Comment by actualham: Yes. This.

Comment by chrisaldrich: This explanation also highlights an additional idea of open itself. I have heard many in the W3C space criticize the open standard of web annotation arrived at because of the ultimate monoculture of the space. Most of the participants of the process were all related to Hypothes.is in some way and the result was a single product that implemented the standard. To my knowledge no other companies, groups, or individual programmers have separately implemented the standard.

In this sense, while the "standard" is openly defined, it isn't as open as other standards which were mote slowly evolved and implemented gradually and more broadly by various programming languages and disparate groups.

Comment by BMBOD: I think there are particular personal epistemological assumptions tied up in this, that impact not only how we wish web annotation to be used, but how it functionally can and will primarily be used. If you approach knowledge as something coming from an authority, it is very hard to fathom being able to create it yourself, or talk back to it, even if those platforms exist. Conversely, if you think any opinion is valid, because knowledge is completely subjected as individual "truths" then I think you end up with what we see in a majority of places on the internet that allow discourse... I wonder if, and suspect that, hypothes.is could a powerful tool in shifting personal epistemology - especially where the text creators or "authorities" engage with annotators and the comments they pose...

...forgive me, I bring everything back to personal epistemologies

Comment by BMBOD: As always, so glad you did.

Comment by chrisaldrich: One of the pieces missing from Hypothes.is is the curateable notebook which more easily allows one to create new content from one's annotations.

Search is certain there, but being able to move the pieces about and re-synthesize them into new emergent pieces is the second necessary step.

Comment by chrisaldrich: Not to mention the fact that the experiment will continue on into the future past the official publication.

Comment by chrisaldrich: I personally identify with this since I'm porting my annotations and thoughts to a notebook as part of a process for active thinking, revision, writing, and eventual publication.

Comment by actualham: Interesting phrase. Thinking about how all spaces are social (I think?) and all are built (I think?), and so what exactly makes the sociality or structure of a space "open"? Could it have to do not just with inclusivity, but also with the decentering effect that resists boundaries and borders-- and maybe therefore meaning? Invoking Homi Bhabha here in thinking about how margins define centers (not vice versa) and wondering if part of what feels politically important about web annotation is the way that it decenters the text itself, makes it incomplete, multi-authored, dynamic. And that will both challenge master-narratives and maybe also challenge meaning itself, which can be alienating to participants and a challenge to community building. Sorry-- I used to teach Intro Lit Crit. :)

Comment by chrisaldrich: There's something discordant here in a scholarly article about having academic participants with names like SenorG and actualham. It's almost like a 70's farce starring truckers with bizarre CB handles. It's even more bizarre since I know some of the researchers behind these screennames.

Is the pseudonymous nature of some of these handles useful in hiding the identity of the participants and thereby forcing one to grapple only with their ideas and not the personas, histories and contexts behind them?

Comment by chrisaldrich: Something about this is reminiscent of WordPress' mission to democratize publishing. We can also compare it to Facebook whose (stated) mission is to connect people, while it's actual mission is to make money by seemingly radicalizing people to the extremes of our political spectrum.

This highlights the fact that while many may look at content moderation on platforms like Facebook as removing their voices or deplatforming them in the case of people like Donald J. Trump or Alex Jones as an anti-democratic move. In fact it is not. Because of Facebooks active move to accelerate extreme ideas by pushing them algorithmically, they are actively be un-democratic. Democratic behavior on Facebook would look like one voice, one account and reach only commensurate with that person's standing in real life. Instead, the algorithmic timeline gives far outsized influence and reach to some of the most extreme voices on the platform. This is patently un-democratic.

Comment by BMBOD: What sort of digital literacy does this require?

Comment by SenorG: Just as there is ambiguity around defining a word as simple as open this conversation hints at an emerging struggle with a shared meaning of annotation. As Remi points out, the traditional view of annotation is as a tool acting in service of reading comprehension. What we are talking about goes beyond that.

While Merriam Webster and other disseminators of meaning can add extra definitions to their dictionaries under the word 'annotation', I wonder if an easier path to meaning making might be through using different words. There's a risk that it only leads to complications and muddying the waters over semantics but is it worth considering? I describe it as uptexting (not sure if I thought of that on my own or borrowed from someone else). I've heard University of Texas' Carl Blyth describe this as Social Reading and am drawn to this term because it captures the community aspect and might be less confusing because it doesn't seek to reappropriate an existing, commonly held definition.

Comment by onewheeljoe: In that way, using a digital, sharable sticky note matters. Anyone can make a mess with sticky notes and a more skilled respondent can support another's meaning making with sticky notes.

Comment by onewheeljoe: What do annotations in an edited volume of Shakespeare communicate to a struggling 9th grade reader? It strikes me that reader-text interactions always leave meaning negotiable, messy and interaction dependent.

Does this question attempt to rubric-ize the notes we'd put in margins?

Comment by onewheeljoe: This question seems like it should be more specific to disciplines. What is annotation in the legal world? How about for scientists? For beginning readers?

If I'm annotating a text to make meaning, that's different than if I'm a prof annotating a historical text to provide relevant background. The two notes have only their "noteness" in common, I'd say.

Comment by onewheeljoe: Neatness matters for teachers who have to keep track of the artifacts students create as writers. If my students are doing great work but I can't see it, I'm disorganized as a teacher. In the online instructional space it is even more important that teachers can see a footprint. If a tool leaves it to chance whether a student's work will be found by the teacher or a stranger, it is a messy tool, from a teacher's perspective.

Comment by SenorG: Allow me to push back a bit here. While the "canned responses" could, in some environments, build fences around student creativity and expression, it does not have to. From my limited play in the Ponder sandbox, I noticed that students could click on the canned responses OR offer their own annotation just like with Hypothes.is (though I am not sure if the annotation is limited to text). Also, I perceive the canned responses as allowing for scaffolding for younger readers and second language learners.

Comment by silvertwin: Does this, then, also do something to disrupt the discourse of 'impact' as currently articulated / imposed by REF etc?

Comment by Whippo: It may be a challenge, but it shouldn't be very hard. The hypothe.is R package is a good start in creating a data science workflow. This workflow would have a clear advantage in higher education... especially in academic disciplines that use R.

Comment by onewheeljoe: I'd be interested to know how you test this theory, or hypothesis. What does user feedback look like? How is it analyzed?

Comment by actualham: I know I am basically just another lit crit perspective here, but honestly that background-- particularly as a poststructuralist and probably as a postmodernist--inflects so much of my thinking about what the web is and can be. I think moving from English to Interdisciplinary Studies has also made me value the perspective that sees all knowledge as always incomplete; I love that my new field has a core value of noting that there is always another perspective, even if it's not visible or known yet. So when I work with students on the web-- especially in "intro" courses that are supposed to indoctrinate them to the core principles and theories of Interdisciplinary Studies-- I like to present the web as a space that allows and supports dynamism rather than stasis (process over product). But this is so out of line with so much about how teachers think about "public" writing and projects; we want them to be "portfolio" worthy, tidy, complete. When student work is flawed, I think it's a reminder to us about how we can think of all scholarly work. I love that H lets us focus on critique without a requirement that we devalue the work-- in fact, quite the opposite (we critique what has value and potential and impact and utility...). Just thinking out loud, but I think this aspect of "open-endedness" is really the core (ha ha-- irony) of so many of my areas of interest right now.

Comment by BMBOD: oh right, authoritative scholarly voice... guess I should leave out all those "I think"s... ;)

now I am just adding noise; this can be deleted.

Comment by actualham: Thinking about this term, and about preceding it with "everyday." Wondering how one becomes this, whether it can ever be mundane without erasing the privilege inherent in the status.

Comment by ndsteinmetz: An important establishment in learning from text. How often we presume the author to be the authority. It's important to be open and willing to listen to the ideas of others if we are really seeking expertise. Growing from feedback and criticism is one of my greatest achievements.

Comment by ndsteinmetz: It is my hope to see this in all learning environments, too often it is pre-established or determined without respect to learners' needs and interests.

Comment by BMBOD: Are there standards for citing web annotations? How do we acknowledge and credit this shared authorship?

Comment by BMBOD: +1

Comment by actualham: So much of this resonates after reading Martin Weller's wonderful little post today (and the awesome PPT embedded therein): http://blog.edtechie.net/openness/the-paradoxes-of-open-scholarship/

Comment by onewheeljoe: Great question because it shows how our language evolves as we learn in much the same way we do.

Comment by BMBOD: I think this is such a powerful motivation for using web annotation as a component of peer-review and academic conversations.

Comment by BMBOD: I love the idea of the public playground, and I think that concept along with the affordances of hypothes.is, say something about the relative safety net of open annotation. Like a public playground, it is not without risk to those participating; however, a degree of anonymity is still offered. You can disconnect your hypothes.is user information from your identity. This can, of course, be both a good and bad thing in generating commentary, but is an important feature of this particular "openness"

I also just love the connection to "play"

Comment by onewheeljoe: Teachers ask for textbooks all the time and insist that student's online work stay inside a walled garden LMS. Profs, too. Empathic listening reveals that they are not novices but professionals with legitimate concerns.

Comment by jeremydean: Burn!

Comment by onewheeljoe: Agreed that tone matters. Open standards are real things but the word open has been evolving since it was first uttered and it predates Linux and the Apache server.

Comment by ndsteinmetz: RK is not the only one, I'm still feeling very new as well and learning each time I annotate. The newness is sometimes intimidating but I proceed nonetheless. How might it become more user friendly and inviting to grow the audience and participants?

Comment by onewheeljoe: This is a conversation I heard as I worked with Karen Fasimpaur on various projects beginning with P2PU. She used to hold to a rigid conception of the word open that prevails in web design communities, before accepting more nuanced definitions of the word as she worked more with learners in open spaces.

Comment by ndsteinmetz: Great point here, when can I use Hypothes.is on my mobile device? I'm falling behind due to the need of being at a keyboarded laptop. How might be promote equitable access to such great tools?

Comment by otterscotter:

Comment by BMBOD: Could we perhaps use tags or groups to functionally sort through the layers of "noise" ? Perhaps things like: content critique, meta, grammatical nuances, etc?

Comment by SenorG: This term is new to me. It really catches my attention while at the same time making me wonder whether there is such a thing as a published text that is not performative. Is the act of publishing - even if as an annotation in the margins of a niche academic journal where it is unlikely to ever be discovered by another set of eyes - inherently performative, regardless of author intent and potentiality for audience?

Comment by amidont: There is, truly, so much potential in these tools and approaches toward asynchronous, distributed reading and writing. One question I have, already, is how such distributed forms of production-consumption further dissolve notions of textuality and authorship so entrenched within traditional notions and practices of scholarship and empirical research. The flattened hierarchies, especially, threaten the institutionalized power structures which have tightly controlled the design, review, and dissemination of scholarship and research.

Comment by onewheeljoe: The analytics of this article as inquiry are to some degree plain to interested readers. If a reader wants to test out the hypothesis that the conversation will be "interrupted," all they have to do is check the margins. I'm curious about the choice of the word interrupted, tho. Won't bookworms in these margins build on the conversation, the way kids in a sandbox build with what they find? Do annotations interrupt or do they make plain the reader-text interactions?

Comment by BMBOD: Interrupted seems like such a harsh word here. Perhaps punctuated fits better? You don't have to interrupt reading the conversation with the annotations, but you can. Of course in a journal of disruptive media, maybe interruption is exactly the disruption desired...

Comment by onewheeljoe: This reminds me of Paul Allison's LRNG playlist in which youth have to choose keywords associated with their own inquiry questions.

Comment by silvertwin: General point - I've been asked to join in this dialogue but it is already very rich and substantial so opting to add only a couple of comments rather than reinvent the wheel.

Comment by bali: Never seen this word before

Comment by Gary_Hall: The oldest university in Europe is the University of Bologna. It was founded in 1088. Isn't this before the Enlightenment?

Comment by bali: Small point. Rose et al's italics or yours, Adam? If Adam then you should claim that after the citation, i think

Comment by Gary_Hall: Isn't Matthew Fuller working on this kind of grey literature? Might be worth checking out.

Comment by bali: That's very interesting. Could you give examples of how resistance might be represented or portrayed in the real building vs the architectural representation of it before it exists physically? Are we talking graffiti? People using space in ways it wasn't intended for?

Comment by bali: Wow, I love this point! Making me think in many directions!

