direct hermaphrodite healthspan comparisons, and the reliance on a single mutant allele leaves open the possibility that background mutations, rather than daf-2 loss-of-function, drive the phenotype. Methodological details critical for reproducibility are also lacking, particularly regarding male housing density, censoring of plate-leaving animals, and the adequacy of replication for the key epistasis experiment. The work could be substantially strengthened by a targeted set of additional experiments and fuller engagement with the existing literature on sex-specific aging in C. elegans.

Reviewer #1 (Public review):

Summary:

This paper provides interesting observations about the effects of a classical mutation in the daf-2 insulin-like receptor in male C. elegans. The observations are a contribution in and of themselves; however, the conclusions reached about these observations are not supported by the work presented. Most importantly, male-specific effects on healthspan measures are asserted without direct comparison to hermaphrodites. Perhaps more fundamentally, essential features of the methods and experimental design are lacking, which makes formal assessment of the results impossible, especially given our knowledge of negative male-male interactions, which have gone completely unacknowledged here. Indeed, there is a general lack of context for known sex differences in C. elegans, especially in terms of the core elements of longevity, which are presented here as entirely novel but in fact are not.

Major comments:

(1) The main overall criticism of the premise of the paper is that it lacks a clear hypothesis that would lead to explicit experimental tests. Instead, many of the results are observational, and the conclusions reached go beyond the actual experiments conducted. The goal should be explicit and consistent between the introduction/ discussion, and the findings should directly address the goal.

The overall focus appears to be that daf-2 males have an extended lifespan for reasons that are different from hermaphrodites. This conclusion is apparently based on the observation of lipid reserves in mutant animals. However, none of the healthspan measures were conducted in parallel with identical measures in hermaphrodites. How can the authors then claim that males are unique? This is especially problematic since other studies have demonstrated that daf-2 hermaphrodites also have altered lipid composition (Vrablik 2015 Biochim Biophys Acta; Horikawa 2010 Mol Cell Endocrin).

(2) The authors make unwarranted claims about causation from observational data that is correlative in nature. Again, they claim that male longevity is caused by increased lipid reserves. This may in fact be the case, but there is no evidence to show that this is causal, only that lipid reserves are increased in mutant animals. Causation requires an actual experiment, in this case, disrupting lipid maintenance in daf-2 males (e.g., Lapierre 2013 Autophagy). Their conclusions are consistent with their results, but their conclusions are much too strong given the nature of the evidence, especially given the concerns about proper comparisons to hermaphrodites.

(3) With these concerns in mind, all conclusions related to male-specific effects should be statistically tested using a sex-by-treatment interaction term in the statistical model. This is obviously impossible for the healthspan data, but for lifespan, this can be directly tested using (genotype x sex interaction in the CPH analysis). Further, it is unclear why each of the replicates is shown separately in Figure 1.

It is nice that the authors do not directly pool them, as most longevity studies do, but the replicate effects can be included in a more comprehensive model, which would yield an appropriate "average" effect curve.

(4) There is an inadequate review of pre-existing literature and findings that predate the observations presented here. While this is not an issue in general, the authors present their work as entirely novel when it is not.

In addition to Gems and Riddle (2000), which is tangentially cited in the discussion, the following papers should be cited and discussed in the introduction to clarify what is currently known and what remains to be explored:

Partridge and Gems (2002) Mechanisms of aging: public or private?

McCulloch and Gems (2007) Sex‐specific Effects of the DAF‐12 Steroid Receptor on Aging in Caenorhabditis elegans

Hotzi et al (2018) Sex‐specific regulation of aging in Caenorhabditis elegans

Al-Saadi et al (2025) Disruption of the insulin signaling pathway in C. elegans dramatically increases male longevity and enhances reproductive health late in life

In addition, the authors assert that the study of sex differences is unstudied. If the authors are specifically referring to the sex differences in aging research, they should explicitly state that and revise their language to reflect that it is "understudied" rather than "unstudied". But as stated below, there are many studies that look at sex-specific differences in behavior, physiology, development, etc. This is most important in the context of sexual conflict, of which there are many studies that are directly relevant to the work presented here. The authors are encouraged to review some of these papers.

(5) This is particularly important in the context of how the experiments presented here were actually conducted. The methods are inadequate to assess this, and the results would therefore be impossible to replicate in the absence of additional details. Exactly how many individuals were raised on each plate during the longevity assays (and other work) is critical to understanding the results of this study. This is because males have direct, chemically and physically mediated negative impacts on one another (see many papers from the Brunet and Murphy labs). Further, it is not even clear whether males and hermaphrodites were reared separately from one another. Males are known to leave plates without hermaphrodites, which requires appropriate inclusion of censoring criteria in studies such as these. It is unclear whether and how this was handled. Censoring is an essential feature of any longevity study and so needs to be explicitly described in the statistical methods.

The methods describe the use of heat shock to induce the production of males, but it is unclear which generation is being used here. Ordinarily, males would be induced, and then male populations would be maintained by forced mating (picking to ensure that there is a high relative frequency of males) for several generations to eliminate any carryover effects of the heatshock itself. Were the heatshock males put directly into the longevity assays? If so, were hermaphrodites subject to identical treatment? This is confusing, and a potentially critical confound is not performed correctly.

Reviewer #2 (Public review):

Summary:

The manuscript presents interesting observations regarding the exceptional longevity and improved healthspan of male daf-2 mutants. Given the comparatively limited focus on male aging in C. elegans, the study provides a potentially useful characterization of sex-specific effects associated with reduced IIS signaling.

Strengths:

The 4-fold increase in lifespan of male daf-2 mutants is a striking and unexpected observation. The altered fat metabolism between older daf-2 mutant males and hermaphrodites provides further evidence of sex-specific effects.

Weaknesses:

(1) A major limitation of the current study is that the conclusions rely primarily on a single daf-2 allele. It would strengthen the manuscript to validate at least the major observations using an independent daf-2 allele or through daf-2 RNAi. This is particularly relevant for the proposed male-specific enhancement of longevity and healthspan, as it remains unclear whether the observed effects broadly reflect reduced IIS signaling or may be influenced by allele-specific effects or background mutations.

(2) The methods for male lifespan assays require additional detail. Although the authors state that males were generated and transferred every three days, it is not clear whether males were maintained singly or in groups, how many males were placed per plate, or how many were censored by fleeing. These details are particularly important for male aging assays, as male lifespan in C. elegans is known to be influenced by social interactions. Factors such as population density can affect survival and healthspan measurements. Clarifying these procedures would improve reproducibility and interpretation of the reported male-specific lifespan effects.

(3) Because reduced IIS signaling in daf-2 mutants is known to alter metabolism, physiology, and potentially male-derived signaling, it would be interesting to determine whether the enhanced longevity of daf-2 males is influenced by altered male-male interactions or resistance to male-associated toxicity. In this context, clarification of whether lifespan assays were performed with grouped or individually maintained males would be valuable. If not already tested, lifespan analysis under isolated single-male conditions could help distinguish intrinsic longevity effects from potential contributions of population-dependent signaling or social interactions.

(4) In Figure 2D, the body-length measurements in WT males appear somewhat unexpected, particularly the apparent increase between Day 14 and Day 20. Since adult worms are not typically expected to exhibit substantial growth at advanced ages, additional clarification regarding the measurement methodology would be helpful, including confirmation that the scale bars and image scaling were applied consistently across conditions.

(5) The use of palmitic acid barriers following Beydoun et al. (2024) is appropriate; however, it would be helpful to clarify whether WT and daf-2 males exhibited comparable fleeing behavior under these assay conditions. Because male worms are highly prone to plate leaving and censoring, genotype-dependent differences in fleeing behavior could potentially influence survival analyses and the number of censored animals. In addition, as Beydoun et al. primarily characterized these barrier conditions using hermaphrodites, it would be useful to clarify whether comparable barrier effectiveness was observed in male lifespan assays.

(6) Separately, Beydoun et al. (2024) also reported that palmitic acid barrier conditions can influence body-size measurements, whereas PEG-based barriers did not show similar effects on body size. It would therefore be useful to know whether comparable body-length trends were observed under alternative barrier conditions, particularly given the unexpected increase in WT male body length at later ages.

Reviewer #3 (Public review):

This manuscript reports a striking sex-specific effect of the daf-2(e1370) mutation on C. elegans lifespan. The authors show that male daf-2 mutants exhibit dramatically extended lifespan relative to wild-type males, wild-type hermaphrodites, and daf-2 hermaphrodites. The study also demonstrates increased lipid accumulation in these long-lived males, which is increased further over time, improved late-life motility, enhanced oxidative stress resistance, and a requirement for the downstream effector of daf-2, daf-16, for the longevity phenotype.