Comment by bali: This paragraph seems to be coming out of nowhere. Like it isn't clear why we are now talking about your labor and mechanisation. But I am sure you have a point there and a reason why you inserted this here. But you need to help the reader see that connection.

Comment by Gary_Hall: Is the bureaucratic control you refer to really as total as you seem to be suggesting?

Comment by bali: I would clarify to readers what is different or special about CGI renderings here

Comment by bali: Again, really liking how you are making this point explicit

Comment by Gary_Hall: This is interesting. Can you say more about how the ideas of these undergraduates etc. constitute unrepresented knowledges?

Comment by Gary_Hall: Is it just capital? Is there nothing in your work that escapes capture by capital?

Comment by bali: You may want to expand on these points. The multidisciplinary nature of your writing means that most of it is understandable to people from a critical stance (so far) but some terms will be completely new. To me, dreamwork is completely new.

Comment by Gary_Hall: This is one understand of what a university is. But there are others. For example, the UK government's emphasis on 'impact' is surely about making universities produce knowledge that is very tangible and instrumental-- certainly enough for it to be capable of being measured. Hence also the REF. Do you need to articulate which understanding of the university you are working with here?

Comment by postoccupant: There is a huge architectural metaphor I have yet to make the most of - if later I draw on the caricature of contemporary attitudes to research as the accumulation of factual 'bricks' (Bernard Forscher (1962) quoted in Gary Rolfe, see references) and oppose it to Bill Readings' notion that the University is in ruins, and that this is something that can be seen as productive, against attempts to aestheticize these ruins. Which is something the CGI University definitely attempts to do! He writes about 'the University as a space for a ... structurally incomplete practice of thought... (I)t is imperative to accept that the University cannot be understood as the natural or historically necessary receptacle for such activities, that we need to recognize the University as a ruined institution, one that has lost its historical raison d'etre. At the same time, the University has, in its modern form, shared modernity's paradoxical attraction to the idea of the ruin, which means that considerable vigilance is required in disentangling this ruined status from a tradition of metaphysics that seeks to re-unify those ruins, either practically or aesthetically.' (Readings, p. 19) CGI as the continuation of metaphysics by other means? Much to do here...

Comment by Gary_Hall: Do you need to establish that the university is a space designed to contain absolute uncertainty.? At the moment you are just asserting that it is. Don't you need to make a persuasive argument to this effect?

Comment by bali: Love this question! I had never thought of it before.

Comment by Gary_Hall: OHP used to, during the initial five years of our OHP monograph project, which began in 2009. We are not working with Michigan now, though.

Comment by Gary_Hall: This section on open access seems almost to be positioned as a solution to some of the problems discussed previously in relation to Elsevier. Yet can't Elsevier in some respects be understood as one of the largest of open access publishers? (For more, see the recent discussion of 'Elsevier As An Open Access Publisher' on the GOAL email list.)

Comment by samoore_: This is related to the point above. Harvard has an endowment of $35.7 billion. (http://www.hmc.harvard.edu/investment-management/performance-history.html.) and can clearly afford all the resources it needs.

The reason I say this is because such institutions may be in a position to take the publishing process in-house, away from large corporations, and compensate the editorial labour mentioned above.

Comment by samoore_: While I agree that they have already seen the writing on the wall for the subscription system, they have rapidly moved into APC-based publishing and ‘vertical integration’ of its products by trying to access the data for all aspects of the publishing process (see their purchase of SSRN for example). It’s the data that is most valuable here and allows them to sell services to universities and other institutions.

Comment by samoore_: It might be worth briefly summarising Schmitt's argument here.

Comment by samoore_: In fact, the pull of high-reputation is so strong that roughly a quarter of those who do sign the boycott still end up publishing in an Elsevier journal http://journal.frontiersin.org/article/10.3389/frma.2016.00007/full

Comment by Gary_Hall: I'm not sure about this. One of the requests we often get at Open Humanities Press, for example, concerns whether we can provide people who want to start an open access journal with free or at least affordable server space.

Comment by samoore_: This is true, but does it not somewhat diminish your argument above about the workload pressures of academics? Universities require academics to publish more and more, for fewer career opportunities, and at the same time publishers are pushing increasing amounts of the labour onto academics. Would a disruptive approach be one in which universities provide time and space for editorial/publishing responsibilities?

Is this true within an academic publishing context? First copy costs remain substantially high: editing, review, typesetting, server and band with costs, marketing etc. are still part of a digital only model. One thing that is important to explore though is what these first copy costs consist off in a digital context, to explore from there what the profit margin is that publishers put on top (plus cost for printing etc.). This could lead to the development of perhaps a more fair and transparent system based on actual costs. For books the OAPEN-NL project tried to do exactly this: https://openreflections.files.wordpress.com/2011/03/oapen-nl-final-report.pdf

One of the complaints I hear more and more often from academics is that traditional publishing houses are actually no longer doing this work. Editing and formatting are increasingly outsourced to academics themselves (as are indexing etc.) and even marketing is something publishers ask authors to so themselves using their social media profiles and academic brands. This is one of the issues many scholar-led publishing initiatives are trying to address, by highlighting for example the various processes that go into creating a scholarly publication and giving these due recognition. Mattering Press is at the forefront of this:https://www.matteringpress.org/

http://www.csisponline.net/2014/06/18/from-openness-to-openings-reflections-on-the-experiments-in-knowledge-production-workshop/

Comment by samoore_: This point is completely true, but is it not also the fault of universities who perpetuate this system? Publishers merely exploit it. I feel it's quite difficult to talk about disrupting the journal without also talking about disrupting the increasingly privatised university too.

Comment by Gary_Hall: Yes, following on from Sam's comment, do you need to provide some evidence to support this claim? What about journals published by scholarly societies, or by university presses? Aren't they valued even more than those owned by large corporations precisely because the latter are seen as possibly being motivated more by money than an interest in supporting high-quality scholarship?

Comment by samoore_: I'm not sure how true this clause is. I think large corporations own a disproportionate number of journals but I think they're valued just as much as those owned by smaller commercial publishers (e.g., Sage) or not-for-profits (university presses, PLOS, etc.)

If this special issue of Ephemera ever comes out (I have been keeping an eye out for it but nothing as yet) it might be highly relevant for this discussion: http://www.ephemerajournal.org/content/labour-academia-0 Back issues of Ephemera do cover topics related to this discussion too though, so might be a relevant resource anyway (and OA!)

Comment by samoore_: This section could do with fleshing out. There are a number of similar instances around accessibility for disadvantaged communities (https://jhatkaa.org/oxford-cambridge-university-press-drop-the-lawsuit/); Publishers attacking fair use for universities https://en.wikipedia.org/wiki/Cambridge_University_Press_v._Patton); Commercial publishers in the UK seeking to control public access to research with 'walk-in' services https://www.elsevier.com/connect/free-access-to-research-at-your-local-uk-public-library

Comment by LucaMorini: And most of what actually comprises our work in the 21st Century, such as this kind of discussions, will never give us any "points" in the "game" of publishing, where only the final outcomes (that are still produced under a pre-digital, Gutenbergian paradigm) count for something. A system oriented to quantification and control over 19th century media. Please note I am not saying that I would prefer that my tweets counted toward my career, but indeed further highlighting the inconsistencies and absurdity of the current academic publishing policies (and market).

Comment by LucaMorini: It is interesting, in comparing foucauldian and cybernetic conceptualisations of power (Wiener, 1948, and especially Von Foerster, 2002) to highlight similarities and differences. While both constructions are deeply relational, showing how power is not simply imposed but co-constructed, the cybernetic ideal is markedly (and maybe deceivingly) more optimistic, by focusing on the mutuality of this relationship. Maybe only a plurality of epistemologies and points of view can help us successfully navigate and resist the pervasivity of power.

Comment by Gary_Hall: Isn't it more often the other way round? Many authors are currently writing about the power associated with digital capitalist companies such as Google, Twitter and WhatsApp (Facebook). But they are doing so in 'ink-printed' books, copyrighted to for-profit presses (such as Polity, who distribute their titles through John Wiley and & Sons Ltd., one of the 'big four,' profit-maximising, scholarly publishing mega-corporations, along with Reed Elsevier, Springer, and Taylor & Francis/Informa), and published on an all-rights-reserved basis, with little appreciation of the issues of power (or hypocrisy) involved in doing so. And this includes many media archaeologists and software theorists. They, too, have yet to learn the lesson you attribute in your conclusion to Derrida and Kittler: that there is no outside of media.

Comment by Gary_Hall: Is this only the case if you adhere to the Anglo-American copyright tradition? I know this is the understanding of copyright that is discussed most often. But is there not also an understanding of copyright connected with what is known as author’s or moral rights, which is understood as having originated in the culture of Western Europe?

Comment by Gary_Hall: Doesn't academic journal publishing have other stated goals besides this one? The establishment of trust, for example, and of a permanent record. Hence all the concern with the fixed and finished, 'final' published version of a text.

Comment by Gary_Hall: The issue of paywalls seems highly relevant to to the content-driven world of profit-maximising academic publishers such as Reed Elsevier, Springer, Wiley-Blackwell, and Taylor & Francis/Informa. It seems less relevant to the the data-driven world of search engines, social media, and social networking, and of digital capitalist companies such as Google and Twitter. In the latter world, who gate-keeps access to (and so can extract maximum value from) content is less important, because that access is already free, than who gate-keeps (and so can extract maximum value from) the data generated around the use of that content, which is used more because access to it is free.

All of which raises interesting questions for the issue of JMP and its open/public peer review. Reviewers are encouraged to use Google Chrome to install the Hypothes.is plug-in needed to make these public annotations. Which means the work is no longer behind a paywall, yes. But haven't we've just swapped the content-driven world of paywalls for the data-driven world of Google, with its emphasis on open and free content?

So how much is disruption a matter of coming out from behind paywalls? And how disruptive is this issue of JMP?

Comment by Gary_Hall: Do you need to say something about how you are understanding 'disruptive' here and throughout this piece. Is it along the lines of Silicon Valley rhetoric; of Clayton Christensen and his colleagues at the Harvard Business School; of the affirmative disruption some of us at Coventry have talked about?

Comment by lskains: They shouldn't be! After all, in a lot of ways, no matter what our purpose in creative practice -- whether for research or not -- it nonetheless is a form of research. We are experimenting with art, trying to be better, get better. It's always research in an implicit sense. What makes it explicitly research is when it is incorporated into a defined methodology that allows us to explore and respond to specific research questions, and to communicate how the practice helps us answer those questions. Ideally, it should be a symbiotic relationship.

Also, who defines what is "good"? The academy? Research councils? Consumers? Prize committees?

Comment by lskains: Depends on the research question, and whether or not it is applicable as research.

Comment by lskains: I think this is a key element in practice-based research in media in general - if we look at the creative practice as analogous to data (in the sciences, for example), then we still have to make the contribution to knowledge explicit through a statement of research and/or exegesis. The sciences don't just throw raw data at each other and ask one another to figure out what its contribution is - that's what papers and reports are for. I can tell you a lot of them don't like writing it up either! But at its core, isn't that what research is -- collecting data, analyzing it, and communicating it explicitly to others in the field (and even outside the field)?

Comment by NealW: Really interesting - as well as knowing, there is not knowing, about a subject or an area, which leads to research processes being developed. These new approaches or methods being the actual area in which new knowledge lies, can also be transformative, or lead to epistemic things (Rheinberger, Schwab, White etc) - many in the field of art research, that do not seek to explain a subject, might use such as distinction which also speaks of methods, or 'models of research practice'.

Comment by NealW: Impact in UK - which is part of our beloved REF is not the same as dissemination. For Impact to have value, its qualities must be transformative, to an audience, a policy, or to a group in terms of distinct categories, such as; health and wellbeing, cultural enrichment. Providing evidence of the pathways, or in areas such as public engagement, has been under scrutiny for some time, but the arts / creative areas have been looking at this for years too - so Arts & Humanities fared well relatively in 2014.

Comment by NealW: The challenge for institutions is very real, as to differentiate between scholarly activity, practice which is also research, and practice which is not, might mean recognising activity which is also not paid for, practice outside the academy. Then we have issues of ownership etc too, along with many others. Again, if there are practical approaches that have been researched as part of your previous activity, and which are of use to other practitioner / researchers, this would be valuable to readers.

Comment by NealW: I would question this - many papers in science are riddled with all kinds of language, material, graphs, mathematics, that require a high degree of a priori knowledge. I am sometimes concerned within non-science areas, the demand for dissemination/enhanced understanding (category alongside new knowledge of increasing importance in UK) overrides the expediency by which a range of communications can be utilised. In this sense, do we have, or only make use of a narrower lexicon?

Comment by NealW: Can you reference your research in some form. Endnotes?

Comment by silvertwin: As we've heard from, in the UK context, REF sub-panel reports and consecutive editorial pieces in JMP, there is a vicious circle here in that the REF panel are calling for more practice researchers to 'step up' to be on such evaluative platforms but also practice researchers may need to be more coherent as a community about criteria for rigour. So posing the question again within an 'output' is part of the problem, not the solution, perhaps? In which case, can you articulate how the informal conversations to camera, this piece and SIghtlines work as a research 'output'?

Comment by silvertwin: I cannot access these links, they are pretty important context I think, so please try to resolve.

Comment by silvertwin: Is it helpful to the reader to signpost this approach as practice research by citing aligned studies / previous work in the field taking a similar approach?

Comment by Jessifer: The writing here is so lovely and precise.

Comment by Jessifer: There's a stylistic inconsistency in how each of these sections is presented. I feel like tidying the formatting so that each of these biographical sections has a similar form (and parallel structure) would help keep the reader from veering too far. Honestly, though, I'm loving the mash-up of forms, media, voices. That is working really really well. Just needs a bit more polish as you continue revising.

Comment by Alan_Hook: I think this gives context to the approach. There are many ways that practice researchers position what they do, and this sometimes needs to be foregrounded to help the reader orientate the approach and other researchers understand, and be less resistant too the multiplicity of forms that research can take.

Comment by Alan_Hook: There are issues from a readers perspective in how fragmented this is and how it jumps between people, viewpoints etc. Is there another way of presenting this in the journal which makes the flow easier to navigate. There are a lot of authorial styles and voices which the reader/viewer needs to juggle which cause tensions.

Comment by Alan_Hook: There is a break in the paper to provide a back-story, which maps the researchers relationship to the process which is important and has been outlined as an important process in Design based research. This research narrative positions the research subject, and their bias, in relationship to the research outputs, so that the reader can understand how the new knowledge is shaped, and contorted by the researchers subjective encounters with the research process.

This research process is important for more traditional researchers as there needs to be a solid foundation of this as research for the sceptical.

Comment by Alan_Hook: I had considered this a process of bricolage rather than juxtapositioning. My reading might be wrong, but there is interesting tensions between static images, temporal media such as music, historical context and reconfiguring these to represent something different.

Comment by Alan_Hook: This paragraph shifts the focus from data representation to data source. There needs to be some clarity here, is this a cyclical process. As this sets out some of the bounds of the argument then it becomes a little confusing as to how the authors consider the poems as data. There should be a consideration of the mediating process in this data collection and representation if they are to consider them as data.

Comment by Jessifer: I find myself wanting this to be the lead-off sentence of the piece. It sets the stage in a way that helps the rest flow from this claim, rather than around it.

Comment by Alan_Hook: I think that you need to expand, i would guess as a reader that this has something to do with the transitional nature of the organic material, and its becoming or inbetweeness but i think that you need to explain more about the authorial decisions to help guide the interpretation of the work.

Comment by Alan_Hook: This approach is really important to open up the complexity and 'messiness' (Law 2004) of the generation of new knowledge. It works against the positivism of other research intensive disciplines, but is there a tension in-between the output of research activities opening a space for consideration and reflection and the miscommunication of the new knowledge that has been generated?

Comment by Alan_Hook: I think that there needs to be a recognition of some of the instrumental constraints of research as practice. It would be nice to see the accompanying 300 words for this as a research output as there is a lot of complexity to the work and the authorial process that is not apparent through the practice itself

Comment by markreadman: As Ben says here, the process is opened up to scrutiny, becomes 'part of the work', which means that we have to read these images differently – we're not going to fall into the trap of what Gregory Currie calls 'aesthetic empiricism' and judge the photos as art works in a vacuum. But perhaps there's the danger, lurking, of aestheticising the abject? And (although it's probably the seaside setting that prompts this association) the beach photos taken by the participants just occasionally reminded me of some of Martin Parr's New Brighton photographs. So what kinds of photographic language have the participants internalised? Are they conscious or unconscious rhetoricians of the image?

Comment by markreadman: It strikes me that the soundtrack that accompanies the montage presented here (which is much more limited than the gallery exhibition described in the conversation between Anthony and Ben) is as important as the images; it allows us access to the negotiations behind the camera, the handing over of the cable release so that the subject becomes their own object, the pedagogic conversation, the gratitude. But there is always the awareness of a power differential between artist and participant, and of the editing of the sound, the selection of what to include and what to exclude, despite its vérité quality.

Comment by markreadman: Jo Spence is a great reference point, given how she forged such a powerful voice through images of her vulnerable body. Photography was an autoethnographic tool for her – is there an explicit connection to be made here I wonder?

Comment by markreadman: This apparently oxymoronic term that Ben Burbridge uses, 'photo voice' encapsulates a key issue for this project - how a picture 'enunciates'. Anthony, the artist, understands this debate well, and both he and his interlocutor Ben, are aware of the play of meaning here.

Comment by markreadman: There is something interesting here about the facilitation of the use of professional equipment, rather than, say, a phone camera; this could be empowering (but the access is temporary – a gift then taken away?), or it could be alienating – a self-portrait produced with strange machinery. The technological dimension is not addressed explicitly, despite the power that inheres within 'pro kit', despite the language of democracy and facilitation that the project uses ("invite", "assist"). I wonder what the rationale was for the use of the medium format camera.

Comment by markreadman: Echoes here of the sociological participant observer with, perhaps, the attendant ethical concerns about appropriation. The artist's roles as facilitator, 'voice', 'lens' begin to emerge here.

Comment by postoccupant: Postoccupant took part in a workshop organised by UCL Urban Lab and Centre for Critical Heritage Studies, exploring the impact of University development projects in Newham (London) and Gothenburg (Sweden). 2 days of discussion, and a great series of presentations on subjects as diverse as activist histories of East London, the agonistic politics of university expansion here and overseas, territorial complexity and public space, workshopping architecture for communities in Bangladesh and London, and the V and A's approach to community engagement. Many more, and the conversations ranged far and wide.<br /> See the post on UniverCity here... http://journal.disruptivemedia.org.uk/forum/viewtopic.php?f=2&t=209

Comment by silvertwin: There is a tension here, in the 'in between' status of what's presented for review. On the one hand, the curational and participative practices, partly ethnographic (though not stated as such) are documented as practice research, but on the other witness testimony, mainly written, is presented as 'data' (or equivalent) so the disruption appears to be in the act of curation (ie the un-collecting) but it's less clear how the format of the 'output' proposed here is disrupting the orthodoxies of capturing research evidence?

I would be interested to hear more about issues of academic labor in relationship to OA in specific. One of the issues that we continue to encounter in forms of what we have called more 'radical' open access practices, in specific academic-led projects and experimental publishing endeavours, is that the amount of free labour increases significantly (and this is of course what publishers traditionally offer to researchers, they facilitate many of the publishing processes). So, where there has been a call to only give one's free academic labour to NFP or open access initiatives (and not to Elsevier or Academia.edu for example), although this might be a more ethical use of academic labour, this does not solve the underlying issue of 'un(der)paid labor' in academia as such. So does this mean we need to work towards more recognition for the types of free labor academics do (from reviewing to editing, to board memberships, to what have you?) and to have this included more directly in impact statements etc. Or does this just lead to a further instrumentalisation of academic job specifications? Is there a tension here too between narratives that see this kind of work as part of an academic 'gift economy' versus those that stress 'free and un(der)paid labour'?

Vraag: Is alleen de actuele toestand van het dossier zichtbaar, of is dmv tijdreizen elke actie die op zo'n dossier in de tijd uitgevoerd is te achterhalen?

In essentie staat hier dat je voor een dossier kunt toevoegen, wijzigen en inzien. Je kunt een dossier dus niet verwijderen? Maar dmv wijzigen kun je een dossier wel 'leeg' maken?

Wie mag wat? Elke partij mag toevoegen, wijzigen en inzien of bepaalde partijen mogen alle acties uitvoeren en sommige partijen een deel van de acties?

samenwerkdossier

behandelen

behandelen

Prices are the same as Villa California, I believe, considering the perks, that we should be between Villa Miami and Villa California.

Put a photo of the home cinema in 4th so it is directly visible when you land on the page without clicking on "see more photos"

15 minute

there is also a ceiling fan in this bedroom

a professional dumbell set (6kg to 36kg) as well as barbels and 100 kg in plates for squatts and other strength exercises.

Multi-exercise cable machine

Technogym step machine

Top grade home cinema, 4k and Dolby Atmos connected to a Chromecast 4k (you can use your personal Netflix, Prime Video or Disney accounts, as well as cast from your phone).

All bedrooms except one have 180 views other the sea

This bedroom has view over the mountains and no terrace.

There is also a desk and a 600 GB fiber connection. Ethernet (cable) internet is available in this room as well as Wifi.

no, it's a standard fireplace.

Mountains

15 minutes

Precise that the big plus vs. other houses: - Amazing home cinema: 4k + Dolby Atmos - Top Home Gym, with full dumbell set.

Reviewer #1 (Public Review):

Summary:

The study by Pudlowski et al. investigates how the intricate structure of centrioles is formed by studying the role of a complex formed by delta- and epsilon-tubulin and the TEDC1 and TEDC2 proteins. For this, they employ knockout cell lines, EM, and ultrastructure expansion microscopy as well as pull-downs. Previous work has indicated a role of delta- and epsilon-tubulin in triplet microtubule formation. Without triplet microtubules centriolar cylinders can still form, but are unstable, resulting in futile rounds of de novo centriole assembly during S phase and disassembly during mitosis. Here the authors show that all four proteins function as a complex and knockout of any of the four proteins results in the same phenotype. They further find that mutant centrioles lack inner scaffold proteins and contain an extended proximal end including markers such as SAS6 and CEP135, suggesting that triplet microtubule formation is linked to limiting proximal end extension and formation of the central region that contains the inner scaffold. Finally, they show that mutant centrioles seem to undergo elongation during early mitosis before disassembly, although it is not clear if this may also be due to prolonged mitotic duration in mutants.

Strengths:

Overall this is a well-performed study, well presented, with conclusions mostly supported by the data. The use of knockout cell lines and rescue experiments is convincing.

Weaknesses:

In some cases, additional controls and quantification would be needed, in particular regarding cell cycle and centriole elongation stages, to make the data and conclusions more robust.

eLife assessment

The study by Pudlowski et al. shows that a protein complex composed of delta- and epsilon-tubulin together with TEDC1 and TEDC2, which was previously identified, functions in generating centriolar triplet microtubules, and that this is crucial for the proper formation of centriolar subdomains and the stability of centrioles throughout the cell cycle. The findings are valuable for a better understanding of centriole biogenesis and structure and are largely supported by solid evidence based on knockout cell lines, immunoprecipitation, and ultrastructure expansion microscopy. The work is of interest to cell biologists, in particular researchers with interest in centrosome biology.

Reviewer #2 (Public Review):

Summary:

In this article, the authors study the function of TEDC1 and TEDC2, two proteins previously reported to interact with TUBD1 and TUBE1. Previous work by the same group had shown that TUBD1 and TUBE1 are required for centriole assembly and that human cells lacking these proteins form abnormal centrioles that only have singlet microtubules that disintegrate in mitosis. In this new work, the authors demonstrate that TEDC1 and TEDC2 depletion results in the same phenotype with abnormal centrioles that also disintegrate into mitosis. In addition, they were able to localize these proteins to the proximal end of the centriole, a result not previously achieved with TUBD1 and TUBE1, providing a better understanding of where and when the complex is involved in centriole growth.

Strengths:

The results are very convincing, particularly the phenotype, which is the same as previously observed for TUBD1 and TUBE1. The U-ExM localization is also convincing: despite a signal that's not very homogeneous, it's clear that the complex is in the proximal region of the centriole and procentriole. The phenotype observed in U-ExM on the elongation of the cartwheel is also spectacular and opens the question of the regulation of the size of this structure. The authors also report convincing results on direct interactions between TUBD1, TUBE1, TEDC1, and TEDC2, and an intriguing structural prediction suggesting that TEDC1 and TEDC2 form a heterodimer that interacts with the TUBD1- TUBE1 heterodimer.