The interest of the work is the magnitude and consistency of the lifespan effect. The authors report large increases in both median and mean lifespan in daf-2 males across independently replicated experiments. This is further supported by healthspan analyses and their finding that male daf-2 mutants maintain improved motility and stress resistance, which argues against the interpretation that lifespan extension merely reflects prolonged frailty. The genetic epistasis experiment demonstrating loss of the longevity phenotype in daf-2;daf-16 double mutants provides evidence that the effect depends on canonical insulin/IGF-1 signalling.

The main limitation is that at least the first figure is rather an incremental increase on previous work examining the lifespan of daf-2 males, although the authors do indeed show that the effects can be much larger (or more 'plastic') than those previously published. While these findings are potentially important, the manuscript would certainly benefit from a more extensive discussion of how the results compare with prior studies of daf-2 mutants and male longevity, including possible explanations for the apparent discrepancies.

The epistasis experiment shows that this exceptional longevity requires the expression of daf-16. However, in contrast to the initial experiments (Figure 1) that show three replicates of the lifespan experiment (the standard in lifespan work in this model), it appears that the daf-2;daf-16 experiment has only been performed once.

In addition, the lifespan data for hermaphrodite daf-2 mutants appear somewhat unusual. Although the mean lifespan is increased, the median lifespan is reported to be only modestly greater than that of wild-type hermaphrodites. I know that this mutant can give lifespan curves that look like this, but either the use of another allele or of the experimental conditions and how these values compare with previously published daf-2(e1370) datasets would help readers interpret the magnitude of the male-specific effect.

The lipid phenotype is intriguing. It would be interesting to expand this to examine somatic vs embryonic fat. In addition, I noted that in the methods section, the authors use palmitic acid to stop the male worms 'fleeing' the plates; is it possible to rule out the possibility that the daf-2 mutants are simply eating and metabolising/storing this fatty acid barrier differently than their wild-type counterparts? This would be worth considering and controlling for, particularly as male C. elegans have been shown to have dramatically altered metabolic transcriptional profiles. If indeed this increased lipid is responsible for the extreme longevity of the daf-2 mutant males, it would be desirable to try to link this mechanistically to the phenotype.

Overall, the evidence convincingly supports the conclusion that male daf-2(e1370) mutants are exceptionally long-lived under the conditions tested and that this phenotype requires DAF-16. The work has the potential to make an important contribution to understanding sex-specific regulation of ageing, although further contextualisation within the existing literature would strengthen the manuscript.

CS2 en général

Retirer abandonné pour arbres et arbustes

A préciser

This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giag061), which carries out single-anonymized peer review. These reviews are published under a CC-BY 4.0 license and were as follows:

Reviewer 2：

This study uses Anopheles coluzzii as a model to systematically evaluate preservation methods for generating high-quality genomic and Hi-C data, addressing a question of clear practical relevance. The development of approaches that eliminate the need for cold-chain transport is a particularly important step forward for field-based genomics, where maintaining low temperatures is often logistically difficult. In this context, the work provides useful experimental evidence and practical guidance with broad potential value. That said, several aspects of the manuscript would benefit from further clarification and refinement, as outlined below： 1. The study is conducted exclusively on Anopheles coluzzii under controlled laboratory conditions. While the results are promising, the extent to which these preservation strategies can be applied to other arthropods remains unclear. Given the substantial diversity in cuticle structure, body size, and physiological properties across taxa, the authors should more explicitly discuss the limitations of extrapolating their findings. Inclusion of additional taxa would be ideal; alternatively, a more thorough discussion of potential constraints would improve the manuscript. 2. The evaluation of Hi-C data quality is primarily based on scaffolding performance, including analyses using both high-quality and fragmented assemblies. However, the Hi-C datasets were generated at very high sequencing depth (>100×), which may obscure differences among preservation treatments by compensating for variations in data quality. The authors should clarify how they distinguish genuine preservation effects from depth-related compensation. Additional analyses based on downsampled Hi-C datasets would provide a more realistic assessment of performance under typical sequencing conditions. 3. I am particularly interested in the chromosome anchoring rates observed in the Hi-C scaffolding analyses. It would be valuable for the authors to clarify whether Hi-C data generated from different preservation methods lead to differences in chromosome anchoring efficiency, as this is a key indicator of scaffolding quality. 4. The manuscript indicates that sequencing data are not yet publicly available due to their inclusion in a larger ENA project. In line with journal policies, the authors should provide a clear plan for data release, including expected timelines and accession numbers where possible. Furthermore, additional methodological details, particularly regarding genome assembly parameters and Hi-C data processing, would improve reproducibility and transparency. 5. In Table 1, the inclusion of ULI scaffolding appears somewhat abrupt, as ULI sequencing is not sufficiently introduced or contextualized prior to its presentation in the table. Although its role becomes clearer in the Results section, readers may find it difficult to fully understand its relevance at this stage. The authors may consider introducing ULI sequencing earlier in the Methods or Results, or providing a brief explanation in the table caption, to improve clarity and ensure a more coherent presentation.