Weaknesses:

The phenotypes observed in U-ExM on cartwheel elongation merit further quantification, enabling the field to appreciate better what is happening at the level of this structure.

Reviewer #3 (Public Review):

Summary:

Human cells deficient in delta-tubulin or epsilon-tubulin form unstable centrioles, which lack triplet microtubules and undergo a futile formation and disintegration cycle. In this study, the authors show that human cells lacking the associated proteins TEDC1 or TEDC2 have these identical phenotypes. They use genetics to knockout TEDC1 or TEDC2 in p53-negative RPE-1 cells and expansion microscopy to structurally characterize mutant centrioles. Biochemical methods and AlphaFold-multimer prediction software are used to investigate interactions between tubulins and TEDC1 and TEDC2.

The study shows that mutant centrioles are built only of A tubules, which elongate and extend their proximal region, fail to incorporate structural components, and finally disintegrate in mitosis. In addition, they demonstrate that delta-tubulin or epsilon-tubulin and TEDC1 and TEDC2 form one complex and that TEDC1 TEDC2 can interact independently of tubulins. Finally, they show that the localization of four proteins is mutually dependent.

Strengths:

The results presented here are mostly convincing, the study is exciting and important, and the manuscript is well-written. The study shows that delta-tubulin, epsilon-tubulin, TEDC1, and TEDC2 function together to build a stable and functional centriole, significantly contributing to the field and our understanding of the centriole assembly process.

Weaknesses:

The ultrastructural characterization of TEDC1 and TEDC2 obtained by U-ExM is inconclusive. Improving the quality of the signals is paramount for this manuscript.

Comment by Alan_Hook: I think that there needs to be a recognition of some of the instrumental constraints of research as practice. It would be nice to see the accompanying 300 words for this as a research output as there is a lot of complexity to the work and the authorial process that is not apparent through the practice itself

Comment by Alan_Hook: I think that there needs to be a recognition of some of the instrumental constraints of research as practice. It would be nice to see the accompanying 300 words for this as a research output as there is a lot of complexity to the work and the authorial process that is not apparent through the practice itself

I would be interested to hear more about issues of academic labor in relationship to OA in specific. One of the issues that we continue to encounter in forms of what we have called more 'radical' open access practices, in specific academic-led projects and experimental publishing endeavours, is that the amount of free labour increases significantly (and this is of course what publishers traditionally offer to researchers, they facilitate many of the publishing processes). So, where there has been a call to only give one's free academic labour to NFP or open access initiatives (and not to Elsevier or Academia.edu for example), although this might be a more ethical use of academic labour, this does not solve the underlying issue of 'un(der)paid labor' in academia as such. So does this mean we need to work towards more recognition for the types of free labor academics do (from reviewing to editing, to board memberships, to what have you?) and to have this included more directly in impact statements etc. Or does this just lead to a further instrumentalisation of academic job specifications? Is there a tension here too between narratives that see this kind of work as part of an academic 'gift economy' versus those that stress 'free and un(der)paid labour'?

I would be interested to hear more about issues of academic labor in relationship to OA in specific. One of the issues that we continue to encounter in forms of what we have called more 'radical' open access practices, in specific academic-led projects and experimental publishing endeavours, is that the amount of free labour increases significantly (and this is of course what publishers traditionally offer to researchers, they facilitate many of the publishing processes). So, where there has been a call to only give one's free academic labour to NFP or open access initiatives (and not to Elsevier or Academia.edu for example), although this might be a more ethical use of academic labour, this does not solve the underlying issue of 'un(der)paid labor' in academia as such. So does this mean we need to work towards more recognition for the types of free labor academics do (from reviewing to editing, to board memberships, to what have you?) and to have this included more directly in impact statements etc. Or does this just lead to a further instrumentalisation of academic job specifications? Is there a tension here too between narratives that see this kind of work as part of an academic 'gift economy' versus those that stress 'free and un(der)paid labour'?

Not supported. See "Brethren of the Lord"

Reviewer #2 (Public Review):

Summary:

This study looks at sex differences in alcohol drinking behaviour in a well-validated model of binge drinking. They provide a comprehensive analysis of drinking behaviour within and between sessions for males and females, as well as looking at the calcium dynamics in neurons projecting from the anterior insula cortex to the dorsolateral striatum.

Strengths:

Examining specific sex differences in drinking behaviour is important. This research question is currently a major focus for preclinical researchers looking at substance use. Although we have made a lot of progress over the last few years, there is still a lot that is not understood about sex-differences in alcohol consumption and the clinical implications of this.

Identifying the lateralisation of activity is novel, and has fundamental importance for researchers investigating functional anatomy underlying alcohol-driven behaviour (and other reward-driven behaviours).

Weaknesses:

Very small and unequal sample sizes, especially females (9 males, 5 females). This is probably ok for the calcium imaging, especially with the G-power figures provided, however, I would be cautious with the outcomes of the drinking behaviour, which can be quite variable.

For female drinking behaviour, rather than this being labelled "more efficient", could this just be that female mice (being substantially smaller than male mice) just don't need to consume as much liquid to reach the same g/kg. In which case, the interpretation might not be so much that females are more efficient, as that mice are very good at titrating their intake to achieve the desired dose of alcohol.

I may be mistaken, but is ANCOVA, with sex as the covariate, the appropriate way to test for sex differences? My understanding was that with an ANCOVA, the covariate is a continuous variable that you are controlling for, not looking for differences in. In that regard, given that sex is not continuous, can it be used as a covariate? I note that in the results, sex is defined as the "grouping variable" rather than the covariate. The analysis strategy should be clarified.

eLife assessment

This valuable manuscript describes evidence of sex differences in specific corticostriatal projections during alcohol consumption, and this is noteworthy given the increasing rates/levels of drinking in females and the liability for Alcohol Use disorder. They provide solid evidence of the lateralisation of the activity of the circuit, but other evidence is incomplete, particularly with regard to its description of the drinking measure and how this relates to intoxication. The analyses of the histology data are not complete, and there are further inconsistencies that make it difficult to reconcile the photometry and behavioral data. The findings will be of partial interest to researchers investigating functional circuitry underlying alcohol-driven behaviors.

Reviewer #1 (Public Review):

Summary:

This paper uses a model of binge alcohol consumption in mice to examine how the behaviour and its control by a pathway between the anterior insular cortex (AIC) to the dorsolateral striatum (DLS) may differ between males and females. Photometry is used to measure the activity of AIC terminals in the DLS when animals are drinking and this activity seems to correspond to drink bouts in males but not females. The effects appear to be lateralized with inputs to the left DLS being of particular interest.

Strengths:

Increasing alcohol intake in females is of concern and the consequences for substance use disorder and brain health are not fully understood, so this is an area that needs further study. The attempt to link fine-grained drinking behaviour with neural activity has the potential to enrich our understanding of the neural basis of behaviour, beyond what can be gleaned from coarser measures of volumes consumed etc.

Weaknesses:

The introduction to the drinking in the dark (DID) paradigm is rather narrow in scope (starting line 47). This would be improved if the authors framed this in the context of other common intermittent access paradigms and gave due credit to important studies and authors that were responsible for the innovation in this area (particularly studies by Wise, 1973 and returned to popular use by Simms et al 2010 and related papers; e.g., Wise RA (1973). Voluntary ethanol intake in rats following exposure to ethanol on various schedules. Psychopharmacologia 29: 203-210; Simms, J., Bito-Onon, J., Chatterjee, S. et al. Long-Evans Rats Acquire Operant Self-Administration of 20% Ethanol Without Sucrose Fading. Neuropsychopharmacol 35, 1453-1463 (2010).) The original drinking in the dark demonstrations should also be referenced (Rhodes et al., 2005). Line 154 Theile & Navarro 2014 is a review and not the original demonstration.

When sex differences in alcohol intake are described, more care should be taken to be clear about whether this is in terms of volume (e.g. ml) or blood alcohol levels (BAC, or at least g/kg as a proxy measure). This distinction was often lost when lick responses were being considered. If licking is similar (assuming a single lick from a male and female brings in a similar volume?), this might mean males and females consume similar volumes, but females due to their smaller size would become more intoxicated so the implications of these details need far closer consideration. What is described as identical in one measure, is not in another.

No conclusions regarding the photometry results can be drawn based on the histology provided. Localization and quantification of viral expression are required at a minimum to verify the efficacy of the dual virus approach (the panel in Supplementary Figure 1 is very small and doesn't allow terminals to be seen, and there is no quantification). Whether these might differ by sex is also necessary before we can be confident about any sex differences in neural activity.

While the authors have some previous data on the AIC to DLS pathway, there are many brain regions and pathways impacted by alcohol and so the focus on this one in particular was not strongly justified. Since photometry is really an observational method, it's important to note that no causal link between activity in the pathway and drinking has been established here.

It would be helpful if the authors could further explain whether their modified lickometers actually measure individual licks. While in some systems contact with the tongue closes a circuit which is recorded, the interruption of a photobeam was used here. It's not clear to me whether the nose close to the spout would be sufficient to interrupt that beam, or whether a tongue protrusion is required. This detail is important for understanding how the photometry data is linked to behaviour. The temporal resolution of the GCaMP signal is likely not good enough to capture individual links but I think more caution or detail in the discussion of the correspondence of these events is required.

Even if the pattern of drinking differs between males and females, the use of the word "strategy" implies a cognitive process that was never described or measured.

Reviewer #3 (Public Review):

Summary:

In this manuscript by Haggerty and Atwood, the authors use a repeated binge drinking paradigm to assess how water and ethanol intake changes in male in female mice as well as measure changes in anterior insular cortex to dorsolateral striatum terminal activity using fiber photometry. They find that overall, males and females have similar overall water and ethanol intake, but females appear to be more efficient alcohol drinkers. Using fiber photometry, they show that the anterior insular cortex (AIC) to dorsolateral striatum projections (DLS) projections have sex, fluid, and lateralization differences. The male left circuit was most robust when aligned to ethanol drinking, and water was somewhat less robust. Male right, and female and left and right, had essentially no change in photometry activity. To some degree, the changes in terminal activity appear to be related to fluid exposure over time, as well as within-session differences in trial-by-trial intake. Overall, the authors provide an exhaustive analysis of the behavioral and photometric data, thus providing the scientific community with a rich information set to continue to study this interesting circuit. However, although the analysis is impressive, there are a few inconsistencies regarding specific measures (e.g., AUC, duration of licking) that do not quite fit together across analytic domains. This does not reduce the rigor of the work, but it does somewhat limit the interpretability of the data, at least within the scope of this single manuscript.

Strengths:

- The authors use high-resolution licking data to characterize ingestive behaviors.<br /> - The authors account for a variety of important variables, such as fluid type, brain lateralization, and sex.<br /> - The authors provide a nice discussion on how this data fits with other data, both from their laboratory and others'.<br /> - The lateralization discovery is particularly novel.

Weaknesses:

- The volume of data and number of variables provided makes it difficult to find a cohesive link between data sets. This limits interpretability.<br /> - The authors describe a clear sex difference in the photometry circuit activity. However, I am curious about whether female mice that drink more similarly to males (e.g., less efficiently?) also show increased activity in the left circuit, similar to males. Oppositely, do very efficient males show weaker calcium activity in the circuit? Ultimately, I am curious about how the circuit activity maps to the behaviors described in Figures 1 and 2.<br /> - What does the change in water-drinking calcium imaging across time in males mean? Especially considering that alcohol-related signals do not seem to change much over time, I am not sure what it means to have water drinking change.

eLife assessment

Using electrophysiological recordings in freely moving rats, this valuable study investigates the role of different gamma frequency bands in the development of spatial representations in the hippocampus. However, the evidence is incomplete as the methods and data analysis need significant improvement. Critically, alternative interpretations and analyses must be provided, especially regarding the nature of gamma oscillations in the hippocampus and their interaction with neuronal firing dynamics and theta sequence features. This study will be of interest to neuroscientists working in the field of spatial navigation and neuronal dynamics.

in the pipeline

in the process of being planned or developed: the company has three new models in the pipeline.

[in singular] informal an unusual temporary increase in number or size: a bulge in the birth rate.

extremely serious or urgent

(of an event regarded as threatening or significant) about to happen; forthcoming

bring up and care for (a child) until they are fully grown

the excessive display of concern or distress

eLife assessment

This useful study explores the relationship between the sequence of prokaryotic promoter elements and their activity using mutagenesis to generate thousands of mutant sequences. The evidence supporting these findings is incomplete, and would benefit from additional experiments, clarification of methods, and a more detailed discussion of related literature. This work will appeal to those interested in bacterial genetics, genome evolution, and gene regulation.

Reviewer #1 (Public Review):

Summary:

This study by Fuqua et al. studies the emergence of sigma70 promoters in bacterial genomes. While there have been several studies to explore how mutations lead to promoter activity, this is the first to explore this phenomenon in a wide variety of backgrounds, which notably contain a diverse assortment of local sigma70 motifs in variable configurations. By exploring how mutations affect promoter activity in such diverse backgrounds, they are able to identify a variety of anecdotal examples of gain/loss of promoter activity and propose several mechanisms for how these mutations interact within the local motif landscape. Ultimately, they show how different sequences have different probabilities of gaining/losing promoter activity and may do so through a variety of mechanisms.

Major strengths and weaknesses of the methods and results:

This study uses Sort-Seq to characterize promoter activity, which has been adopted by multiple groups and shown to be robust. Furthermore, they use a slightly altered protocol that allows measurements of bi-directional promoter activity. This combined with their pooling strategy allows them to characterize expressions of many different backgrounds in both directions in extremely high throughput which is impressive! A second key approach this study relies on is the identification of promoter motifs using position weight matrices (PWMs). While these methods are prone to false positives, the authors implement a systematic approach which is standard in the field. However, drawing these types of binary definitions (is this a motif? yes/no) should always come with the caveat that gene expression is a quantitative trait that we oversimplify when drawing boundaries.

Their approach to randomly mutagenizing promoters allowed them to find many anecdotal examples of different types of evolutions that may occur to increase or decrease promoter activity. However, the lack of validation of these phenomena in more controlled backgrounds may require us to further scrutinize their results. That is, their explanations for why certain mutations lead or obviate promoter activity may be due to interactions with other elements in the 'messy' backgrounds, rather than what is proposed.

An appraisal of whether the authors achieved their aims, and whether the results support their conclusions:

The authors express a key finding that the specific landscape of promoter motifs in a sequence affects the likelihood that local mutations create or destroy regulatory elements. The authors have described many examples, including several that are non-obvious, and show convincingly that different sequence backgrounds have different probabilities for gaining or losing promoter activity. While this overarching conclusion is supported by the manuscript, the proposed mechanisms for explaining changes in promoter activity are not sufficiently validated to be taken for absolute truth. There is not sufficient description of the strength of emergent promoter motifs or their specific spacings from existing motifs within the sequence. Furthermore, they do not define a systematic process by which mutations are assigned to different categories (e.g. box shifting, tandem motifs, etc.) which may imply that the specific examples are assigned based on which is most convenient for the narrative.

Impact of the work on the field, and the utility of the methods and data to the community:

From this study, we are more aware of different types of ways promoters can evolve and devolve, but do not have a better ability to predict when mutations will lead to these effects. Recent work in the field of bacterial gene regulation has raised interest in bidirectional promoter regions. While the authors do not discuss how mutations that raise expression in one direction may affect another, they have created an expansive dataset that may enable other groups to study this interesting phenomenon. Also, their variation of the Sort-Seq protocol will be a valuable example for other groups who may be interested in studying bidirectional expression. Lastly, this study may be of interest to groups studying eukaryotic regulation as it can inform how the evolution of transcription factor binding sites influences short-range interactions with local regulator elements.

Any additional context to understand the significance of the work:

The task of computationally predicting whether a sequence drives promoter activity is difficult. By learning what types of mutations create or destroy promoters from this study, we are better equipped for this task.

Reviewer #2 (Public Review):

Summary:

Fuqua et al investigated the relationship between prokaryotic box motifs and the activation of promoter activity using a mutagenesis sequencing approach. From generating thousands of mutant daughter sequences from both active and non-active promoter sequences they were able to produce a fantastic dataset to investigate potential mechanisms for promoter activation. From these large numbers of mutated sequences, they were able to generate mutual information with gene expression to identify key mutations relating to the activation of promoter island sequences.

Strengths:

The data generated from this paper is an important resource to address this question of promoter activation. Being able to link the activation of gene expression to mutational changes in previously nonactive promoter regions is exciting and allows the potential to investigate evolutionary processes relating to gene regulation in a statistically robust manner. Alongside this, the method of identifying key mutations using mutual information in this paper is well done and should be standard in future studies for identifying regions of interest.

Weaknesses:

While the generation of the data is superb the focus only on these mutational hotspots removes a lot of the information available to the authors to generate robust conclusions. For instance.

(1) The linear regression in S5 used to demonstrate that the number of mutational hotspots correlates with the likelihood of a mutation causing promoter activation is driven by three extreme points.

(2) Many of the arguments also rely on the number of mutational hotspots being located near box motifs. The context-dependent likelihood of this occurring is not taken into account given that these sequences are inherently box motif rich. So, something like an enrichment test to identify how likely these hot spots are to form in or next to motifs.

(3) The link between changes in expression and mutations in surrounding motifs is assessed with two-sided Mann Whitney U tests. This method assumes that the sequence motifs are independent of one another, but the hotspots of interest occur either in 0, 3, 4, or 5s in sequences. There is therefore no sequence where these hotspots can be independent and the correlation causation argument for motif change on expression is weakened.

(4) The distance between -10 and -35 was mentioned briefly but not taken into account in the analysis.

The authors propose mechanisms of promoter activation based on a few observations that are treated independently but occur concurrently. To address this using complementary approaches such as analysis focusing on identifying important motifs, using something like a glm lasso regression to identify significant motifs, and then combining with mutational hotspot information would be more robust. Other elements known to be involved in promoter activation including TGn or UP elements were not investigated or discussed.

Reviewer #3 (Public Review):

Summary:

Like many papers in the last 5-10 years, this work brings a computational approach to the study of promoters and transcription, but unfortunately disregards or misrepresents much of the existing literature and makes unwarranted claims of novelty. My main concerns with the current paper are outlined below although the problems are deeply embedded.

Strengths:

The data could be useful if interpreted properly, taking into account i) the role of translation ii) other promoter elements, and iii) the relevant literature.

Weaknesses:

(1) Incorrect assumptions and oversimplification of promoters.

- There is a critical error on line 68 and Figure 1A. It is well established that the -35 element consensus is TTGACA but the authors state TTGAAA, which is also the sequence represented by the sequence logo shown and so presumably the PWM used. It is essential that the authors use the correct -35 motif/PWM/consensus.

-Likely, the authors have made this mistake because they have looked at DNA sequence logos generated from promoter alignments anchored by either the position of the -10 element or transcription start site (TSS), most likely the latter. The distance between the TSS and -10 varies. Fewer than half of E. coli promoters have the optimal 7 bp separation with distances of 8, 6, and 5 bp not being uncommon (PMID: 35241653). Furthermore, the distance between the -10 and -35 elements is also variable (16,17, and 18 bp spacings are all frequently found, PMID: 6310517). This means that alignments, used to generate sequence logos, have misaligned -35 hexamers. Consequently, the true consensus is not represented. If the alignment discrepancies are corrected, the true consensus emerges. This problem seems to permeate the whole study since this obviously incorrect consensus/motif has been used throughout to identify sequences that resemble -35 hexamers.

- An uninformed person reading this paper would be led to believe that prokaryotic promoters have only two sequence elements: the -10 and -35 hexamers. This is because the authors completely ignore the role of the TG motif, UP element, and spacer region sequence. All of these can compensate for the lack of a strong -35 hexamer and it's known that appending such elements to a lone -10 sequence can create an active promoter (e.g. PMIDs 15118087, 21398630, 12907708, 16626282, 32297955). Very likely, some of the mutations, classified as not corresponding to a -10 or -35 element in Figure 2, target some of these other promoter motifs.

- The model in Figure 4C is highly unlikely. There is no evidence in the literature that RNAP can hang on with one "arm" in this way. In particular, structural work has shown that sequence-specific interactions with the -10 element can only occur after the DNA has been unwound (PMID: 22136875). Further, -10 elements alone, even if a perfect match to the consensus, are non-functional for transcription. This is because RNAP needs to be directed to the -10 by other promoter elements, or transcription factors. Only once correctly positioned, can RNAP stabilise DNA opening and make sequence-specific contacts with the -10 hexamer. This makes the notion that RNAP may interact with the -10 alone, using only domain 2 of sigma, extremely unlikely.

(2) Reinventing the language used to describe promoters and binding sites for regulators.

- The authors needlessly complicate the narrative by using non-standard language. For example, On page 1 they define a motif as "a DNA sequence computationally predicted to be compatible with TF binding". They distinguish this from a binding site "because binding sites refer to a location where a TF binds the genome, rather than a DNA sequence". First, these definitions are needlessly complicated, why not just say "putative binding sites" and "known binding sites" respectively? Second, there is an obvious problem with the definitions; many "motifs" with also be "bindings sites". In fact, by the time the authors state their definitions, they have already fallen foul of this conflation; in the prior paragraph they stated: "controlled by DNA sequences that encode motifs for TFs to bind". The same issue reappears throughout the paper.

- The authors also use the terms "regulatory" and non-regulatory" DNA. These terms are not defined by the authors and make little sense. For instance, I assume the authors would describe promoter islands lacking transcriptional activity (itself an incorrect assumption, see below)as non-regulatory. However, as horizontally acquired sections of AT-rich DNA these will all be bound by H-NS and subject to gene silencing, both promoters for mRNA synthesis and spurious promoters inside genes that create untranslated RNAs. Hence, regulation is occurring.

- Line 63: "In prokaryotes, the primary regulatory sequences are called promoters". Promoters are not generally considered regulatory. Rather, it is adjacent or overlapping sites for TFs that are regulatory. There is a good discussion of the topic here (PMID: 32665585).

(3) The authors ignore the role of translation.

- The authors' assay does not measure promoter activity alone, this can only be tested by measuring the amount of RNA produced. Rather, the assay used measures the combined outputs of transcription and translation. If the DNA fragments they have cloned contain promoters with no appropriately positioned Shine-Dalgarno sequence then the authors will not detect GFP or RFP production, even though the promoter could be making an RNA (likely to be prematurely terminated by Rho, due to a lack of translation). This is known for promoters in promoter islands (e.g. Figure 1 in PMID: 33958766).

- In Figure S6 it appears that the is a strong bias for mutations resulting in RFP expression to be close to the 3' end of the fragment. Very likely, this occurs because this places the promoter closer to RFP and there are fewer opportunities for premature termination by Rho

(4) Ignoring or misrepresenting the literature.

- As eluded to above, promoter islands are large sections of horizontally acquired, high AT-content, DNA. It is well known that such sequences are i) packed with promoters driving the expression on RNAs that aren't translated ii) silenced, albeit incompletely, by H-NS and iii) targeted by Rho which terminates untranslated RNA synthesis (PMIDs: 24449106, 28067866, 18487194). None of this is taken into account anywhere in the paper and it is highly likely that most, if not all, of the DNA sequences the authors have used contain promoters generating untranslated RNAs.

- The authors state that GC content does not correlate with the emergence of new promoters. It is known that GC content does correlate to the emergence of new promoters because promoters are themselves AT-rich DNA sequences (e.g. see Figure 1 of PMID: 32297955). There are two reasons the authors see no correlation in this work. First, the DNA sequences they have used are already very AT-rich (between 65 % and 78 % AT-content). Second, they have only examined a small range of different AT-content DNA (i.e. between 65 % and 78 %). The effect of AT-content on promoter emerge is most clearly seen between AT-content of between around 40 % and 60 %. Above that level, the strong positive correlation plateaus.

- Once these authors better include and connect their results to the previous literature, they can also add some discussion of how previous papers in recent years may have also missed some of this important context.

(5) Lack of information about sequences used and mutations.

- To properly assess the work any reader will need access to the sequences cloned at the start of the work, where known TSSs are within these sequences (ideally +/- H-NS, which will silence transcription in the chromosomal context but may not when the sequences are removed from their natural context and placed in a plasmid). Without this information, it is impossible to assess the validity of the authors' work.