This manuscript presents a systematic evaluation of preservation methods for generating high-quality genomic and Hi-C data using Anopheles coluzzii as a model organism. The study addresses an important practical challenge in genomics, particularly for field-based sample collection where optimal preservation conditions are often difficult to achieve. The experimental design is generally well-structured, and the comparison across multiple preservation treatments provides useful insights for the community. However, several aspects of the study require further clarification and improvement. In particular, concerns remain regarding the generalizability of the findings beyond the focal species, the robustness of the statistical analyses, and the interpretation of Hi-C results under very high sequencing coverage. Additionally, issues related to data availability and methodological transparency should be addressed to ensure reproducibility. Addressing these points would substantially strengthen the manuscript. 1.The study is conducted exclusively on Anopheles coluzzii under controlled laboratory conditions. While the results are promising, the applicability of these preservation strategies to other arthropods remains unclear. Given the diversity in cuticle structure, body size, and physiology across taxa, the authors should clarify the extent to which their findings can be generalized. Inclusion of additional taxa or a more explicit discussion of limitations would strengthen the manuscript. 2.The evaluation of Hi-C data quality is based on scaffolding performance, including analyses using both high-quality and fragmented assemblies. However, the Hi-C datasets were generated at very high coverage (>100×), which may mask differences in preservation efficiency. The authors should clarify how they distinguish true preservation effects from sequencing depth-related compensation. Additional analyses using downsampled Hi-C data would provide a more realistic assessment of performance under typical conditions. 3.I am particularly interested in the chromosome anchoring rate of the genome assemblies in the Hi-C scaffolding analysis. It would be valuable for the authors to clarify whether Hi-C data generated using different preservation methods result in differences in chromosome anchoring efficiency. 5. The column headers “Self scaffolding” and “ULI scaffolding” in Table 1 are not sufficiently clear, making it difficult for readers to fully understand the intended meaning.

1. The manuscript indicates that sequencing data are not yet publicly available due to their inclusion in a larger ENA project. In line with journal policies, the authors should provide a clear plan for data release, including expected timelines and accession numbers if available. Furthermore, greater detail in the Methods section—particularly regarding assembly parameters and Hi-C processing—would improve reproducibility.

This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giag061), which carries out single-anonymized peer review. These reviews are published under a CC-BY 4.0 license and were as follows:

Reviewer 1：

This is a straightforward and valuable study. Demonstrating that breaking the cold chain is feasible has strong potential to transform how genomes can be obtained across much of biodiversity. In my view, the broader significance of this advance is somewhat underemphasized in the Introduction (Really just a short mention around lines 72-75). Based on firsthand experience in large field campaigns designed specifically to generate genomes from very small insects, I can attest that much of arthropod biodiversity (especially within small-bodied, hyperdiverse "dark taxa") stands to benefit substantially from approaches like this. The practical importance of scalable, field-friendly preservation solutions cannot be overstated.

Overall, the study is well designed, clearly presented, and addresses a real logistical bottleneck in biodiversity genomics. The conclusions are generally well supported by the data presented. I have a few suggestions that I believe would further strengthen the manuscript and increase its practical value and reproducibility.

Major / Substantive Comments

1. Framing and impact As above, I recommend strengthening the Introduction's framing of downstream biodiversity applications, especially for small-bodied and taxonomically challenging groups where cold-chain logistics are often the primary limiting factor. The method has particularly high relevance for large-scale efforts targeting hyperdiverse insect groups ("dark taxa"), and this applied significance could be more explicitly highlighted to broaden the paper's audience and appeal.

2. Practical protocol clarity and reproducibility Because this method is likely to be adopted by field teams, including non-specialists, it would be very helpful to include a concise protocol-style summary or workflow outlining the recommended handling steps, specimen size considerations, and timing constraints. A short practical guide or decision framework would improve reproducibility and uptake.

Relatedly, laying out any known tolerance ranges in this fashion would strengthen the paper, for example, how sensitive outcomes are to delays in processing, temperature variation, or specimen size differences under field conditions.

1. Preservation benchmarking I suggest including a summary comparison (table or figure) of yield/quality metrics across preservation treatments relative to standard cold-chain approaches. This would make performance differences easier for readers to interpret and apply.