- The authors do not account for the possibility that DNA sequences in the plasmid, on either side of the cloned DNA fragment, could resemble promoter elements. If this is the case, then mutations in the cloned DNA will create promoters by "pairing up" with the plasmid sequences. There is insufficient information about the DNA sequences cloned, the mutations identified, or the plasmid, to determine if this is the case. It is possible that this also accounts for mutational hotspots described in the paper.

(6) Overselling the conclusions.

Line 420: The paper claims to have generated important new insights into promoters. At the same time, the main conclusion is that "Our study demonstrates that mutations to -10 and -35 boxes motifs are the primary paths to create new promoters and to modulate the activity of existing promoters". This isn't new or unexpected. People have been doing experiments showing this for decades. Of course, mutations that make or destroy promoter elements create and destroy promoters. How could it be any other way?

formative assessment

This is even more important in the age of AI when you can get your answers readily but you need to be able to form a good question

https://www.derstandard.at/story/3000000226089/regierung-einigt-sich-auf-regeln-fuer-co2-speicherung

Replication--getting the same result from doing the whole study again--is logically independent from whether there even exist any code/analysis from the original paper. Recommend revising and instead talking about how they are related and often conflated.

Neat idea. I think readers would benefit if you also described that the same check for exact reproducibility is achieved with zero changes to certain workflows (if using VCS like git) when analyst uses Quarto with the freeze: auto option.

https://quarto.org/docs/projects/code-execution.html#freeze

eLife assessment

This useful work provides a risk-prediction tool, in the form of a nomogram, for practitioners and elderly patients with non-metastatic colon cancer using data from the SEER registry. The unique contribution of this work is the focus on conditional survival. However, the underlying statistical approach is suboptimal and therefore incomplete, which substantially lessens the potential impact of this work. The analysis could use a more rigorous consideration of competing risks.

Reviewer #1 (Public Review):

Summary:

This study assessed conditional survival in elderly patients with non-metastatic colon cancer who underwent colectomy. The study found that 5-year conditional overall survival rates exhibited a slight increase initially, followed by a decrease over time. In contrast, 5-year conditional colon-specific survival rates consistently improved over the same period. Nomograms were developed to predict survival probabilities at baseline and for patients surviving 1, 3, and 5 years post-diagnosis, with good predictive performance. The study concludes that conditional survival offers valuable insights into medium- and long-term survival probabilities for these patients.

Strengths:

The strengths of this study include robust study design, methodology, statistical analysis, and interpretation of the findings. Utilizing a well-known database for the analysis is another strength. Differentiating overall survival and colon-specific survival rates could be another one. Focusing on elderly patients with this condition is another major point. Providing nomograms for an easier implication of the findings in real-world clinical practice is a major strength of the study.

Weaknesses:

Relying on only one database of patients and narrowing down the population to only elderly patients who underwent colectomy could be mentioned as a weakness. Less generalizability of the findings for other populations and not including more diverse databases is a major weakness of this study. The good predictive capabilities of the developed tools are another weakness that could be improved to be excellent.

Reviewer #2 (Public Review):

Summary:

The authors assessed the conditional survival of elderly patients with non-metastatic colon cancer who had survived a certain length of time after colectomy. They used data from the Surveillance, Epidemiology, and End Results (SEER) registry to conduct a conditional survival analysis providing estimates of conditional survival rates as well as an analysis of which variables were most important for survival at baseline, one year, three years, and five years.

Strengths:

- The authors used SEER data, providing them with long-term follow-up, and thoroughly considered a wide range of variables related to cancer mortality.<br /> - The authors did a thorough job of assessing the predictive ability of their models.<br /> - The authors used conditional survival, providing estimates of survival that are meaningful for patients/physicians, making them useful for clinical practice.

Weaknesses:

- The paper would have benefited from a more thorough explanation of why the methods were improvements on existing approaches.

- This study was primarily interested in cancer mortality, and compared it to the secondary outcome of death from any cause. The study would have benefited from modeling death from non-cancer causes (the competing risk) in addition to death from colon cancer, rather than comparing only to the composite endpoint of death from any cause.

- When considering a cause-specific hazard, as done with cancer survival in this paper, it would be better to consider the cumulative incidence function rather than Kaplan Meier, since it does not assume the independence of the events like Kaplan Meier does. For this reason, the paper would benefit from focusing on the results of the adjusted cause-specific hazard models (rather than the unadjusted conditional survival estimates done using Kaplan Meier estimates shown in Figure 1 and conducting a parallel analysis for death from other causes.

- The authors mention that they consider disparities using a log-rank test. For the same reason as above, is not the best approach when dealing with competing risks as it depends on Kaplan Meier curves. The log-rank test may be fine if there is no strong dependence between the two causes of death, but the paper would benefit from some discussion of that choice, or sensitivity analysis by comparison to other approaches.

- The variables for the adjusted models were chosen with univariate Cox regression analysis, with any variables having a p-value less than 0.05 being included in the adjusted. Another approach, which may have made the models more easily comparable, would be to choose the variables that are relevant based on prior literature and include them in the multivariate model regardless of significance. The paper would benefit from a discussion of what is gained by excluding some variables from some models.

Reviewer #3 (Public Review):

Summary:

This article uses a subset of data from the SEER cancer registry to develop nomograms, a patient-facing risk prediction tool, for predicting overall and cancer-specific survival in elderly patients who underwent colectomy for the treatment of non-metastatic colon cancer. A unique contribution is the intent to provide conditional predictions, i.e. given that you have survived for x years from your diagnosis, what is your probability of survival for an additional y years? Although the goal is a useful one, the approach is unfortunately hampered by some important weaknesses.

Strengths:

Predicting conditional overall survival is a useful, patient-oriented goal.

The data source is the high-quality SEER cancer registry.

Weaknesses:

Using Kaplan-Meier methodology to estimate the survival distribution for a time-to-event in the presence of another competing time-to-event (in this case: estimating colon-specific survival in the presence of death from other causes) will generally over-estimate the event rate. The reported colon-specific survival probabilities are probably biased downwards from their true values. See https://pubmed.ncbi.nlm.nih.gov/10204198/

A similar concern would apply to the use of the cause-specific Cox model, and thus also the nomogram, to predict absolute (conditional) survival.

The p-value-based methodology for determining which predictors should be included in the nomogram is rudimentary. More modern variable selection methods, e.g. the Lasso, would have been preferred.

Related to the above comment, some predictors are present for the conditional survival nomogram for time t, then absent for time t+1, then present again for time t+2. A cancer site is an example of such a predictor. From a face validity perspective, this doesn't really make sense. Ideally, predictors would not enter, then leave, and then re-enter a model.

Many observations were excluded due to missingness in predictors, e.g. >10000 were excluded to due unknown CEA (Supplementary Figure 1). Given the number of observations dropped due to missingness in the predictors, ideally an attempt would have been made to incorporate the partial information available in these data.

Details are lacking on how the AUCs and Brier scores were calculated in the presence of censoring / competing events, which limits the reader's understanding of the results.

It is not clear why a nomogram would be preferred to an online risk prediction calculator.

eLife assessment

The work is important and of potential value to areas other than the bone field because it supports a role and mechanism for beta-catenin that is novel and unusual. The findings are significant in that they support the presence of another anabolic pathway in bone that can be productively targeted for therapeutic goals. The data for the most part are convincing. The work could be strengthened by better characterizing the osteoclast KO of Malat1 related to the Lys cre model and by including biochemical markers of bone turnover from the mice.

Reviewer #1 (Public Review):

Summary

The authors were trying to discover a novel bone remodeling network system. They found that an IncRNA Malat1 plays a central role in the remodeling by binding to β-catenin and functioning through the β-catenin-OPG/Jagged1 pathway in osteoblasts and chondrocytes. In addition, Malat1 significantly promotes bone regeneration in fracture healing in vivo. Their findings suggest a new concept of Malat1 function in the skeletal system. One significantly different finding between this manuscript and the competing paper pertains to the role of Malat1 in osteoclast lineage, specifically, whether Malat1 functions intrinsically in osteoclast lineage or not.

Strengths:

This study provides strong genetic evidence demonstrating that Malat1 acts intrinsically in osteoblasts while suppressing osteoclastogenesis in a non-autonomous manner, whereas the other group did not utilize relevant conditional knockout mice. As shown in the results, Malat1 knockout mouse exhibited abnormal bone remodeling and turnover. Furthermore, they elucidated molecular function of Malat1, which is sufficient to understand the phenotype in vivo.

Weaknesses:

Discussing differences between previous paper and their status would be highly informative and beneficial for the field, as it would elucidate the solid underlying mechanisms.

Reviewer #2 (Public Review):

Summary:

The authors investigated the roles of IncRNA Malat1 in bone homeostasis which was initially believed to be non-functional for physiology. They found that both Malat1 KO and conditional KO in osteoblast lineage exhibit significant osteoporosis due to decreased osteoblast bone formation and increased osteoclast resorption. More interestingly they found that deletion of Malat1 in osteoclast lineage cells does not affect osteoclast differentiation and function. Mechanistically, they found that Malat1 acts as a co-activator of b-Catenin directly regulating osteoblast activity and indirectly regulating osteoclast activity via mediating OPG, but not RANKL expression in osteoblast and chondrocyte. Their discoveries establish a previously unrecognized paradigm model of Malat1 function in the skeletal system, providing novel mechanistic insights into how a lncRNA integrates cellular crosstalk and molecular networks to fine-tune tissue homeostasis, and remodeling.

Strengths:

The authors generated global and conditional KO mice in osteoblast and osteoclast lineage cells and carefully analyzed the role of Matat1 with both in vivo and in vitro systems. The conclusion of this paper is mostly well supported by data.

Weaknesses:

More objective biological and biochemical analyses are required.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Qin and colleagues study the role of Malat1 in bone biology. This topic is interesting given the role of lncRNAs in multiple physiologic processes. A previous study (PMID 38493144) suggested a role for Malat1 in osteoclast maturation. However, the role of this lncRNA in osteoblast biology was previously not explored. Here, the authors note osteopenia with increased bone resorption in mice lacking Malat1 globally and in osteoblast lineage cells. At the mechanistic level, the authors suggest that Malat1 controls beta-catenin activity. These results advance the field regarding the role of this lncRNA in bone biology.

Strengths:

The manuscript is well-written and data are presented in a clear and easily understandable manner. The bone phenotype of osteoblast-specific Malat1 knockout mice is of high interest. The role of Malat1 in controlling beta-catenin activity and OPG expression is interesting and novel.

Weaknesses:

The lack of a bone phenotype when Malat1 is deleted with LysM-Cre is of interest given the previous report suggesting a role for this lncRNA in osteoclasts. However, to interpret the findings here, the authors should investigate the deletion efficiency of Malat1 in osteoclast lineage cells in their model. The data in the fracture model in Figure 8 seems incomplete in the absence of a more complete characterization of callus histology and a thorough time course. The role of Malat1 and OPG in chondrocytes is unclear since the osteocalcin-Cre mice (which should retain normal Malat1 levels in chondrocytes) have similar bone loss as the global mutants.

eLife assessment

In this valuable study, Gue, Hue et al. describe how two poorly understood rhabdomyosarcoma fusion-oncogenes, VGLL2::NCOA2 and TEAD1::NCOA2, function at the genomic, transcriptional, and proteomic levels in multiple systems. They generated solid data that support that these fusion-oncogenes leverage TEAD transcriptional signatures, in a mechanism that is independent of YAP/TAZ, and that this activity potentially contributes to tumorigenesis. This work offers new mechanistic insights into oncogenic gene fusion events identified in cancer patients and reveals potential therapeutic strategies for the treatment of rhabdomyosarcomas.

Reviewer #1 (Public Review):

Summary:

Guo, Hue et al. focused on understanding the epigenetic activity and functional dependencies for two different fusions found in infantile rhabdomyosarcoma, VGLL2::NCOA2, and TEAD1::NCOA2. They use a variety of models and methods; specifically, ectopic expression of the fusions in human 293T cells to perform RNAseq (both fusions), CUT&RUN (VGLL2::NCOA2), and BioID mass spec (both fusions). These data identify that the VGLL2::NCOA2 fusion has peaks that are enriched for TEAD motifs. Further, CPB/p300 CUT&RUN support an enrichment of binding sites and three TEAD targets in VGLL2::NCOA2 and TEAD1::NCOA2 expressing cells. They also functionally evaluated genetic and chemical dependencies (TEAD inhibition), and found this was only effective for the VGLL2::NCOA2 fusion, and not for TEAD1::NCOA2. Using complementary biochemical approaches they suggest (with other supporting data) that the fusions regulate TEAD transcriptional outputs via a YAP/TAZ independent mechanism. Further, they expand into a C2C12 myoblast model and show that TEAD1::NCOA2 is transforming in colony formation assays and in mouse allografts. This is consistent with previously published strategies using VGLL2::NCOA2. Importantly, they show that a CBP/p300 (a binding partner found in their BioID mass spec) small molecule inhibitor suppresses tumor formation using this mouse allograft model, that the tumors are less proliferative, and have a reduction in transcriptional of three TEAD target genes. Generally, the data is interesting and suggests new biology for these fusion-oncogenes. However, the choice of 293T for the majority of the transcriptional, epigenetic, and proteomic studies makes the findings difficult to interpret in the context of the human disease, and the rationale for the choice of an epithelial-like kidney cell line is not discussed. Further, details are missing from the figures, figure legends, and methods that make the data difficult to interpret, and should be added to improve the reader's understanding. Overall, the breadth of methods used in this study, and the comparison of the two fusion-oncogene's biology is of interest to the fusion-oncogene, pediatric sarcoma, and epigenetic therapeutic targeting fields.

Strengths:

(1) Multiple experimental approaches were used to understand the biology of the fusion-oncogenes, including genomic, proteomic, chemical, and genetic inhibition. These approaches identify potential new mechanisms of convergent fusion-oncogene activity, around TEAD transcriptional targeting (that is YAP/TAZ independent) and reveal CBP/p300 as a functional dependency.

(2) Complementary models were used, including cell-based assays and mouse allograft models to show the dependency on CBP/P300.

(3) Co-IPs were clear and convincing and showed direct interaction of the fusion-oncogene with ectopic and endogenous TEAD1/pan-TEAD, but not YAP/TAZ.

(4) Potential to follow-up on additional targets/mechanisms of tumorigenesis. For example, in the BioID proteomics screen, a unique VGLL2::NCOA2 and TEAD::NCOA2 interactor is P53, which also is an enriched pathway in Figure 4C in the p300 CUT&RUN peaks in the VGLL2::NCOA2 and TEAD1::NCOA2 expressing cells - is this indicative of the toxicity of the fusion-oncogenes or do you think this informs potential mechanisms for transformation.

Weaknesses:

(1) The rationale for performing genomics, transcriptional, and proteomics work in 293T cells is not discussed. Further, there are no functional readouts mentioned in the 293T cells with expression of the fusion-oncogenes. Did these cells have any phenotypes associated with fusion-oncogene expression (proliferation differences, morphological changes, colony formation capacity)? Further, how similar are the gene expression signatures from RNA-seq to rhabdomyosarcoma? This would help the reader interpret how similar these cell models are to human disease.

(2) TEAD1::NCOA2 fusion-oncogene model was not credentialed past H&E, and expression of Desmin. Is the transcriptional signature in C2C12 or 293T similar to a rhabdomyosarcoma gene signature?

(3) For the fusion-oncogenes, did the HA, FLAG, or V5 tag impact fusion-oncogene activity? Was the tag on the 3' or 5' of the fusion? This was not discussed in the methods.

(4) Generally, the lack of details in the figures, figure legends, and methods make the data difficult to interpret. A few examples are below:

a. Individual data points are not shown for figure bar plots (how many technical or biological replicates are present and how many times was the experiment repeated?).<br /> b. What exons were included in the fusion-oncogenes from VGLL2 and NCOA2 or TEAD1 and NCOA2?<br /> c. For how long were the colony formation experiments performed? Two weeks?<br /> d. In Figure 2D, what concentration of CP1 was used and for how long?<br /> e. How was A485 resuspended for cell culture and mouse experiments, what is the percentage of DMSO?<br /> f. How many replicates were done for RNA-seq, CUT&RUN, and ATACseq experiments?

Reviewer #2 (Public Review):

In the manuscript entitled "VGLL2 and TEAD1 fusion proteins drive YAP/TAZ-independent transcription and tumorigenesis by engaging p300", Gu et al. studied two Hippo pathway-related gene fusion events (i.e., VGLL2-NCOA2, TEAD1-NCOA2) in spindle cell rhabdomyosarcoma (scRMS) and showed that their fusion proteins can activate Hippo downstream gene transcription independent of YAP/TAZ. Using the BioID-based mass spectrometry analysis, the authors revealed histone acetyltransferase CBP/p300 as specific binding proteins for VGLL2-NCOA2 and TEAD1-NCOA2 fusion proteins. Pharmacologically targeting p300 inhibited the fusion proteins-induced Hippo downstream gene transcription and tumorigenic events.

Overall, this study provides mechanistic insights into the scRMS-associated gene fusions in tumorigenesis and reveals potential therapeutic targets for cancer treatment. The manuscript is well-written and easy to follow.

Here, several suggestions are made for the authors to improve their study.

Main points

(1) The authors majorly focused on the Hippo downstream gene transcription in this study, while a significant portion of genes regulated by the VGLL2-NCOA2 and TEAD1-NCOA2 fusion proteins are non-Hippo downstream genes (Figure 3). The authors should investigate whether the altered Hippo pathway transcription is essential for VGLL2-NCOA2 and TEAD1-NCOA2-induced cell transformation and tumorigenesis. Specifically, they should test if treatment with the TEAD inhibitor can reverse the cell transformation and tumorigenesis caused by VGLL2-NCOA2 but not TEAD1-NCOA2. In addition, it is important to examine whether YAP-5SA expression can rescue the inhibitory effects of A485 on VGLL2-NCOA2 and TEAD1-NCOA2-induced colony formation and tumor growth. This will help clarify whether Hippo downstream gene transcription is important for the oncogenic activities of these two fusion proteins.

(2) Rationale for selecting CBP/p300 for functional studies needs to be provided. The BioID-MS experiment identified many interacting proteins for VGLL2-NCOA2 and TEAD1-NCOA2 fusion proteins (Table S4). The authors should explain the scoring system used to identify the high-interacting proteins for VGLL2-NCOA2 and TEAD1-NCOA2 fusion proteins. Was CEP/p300 the top candidates on the list? Providing this information will help justify the focus on CBP/p300 and validate their importance in this study.

(3) p300 was revealed as a key driver for the VGLL2-NCOA2 and TEAD1-NCOA2 fusion proteins-induced transcriptome alteration and tumorigenesis. To strengthen the point, the authors should identify the p300 binding region on VGLL2-NCOA2 and TEAD1-NCOA2 fusion proteins. Mutants with defects in p300 binding/recruitment should be generated and included as a control in the related q-PCR and tumorigenic studies. This work will help confirm the crucial role of p300 in mediating the oncogenic effects of these two fusion proteins.

(4) Another major issue is the overexpression system extensively used in this study. It is important to determine whether the VGLL2-NCOA2 and TEAD1-NCOA2 fusion genes are also amplified in cancer. If not, the expression levels of the VGLL2-NCOA2 and TEAD1-NCOA2 fusion proteins should be adjusted to endogenous levels to assess their oncogenic effects on gene transcription and tumorigenesis. This approach would make the study more relevant to the pathological conditions observed in scRMS cancer patients.

Résumé de la vidéo [00:00:00][^1^][1] - [00:31:36][^2^][2]:

Cette vidéo présente une session académique sur le grand oral du baccalauréat français, avec des conseils et des témoignages d'étudiants et de formateurs. Les intervenants discutent de la préparation, de la gestion du stress, de la posture, de l'interaction avec le jury et de la présentation du support visuel.

Points forts: + [00:00:09][^3^][3] Introduction et présentation des intervenants * Raphaëlle Boss de Renois, enseignante et formatrice, introduit la session * Claire Rain, enseignante en lettres, spécialisée dans la posture et la voix + [00:01:01][^4^][4] Témoignages d'étudiants sur le grand oral * Des étudiants partagent leur expérience et leurs stratégies de préparation * Conseils sur la gestion du stress et l'importance de ne pas apprendre par cœur + [00:02:18][^5^][5] Cadre réglementaire du grand oral * Explication des dates, des coefficients et de la structure de l'épreuve * Importance de la question posée et de l'argumentation + [00:04:49][^6^][6] Conseils sur la posture et la voix * Importance de la stabilité, de la respiration et de l'articulation * Utilisation des mains pour animer le discours + [00:07:56][^7^][7] Déroulement de l'épreuve * Processus de sélection de la question par le jury et de la préparation * Utilisation de supports visuels et interaction avec le jury + [00:14:12][^8^][8] Gestion du stress et techniques de mémorisation * Astuces pour gérer le stress et renforcer la confiance en soi * Techniques de répétition et d'interaction pour mémoriser le contenu

eLife assessment

Approaches for quantifying synaptic activity events are currently limited, and recent advances in AI and deep learning provide an opportunity to develop powerful new ways to automate this process. In this study, the authors have generated a valuable tool, miniML, that uses open-source software that convincingly enables rapid, automated, and accurate quantification of synaptic events from a variety of systems and approaches. This software will be of significant utility to a variety of neuroscience researchers.

eLife assessment

This study provides a novel and promising NPRL2 gene therapy for enhanced immunotherapy response in a KRAS/STK11 mutant anti-PD1 resistant metastatic NSCLC humanized mouse model. Overall, the authors presented a large amount of convincing in vivo data to demonstrate that NPRL2 gene therapy induces antitumor activity through DC-mediated antigen presentation and cytotoxic immune cell activation. This work will be of interest and useful to medical biologists and oncologists in the research field of KRAS-mutant NSCLC.

Reviewer #1 (Public Review):

This study excellently complements the previous one by unveiling the properties of NPRL2 in augmenting the effect of immune checkpoint inhibitors such as pembrolizumab in KRAS mutant lung cancer models.

The following points should be clarified:

(1) In KRAS mutant cell lines with LKB1 co-mutations or deletions, such as A549 cells, does treatment with NPRL2 not increase the efficacy of immunotherapy? Is this correct? Similarly, does the delivery of NPRL2 only potentiate the effect of immunotherapy in KRAS mutant cell lines without associated LKB1 mutations?

(2) Do the authors analyze by western blot if NPRL2 influences or restores STING and LKB1 in the A549 cell line that lacks LKB1 and STING?

(3) Mechanistically, is there any explanation as to why NPRL2 delivery increases the efficacy of immunotherapy? Is there any effect on FUS or MYC?

(4) Is there any way to carry out a clinical study of systematically delivering NPRL2 in KRAS lung cancer patients?

Reviewer #2 (Public Review):

Summary:

NPRL2 gene therapy induces effective antitumor immunity in KRAS/STK11 mutant anti-PD1 resistant metastatic non-small cell lung cancer (NSCLC) in a humanized mouse model by Meraz et al investigated the antitumor immune responses to NPRL2 gene therapy in aPD1R / KRAS/STK11mt NSCLC in a humanized mouse model, and found that NPRL2 gene therapy induces antitumor activity on KRAS/STK11mt/aPD1R tumors through DC-mediated antigen presentation and cytotoxic immune cell activation.

Strengths:

The novelty of the study.

Weaknesses:

(1) The inconsistent effect of NPRL2 combined with pembrolizumab. Figure 2I-K, showed a similar tumor intensity in the NPRL2 group and combination group. However, NPRL2 combined with pembrolizumab was synergistic in the KRASwt/aPD1S H1299 tumors in Figure 4.

(2) The authors stated that NPRL2 combined with pembrolizumab was not synergistic in the KRAS/STK11mt/aPD1R tumors but was synergistic in the KRASwt/aPD1S H1299 tumors. How did the synergistic effect defined in the study, more details need to be provided here.

(3) Nearly all of the work was performed pre-clinically. Validation in the clinical setting would provide more strong evidence for the conclusion.

(4) Figure 5 and Figure 6 have the same legend. These 2 figures could be merged as a new one.

(5) Figure 5B & C, n=9 in the Figure 5B. However, the detail number in Figure 5C was less than 9.

Reviewer #3 (Public Review):

Summary:

NPRL2/TUSC4 is a tumor suppressor gene whose expression is reduced in many cancers including NSCLC. This study presents a novel finding on NPRL2 gene therapy, which induces antitumor activity on aPD1-resistant tumors. Since KRAS/STK11 mutant tumors were reported to be less benefited from ICIs, this study has potential clinical application value.