2. Voucher integrity and downstream taxonomic usability Given that many target organisms will come from taxonomically difficult groups, a short discussion of voucher integrity after treatment would be valuable. Guidance on expected morphological preservation and suitability for downstream taxonomic work would significantly increase the method's usefulness for specimen-based biodiversity genomics workflows.

Methodological Clarification

The instruction to "lightly squish" specimens to compromise the cuticle is practical, but could benefit from additional detail. Does the location of compression affect outcomes? For example, is thoracic compression preferable (to access muscle tissue), or is abdominal disruption sufficient? This may seem like a fine detail, but it matters in practice, particularly because damage to thoracic characters or terminalia can reduce taxonomic value. If there is an optimal or recommended compression location, it would be useful to specify it.

This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giag059), which carries out single-anonymized peer review. These reviews are published under a CC-BY 4.0 license and were as follows:

Reviewer 2：

This study presents an analysis of gene co-expression modules in Alzheimer's disease (AD) and Parkinson's disease (PD), utilizing a pattern learning framework predicated on single-nucleus RNA sequencing data. The research uncovered shared molecular mechanisms between the two diseases within neurons, microglia, oligodendrocytes, and astrocytes. These include cytoskeletal dynamics, mitochondrial stress responses, T cell activation processes, dysregulated myelin synthesis pathways, and abnormal heavy metal handling mechanisms. These findings illustrate common genetic underpinnings of AD and PD in specific cellular contexts, thereby providing novel insights into their pathological overlaps. 1. It is recommended that the author allocate a specific amount of space in the introduction section to elucidating the relationship between neurological disorders and other types of diseases. 2. Detailed validation results using independent AD and PD datasets should be presented in the paper to enhance the credibility of the study. 3. The Methods section should provide a more comprehensive description of the statistical methods used for batch effect correction and control of potential confounding factors. 4. To strengthen the biological relevance of the findings, functional experiments such as in vitro cell assays or animal models should be performed to validate the roles of identified key gene modules in the pathogenesis of AD and PD. 5. The discussion section should further elaborate on the clinical implications of these shared molecular mechanisms, including their potential as therapeutic targets or biomarkers.

This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giag059), which carries out single-anonymized peer review. These reviews are published under a CC-BY 4.0 license and were as follows:

Reviewer 1：

The authors utilized single-nuclei RNA-seq data and GWAS data to study the disease comorbidity between Alzheimer's disease and Parkinson's disease. They also validated their discoveries using independent evaluation cohorts. This is an overall well-conducted and comprehensive study with a solid logical framework and clear presentation of very interesting biological results. I have few points that need clarifications.

1.In section 'Common Mechanisms of microglia's involvement in AD and PD', lines 790-792, the authors mentioned that 'we noted T cell activation in both AD and PD (all 4 datasets).' This is very interesting biological discovery. Based on my knowledge, the amount of T cells extracted from brain snRNA-seq are quite small (I could be wrong). Can authors explain something that could prove the conclusion is not biased due to the small population of T cells?

1. The authors mentioned using PLS-DA method to extract disease modules for AD and PD datasets respectively. if I understand correctly, that the authors apply PLS-DA method to snRNA-seq data instead of for example pseudobulk aggregated for each cell type. Can the authors explain and add some logics in the method section to further clarify why applying PLS-DA to snRNA-seq make sense? As we know snRNA-seq data are quite sparse.

2. In Figure 4D, authors presented multiple gene modules, can the authors also add gene/protein symbols as well, not just circles? Readers can know directly what are related proteins.

3. In the method section, about the 'Differential gene expression', it seems to me that the authors do not mention covariates adjustments when computing DEGs, so does authors consider any covariates for DEG? for example, sex or PMI?

This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giag064), which carries out single-anonymized peer review. These reviews are published under a CC-BY 4.0 license and were as follows:

Reviewer 3：

In the manuscript entitled "4D Single-Cell Spatial Transcriptomics Reveals Dynamic Morphogenetic Gradients and Regenerative Domains in Planarians", Han, Chen, Li et al. use high-resolution Stereo-seq on regenerating planarians to reconstruct a 4D spatiotemporal transcriptomic map of planarian regeneration. In their analysis, they recognize most of the cell types identified in planarians and are able to recover the gene expression dynamics during regeneration of the body axes at the cellular, tissue, and organismal scales. One of the main findings is the identification of injury-induced spatial domains, specifically the Anterior Regenerative Zone (ARZ). Interestingly, the ARZ is enriched in positional control gene expression in several cell types, not only in muscle. The authors identify Mediator 8 as a gene expressed in the ARZ and required for proper blastema formation. The study also provides an interactive web portal with the corresponding data. The analysis of the regeneration process in planarians using Stereo-seq provides a new and very useful strategy to understand the dynamics of gene expression integrated with cell and tissue types. Through this strategy, the authors corroborate the expression patterns of several genes already described as essential for planarian regeneration, and they identify new blastema regions comprising different expression patterns, both anterior and posterior. Among them, the study focuses on the ARZ and performs trajectory analysis, which provides a very informative view of the cellular movements and changes that occur at early stages of regeneration. The finding that Med8 is required to initiate regeneration in both wounds validates the utility of the strategy followed. The publication of an open and interactive web portal with the dataset will be a useful tool for the planarian community and for research on regenerative processes in general. However, in its present form, the study presents several weaknesses and issues that should be addressed before publication.

Main concerns:

The 3 main concerns are 1) the presentation of the strategy of each analysis performed is not detailed and not clear enough; 2) essential data for the present manuscript is supposedly found it Han et al. submitted, when it should be available in the present manuscript; and 3) the conclusions from the functional analysis of med8 are not accurate.

Regarding concern 1: - The authors explain that they analyzed 16 animals (2 per time point) processed into 10 μm thick sections, producing a total of 353 sections. However, they do not specify whether the same number of sections were analyzed per animal, nor do they indicate the total size (or thickness) and cell number of the animals analyzed. In this regard, it may be that the number of sections analyzed per animal is shown in Figure S2A (this is not clear from the figure legend or the text). If so, why is there variation in the number of sections per animal? Is it due to differences in animal size? If so, how were these differences addressed in order to integrate the data from different animals? - In Figure 1F, the different parts of the blastema are divided according to the pigmented/unpigmented area. What were the criteria used to divide each blastema into three parts (proximal, middle, and distal)? Gene expression? Length of the region? This should be clarified. - A schematic overview of each strategy used to obtain the results would help the reader understand the procedures.

Regarding concern 2: - Throughout the study, the authors refer to Han et al., submitted for the custom framework used to create the atlas. Since that study is not currently available, this important information is missing from the current manuscript. - Similarly, from the section "Spatiotemporal dynamics of positional gradients during whole-body regeneration" onward, the study relies on the so-called SBGs (spatially biased genes), also described in Han et al., submitted, which is not published. This is very important data that, if not published elsewhere, must be included in the current manuscript. A description of how the SBGs were obtained, together with a list and explanation, is required. Otherwise, essential information about the SBGs—on which the subsequent analyses depend—is entirely missing. - According to the authors, PCGs are a subset of SBGs. What is the essential difference between PCGs and SBGs?

Regarding concern 3: - Functional analysis of med8. The authors refer to a role of this gene in polarity remodeling. First, this is an unclear concept, because polarity establishment and polarity remodeling are two different processes during regeneration. Second, the RNAi results presented do not support a role for med8 in polarity. The phenotype suggests that med8 is required for cell-fate specification during early stages of regeneration, since neoblasts increase but differentiated cells decrease in general. However, the results do not support a role for med8 in pole formation. The authors report a decrease in sfrp-1, but only at later stages, and no data are shown for notum. The conclusion that med8 regulates blastema growth and positional information is therefore not accurate and does not align with the results presented.

Additional main concerns: - A spatiotemporal transcriptomic atlas of planarian regeneration was already published in Cui et al. 2023. Although the authors cite it in the introduction, they should also compare their results with those published previously. Do they observe similar cell types and gene expression dynamics at the time points analyzed? This comparison should appear in both the Results and Discussion sections. - The authors state that the different clusters of SBGs fluctuate during regeneration and then stabilize. First, which genes belong to each cluster? This information should be included. Second, according to Figure 2A, some clusters fluctuate (e.g., A1 and A2) but others do not (e.g., A5 or M9). A more accurate interpretation is required. Furthermore, the starting point of the analysis is t0, when head and tail are missing, and the endpoint is 14 days of regeneration. How can one assess whether gene expression stabilizes if the starting and ending states are completely different? Importantly, in Figure 2A, the cluster labels in the image do not correspond to the bars, and there are 15 bars but 16 clusters. The figure should be corrected. The colormap labels are also missing. - The authors find that predictions from the Gierer-Meinhardt model are consistent with the expression of some genes (ARNT, Ndk, EGR1…). First, a description and reference for these genes are required. Second, what about other genes involved in Wnt signaling and AP patterning, previously proposed to follow this model in Werner et al. 2015? Third, how can transcription factors such as Hox4b follow the model if they are not secreted? - In general, the manuscript does not refer to specific PCGs known to be critical for regeneration and patterning, such as notum and wnt1. Is this because they could not be identified in the dataset? If so, is it not possible to perform FISH and integrate these results with the Stereo-seq data?

Additional issues to be addressed:

• Lines 161-163: Why do the authors conclude that the increase in dorsal epidermal progenitors, neural progenitors, and pharyngeal lineages indicates an active wound response or the generation of new tissue? This statement is not clear.
• The 4D atlas annotates 36 refined cell types, which is a noteworthy result when compared with published scRNA-seq databases. The authors should compare their results with scRNA-seq in terms of the number and identity of cell types.
• In Figure 1F, the blastema is divided according to the pigmented/unpigmented region. However, in Figure 1G the dividing lines do not follow this curvature. Should they not follow the pigment curvature as well? Additionally, why do the blastemas in Figure 1G show such pronounced lateral deviation? Is this because the incisions were not perpendicular to the AP axis? If so, with this variability, it is difficult to understand how the datasets could be integrated. A detailed explanation is required.
• Lines 202-203: "To test this, we analyzed the spatiotemporal patterns of SBGs by mapping representative samples from each time point onto clusters along the A/P axis using logistic regression (Fig. 2A)." What is meant by "representative samples"? The procedure should be specified more clearly.
• Data S4 is not mentioned in the text.
• smed03831, caveolin3, and smed01640 are the genes enriched in the ARZ domain. However, the authors perform functional analysis only with med8. Is there any specific reason for this?

This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giag064), which carries out single-anonymized peer review. These reviews are published under a CC-BY 4.0 license and were as follows:

Reviewer 2：

In the manuscript '4D single-cell spatial transcriptomics reveals dynamic morphogenetic gradients and regenerative domains in planarians,' Han and colleagues generate a truly stunning spatial transcriptomics dataset of planarian regeneration from the species Schmidtea mediterranea. The authors' dataset includes whole 3D reconstructions of two regenerating planarian fragments at 8 different timepoints during regeneration, a fantastic accomplishment and resource of broad interest to the regenerative biology community. The authors analysis of the dataset includes characterization of spatially biased genes (SBGs) and exploration of an anterior regenerative zone (ARZ) and the role of the gene med8 in its' regulation. While the authors' dataset is remarkable and their analysis of spatially biased genes and med8 function is interesting, I'm not yet convinced that their conclusions are fully tested by the included experiments. In addition, I think that the authors have not included sufficient quality control metrics for their spatial dataset, which makes determining the limitations or caveats of their analysis and conclusions more difficult. However, my concerns could be addressed by additional analysis and minor experiments, or by softening the conclusions of the authors to include alternative models. I've detailed the areas of analysis/discussion that I believe require improvement below:

Major Criticisms: 1. Stereo-seq resolution and capture efficiency: The authors assert that their spatial approach is high enough resolution to resolve cell types and they claim to have characterized 36 cell types in their abstract. However, the 'cell type' in their dataset that they choose to focus on - Clu.31 - has gene markers expressed in three different cell types that have been shown to be distinct in the literature and prior planarian atlases. The authors should analyze gene expression signatures of other stereo-seq 'cell types' to determine if they also show mixed expression signatures. In addition, I am curious if stereo-seq is more likely to capture highly expressed genes (like those expressed in parenchymal cell types) than more lowly expressed genes (like the transcription factors expressed in stem cells). If it exists, this bias could influence annotation of cell types in highly heterogeneous regions of the worm like the parenchyma or parapharyngeal region. Finally, there is very little QC data in the supplementary materials (Size/volume of segmented cells, UMIs and features per cell, variability in features/UMIs per section, per replicate, and per cell type, etc.) I think this analysis would be highly valuable for the reader to interpret the data and the 36 identified 'cell types'.

1. Dynamics of spatially biased genes: The authors analysis on the dynamics of spatially biased genes (SBGs) is very interesting, but the 'oscillations' the authors referred to were not clear to me in the data across all or even most of the pattern clusters in Figure 2A. In general, it seemed more like the pattern cluster was 'noisy' or more broad before stabilizing to its final location. In addition, the PCA analysis in Figure 2B seems to show that Intact and 14dpa transcriptomics is very similar, but 0h, 12h, and 36h timepoints are very distinct from 3, 5, 7, and 10 day fragments. This would suggest that early wound response gene expression is highly distinct (even opposing) the gene expression programs active during late in regeneration. More exploration of this idea, as well as clarified language on exactly what the author means by 'oscillations' and which gene groups follow this pattern would greatly improve this section and better support the author's conclusions.

2. The Cellular/Functional identity of Clu.31: The authors state throughout the manuscript that Clu.31 (the ARZ) is an injury-induced anterior state enriched for SBGs and regulating polarity establishment. However, it is also possible that this spatial state represents the anterior peripheral nervous system (numerous sensory neurons and surface epithelial cells that help sense mechanical and chemical cues). SBGs could be enriched because this combination of cell types is only present in the anterior of the animal. Indeed, the authors show that the ARZ is localized to the anterior in intact animals in the absence of an injury (Figure 3) and enriched genes (S4Aii) strongly indicate that Clu.31 contains gabrg+ mechanosensory neurons. If Clu.31 is regenerating nervous system, this would also explain its ventral bias and expression of tgs-1 and other nb2 genes, since nb2 neoblasts have been suggested to be both an amputation responsive neoblast subset (Zeng et al. Cell) and a neural progenitor state (Raz et al. Cell Stem Cell). Clarifying how the composition of the tri-lineage region changes during regeneration may help distinguish if Clu.31 is truly an injury induced region vs. the regenerating sensory nervous system. For example, it is known that agat-1+ cells transcriptionally responsive and enriched at the wound site a 2-4 days post amputation, but less so at later timepoints (Benham-Pyle et al Nature Cell Biology, Kent et al. Developmental Biology). This shift in composition should be observable in Clu.31 since it contains agat+ epidermal cells. Such a shift in composition or the identification of a regeneration-specific marker expressed in Clu.31 would add support to the author's conclusions. Regardless of the outcome of these experiments/analyses, the discussion and interpretation of the data could be modified to address the hypothesis that Clu.31 represents the cellular neighborhood created when the peripheral nervous system intercalates with the anterior DV boundary epithelium and body wall muscle, which needs to be regenerated in amputated worms. As is, the comparison to the apical epithelial cap considered in the discussion (Line 438) may be pre-mature.

3. Med8 function: Med8 produces a clear phenotype in the authors' experiments, and their data indicates that it is required for ARZ formation. However, I am not sure that the authors data supports the claim that Med8 is directly regulating blastema and PCG expression, as opposed to regeneration of the nervous system (which is highly interconnected with formation of the anterior pole and the size of the anterior blastema) and stem cell function more broadly. The fact that Med8 RNAi also leads to head degeneration in intact worms (Figure S6F) strongly suggests a more fundamental defect in neural differentiation or stem cell function. The strongest evidence presented by the authors supporting a broader function in polarity establishment is the disruption of posterior Wnt expression, (Figure 5F and G), but these in situs are single representative images with no quantitation and could also be explained by a stem cell defect. Additional data could be provided (e.g. visualization of wound-induced gene expression, quantitation of anterior or posterior stem cell numbers and proliferation rates at 2dpa) to support regulation of PCGs or blastema formation. The authors could also leverage their single cell sequencing to determine if Med8 RNAi impacts neural progenitor abundance more than other progenitor cell types. Together, these experiments would determine if Med8 is important for amputation-induced blastema formation and polarity re-establishment vs. stem cell function and neural differentiation more broadly.

Minor Criticism/Feedback: 1. In Figure 1I, the authors show DEGs enriched in each cluster/region. In the blastema regions, I was surprised by the number of DEGs for each time point. It appears that there are ~10K upregulated and 10K downregulated DEGs by the later time points, which suggests that 2/3 of the transcriptome is differentially expressed… The authors should clarify in the text or methods what cutoff they used for the DEGs and how significant the DEGs are in this figure. 2. For readability, I really think that all figures should be on a white background. 3. How do gene expression profiles from the stereo-seq compare to bulk rnaseq at similar timepoints? 4. It is very interesting that there are some cell types that appear to contract and then expand during regeneration (Cluster 0, 23) or that aggregate/become more targeted during regeneration (pharynx pouch, cluster 29). Molecular differences between early and late cells within these cell types would be particularly interesting for understanding different phases of regeneration, but this may be beyond the scope of the current study. 5. The authors frequently reference Han et al. submitted, but this manuscript would need to be pre-printed or published in order for this work to reference it. 6. The Y axis of Figure 2E should be labeled

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