Strengths:

This work uncovers the advantage of NPRL2 gene therapy by using humanized models and multiple cell lines. Moreover, via immune cell depletion studies, the mechanism of NPRL2 gene therapy has focused on dendritic cells and CD8+T cells.

Weaknesses:

A major concern would be the lack of systematic, and logical rigor. This work did not present a link between apoptosis and antigen presenting induced by NPRL2 restoration. There is no evidence proving that the PI3K/AKT/mTOR signaling pathway is related to antigen presenting, which is the major reason of NPRL2 induced antitumor response. Therefore, the two parts may not support each other logically.

Résumé de la vidéo [00:00:00][^1^][1] - [01:05:06][^2^][2]:

La vidéo présente une intervention académique sur l'importance de la médiation artistique et culturelle dans le développement langagier, cognitif et social dès la petite enfance. Elle met en lumière le rôle de la musique et des arts dans ce processus et discute des recherches menées au Baby Lab de Grenoble.

Points forts: + [00:00:05][^3^][3] Introduction de l'événement * Accueil chaleureux et reconnaissance du travail quotidien * Importance de partager théorie et pratique + [00:00:44][^4^][4] Présentation de Christelle Dreyfuss * Directrice du Pôle du spectacle vivant * Engagement dans la médiation artistique et culturelle + [00:03:11][^5^][5] Intervention de Hélène Loevenbruck * Directrice de recherche au laboratoire de psychologie et neurocognition * Études sur l'impact des interventions musicales sur le développement des enfants + [00:04:36][^6^][6] Le Baby Lab de Grenoble * Plateforme de recherche sur la cognition des enfants * Expériences pour étudier le développement cognitif et langagier + [00:06:54][^7^][7] Importance du langage * Fonctions communicatives, cognitives et autonoétiques * Renforcement de la mémoire et de la prise de décision + [00:35:10][^8^][8] Résultats des recherches * Augmentation du vocabulaire chez les enfants exposés à la musique * Projet Baby News confirmant l'importance de la musique dans le développement du langage

not surveys? is that bc opinion-gathering is too much about hypothetical future actions and doesn't validate via existing present action?

I guess the reception to an artifact is inherently a survey—although it constrains the reception to the vehicle of the artifact's form

is this slop-shipping?

i was doing too much of this and ambient input-ingesting hoping that the spark of genius would come to me while just living life

LMAO

it has to be just out of reach

Résumé de la vidéo [00:00:05][^1^][1] - [00:41:59][^2^][2]:

Cette vidéo est un webinaire sur la réforme des lycées professionnels (LP) en France, axé sur la prévention du décrochage scolaire et l'orientation des élèves. Les intervenants discutent des dispositifs mis en place pour soutenir les élèves en risque de décrochage, notamment le parcours aménagé de formation initiale (PAFI TDO) et le parcours ambition emploi (PAE). Ils soulignent l'importance de l'alliance éducative avec les partenaires et du changement de posture de l'enseignant pour accompagner les élèves dans leur parcours.

Points forts: + [00:00:05][^3^][3] Présentation des intervenants * Introduction des responsables de l'orientation et de la lutte contre le décrochage scolaire * Présentation du vice-président du CPLDS (lutte contre le décrochage scolaire) + [00:01:07][^4^][4] Annonce de la généralisation du BTS * Discussion sur la consolidation du BTS dans tous les établissements * Présentation des thématiques des webinaires à venir + [00:02:08][^5^][5] Focus sur le parcours ambition emploi et dispositifs associés * Explication du parcours ambition emploi (PAE) et du dispositif tout droit ouvert (TDO) * Importance de l'alliance éducative et du rôle stratégique de l'enseignant + [00:06:01][^6^][6] Certificat de professionnalisation en matière de lutte contre le décrochage scolaire (CPLDS) * Présentation du CPLDS et de sa structure de formation * Objectifs de la certification et public éligible + [00:09:02][^7^][7] Extension du dispositif avenir pro * Élargissement du dispositif avenir pro pour les élèves de terminale CAP et baccalauréat professionnel * Objectif d'entretiens avec les conseillers pour préparer la projection post-diplôme + [00:22:03][^8^][8] Détails sur le parcours aménagé de formation initiale (PAFI TDO) * Présentation du PAFI TDO comme mesure de prévention du décrochage * Analyse des besoins des élèves et proposition de solutions adaptées + [00:26:22][^9^][9] Durée et mise en œuvre du PAE * Discussion sur la durée limitée du PAE et l'importance de l'anticipation * Analyse des freins et leviers pour une meilleure mise en œuvre + [00:32:41][^10^][10] L'importance du portfolio pour les élèves * L'importance d'apprendre aux élèves à créer et enrichir leur portfolio * Utilisation du portfolio pour la valorisation des compétences et expériences + [00:38:01][^11^][11] Conclusion et contacts pour plus d'informations * Récapitulatif des dispositifs et invitation à contacter les intervenants pour des compléments * Ressources disponibles en ligne pour approfondir les sujets abordés

eLife assessment

This work proposes that positive biodiversity-ecosystem functioning relationships found in experiments have been exaggerated because commonly used statistical analyses are flawed. As an alternative, the authors suggest a new analysis based on species competitive responses. Unfortunately, the presented methods are not reproducibly described, not yet complete, and inadequate for hypothesis testing. The reviewers agreed that the authors have either misinterpreted or chosen not to take into account much of the current research literature in the field of plant competition and biodiversity research.

Reviewer #1 (Public Review):

[Editors' note: this is an overall synthesis from the Reviewing Editor in consultation with the reviewers.]

The three reviews expand our critique of this manuscript in some depth and complementary directions. These can be synthesized in the following main points (we point out that there is quite a bit more that could be written about the flaws with this study; however, time constraints prevented us from further elaborating on the issues we see):

(1) It is unclear what the authors want to do. It seems their main point is that the large BEF literature and especially biodiversity experiments overstate the occurrence of positive biodiversity effects because some of these can result from competition. Because reduced interspecific relative to intraspecific competition in mixture is sufficient to produce positive effects in mixtures (if interspecific competition = 0 then RYT = S, where S is species richness in mixture -- this according to the reciprocal yield law = law of constant final yield), they have a problem accepting NE > 0 as true biodiversity effect (see additive partitioning method of Loreau & Hector 2001 cited in manuscript).

(2) The authors' next claim, without justification, that additive partitioning of NE is flawed and theoretically and biologically meaningless. They misinterpret the CE component as biological niche partitioning and the SE component as biological dominance. They do not seem to accept that the additive partitioning is a logically and mathematically sound derivation from basic principles that cannot be contested.

(3) The authors go on to introduce a method to calculate species-level overyielding (RY > 1/S in replacement series experiments) as a competitive growth response and multiply this with the species monoculture biomass relative to the maximum to obtain competitive expectation. This method is based on resource competition and the idea that resource uptake is fully converted into biomass (instead of e.g. investing it in allelopathic chemical production).

(4) It is unclear which experiments should be done, i.e. are partial-density monocultures planted or simply calculated from full-density monocultures? At what time are monocultures evaluated? The framework suggests that monocultures must have the full potential to develop, but in experiments, they are often performing very poorly, at least after some time. I assume in such cases the monocultures could not be used.

(5) There are many reasons why the ideal case of only resource competition playing a role is unrealistic. This excludes enemies but also differential conversion factors of resources into biomass and antagonistic or facilitative effects. Because there are so many potential reasons for deviations from the null model of only resource competition, a deviation from the null model does not allow conclusions about underlying mechanisms.

Furthermore, this is not a systematically developed partitioning, but some rather empirical ad hoc formulation of a first term that is thought to approximate competitive effects as understood by the authors (but again, there already are problems here). The second residual term is not investigated. For a proper partitioning approach, one would have to decompose overyielding into two (or more) terms and demonstrate (algebraically) that under some reasonable definitions of competitive and non-competitive interactions, these end up driving the respective terms.

(6) Using a simplistic simulation to test the method is insufficient. For example, I do not see how the simulation includes a mechanism that could create CE in additive partitioning if all species would have the same monoculture yield. Similarly, they do not include mechanisms of enemies or antagonistic interactions (e.g. allelopathy).

(7) The authors do not cite relevant literature regarding density x biodiversity experiments, competition experiments, replacement-series experiments, density-yield experiments, additive partitioning, facilitation, and so on.

Overall, this manuscript does not lead further from what we have already elaborated in the broad field of BEF and competition studies and rather blurs our understanding of the topic.

Reviewer #2 (Public Review):

This manuscript is motivated by the question of what mechanisms cause overyielding in mixed-species communities relative to the corresponding monocultures. This is an important and timely question, given that the ultimate biological reasons for such biodiversity effects are not fully understood.

As a starting point, the authors discuss the so-called "additive partitioning" (AP) method proposed by Loreau & Hector in 2001. The AP is the result of a mathematical rearrangement of the definition of overyielding, written in terms of relative yields (RY) of species in mixtures relative to monocultures. One term, the so-called complementarity effect (CE), is proportional to the average RY deviations from the null expectations that plants of both species "do the same" in monocultures and mixtures. The other term, the selection effect (SE), captures how these RY deviations are related to monoculture productivity. Overall, CE measures whether relative biomass gains differ from zero when averaged across all community members, and SE, whether the "relative advantage" species have in the mixture, is related to their productivity. In extreme cases, when all species benefit, CE becomes positive. When large species have large relative productivity increases, SE becomes positive. This is intuitively compatible with the idea that niche complementarity mitigates competition (CE>0), or that competitively superior species dominate mixtures and thereby driver overyielding (SE>0).

However, it is very important to understand that CE and SE capture the "statistical structure" of RY that underlies overyielding. Specifically, CE and SE are not the ultimate biological mechanisms that drive overyielding, and never were meant to be. CE also does not describe niche complementarity. Interpreting CE and SE as directly quantifying niche complementarity or resource competition, is simply wrong, although it sometimes is done. The criticism of the AP method thus in large part seems unwarranted. The alternative methods the authors discuss (lines 108-123) are based on very similar principles.

The authors now set out to develop a method that aims at linking response patterns to "more true" biological mechanisms.

Assuming that "competitive dominance" is key to understanding mixture productivity, because "competitive interactions are the predominant type of interspecific relationships in plants", the authors introduce "partial density" monocultures, i.e. monocultures that have the same planting density for a species as in a mixture. The idea is that using these partial density monocultures as a reference would allow for isolating the effect of competition by the surrounding "species matrix".

The authors argue that "To separate effects of competitive interactions from those of other species interactions, we would need the hypothesis that constituent species share an identical niche but differ in growth and competitive ability (i.e., absence of positive/negative interactions)." - I think the term interaction is not correctly used here, because clearly competition is an interaction, but the point made here is that this would be a zero-sum game.

The authors use the ratio of productivity of partial density and full-density monocultures, divided by planting density, as a measure of "competitive growth response" (abbreviated as MG). This is the extra growth a plant individual produces when intraspecific competition is reduced.

Here, I see two issues: first, this rests on the assumption that there is only "one mode" of competition if two species use the same resources, which may not be true, because intraspecific and interspecific competition may differ. Of course, one can argue that then somehow "niches" are different, but such a niche definition would be very broad and go beyond the "resource set" perspective the authors adopt. Second, this value will heavily depend on timing and the relationship between maximum initial growth rates and competitive abilities at high stand densities.

The authors then progress to define relative competitive ability (RC), and this time simply uses monoculture biomass as a measure of competitive ability. To express this biomass in a standardized way, they express it as different from the mean of the other species and then divide by the maximum monoculture biomass of all species.

I have two concerns here: first, if competitive ability is the capability of a species to preempt resources from a pool also accessed by another species, as the authors argued before, then this seems wrong because one would expect that a species can simply be more productive because it has a broader niche space that it exploits. This contradicts the very narrow perspective on competitive ability the authors have adopted. This also is difficult to reconcile with the idea that specialist species with a narrow niche would outcompete generalist species with a broad niche. Second, I am concerned by the mathematical form. Standardizing by the maximum makes the scaling dependent on a single value.

As a final step, the authors calculate a "competitive expectation" for a species' biomass in the mixture, by scaling deviations from the expected yield by the product MG ⨯ RC. This would mean a species does better in a mixture when (1) it benefits most from a conspecific density reduction, and (2) has a relatively high biomass.

Put simply, the assumption would be that if a species is productive in monoculture (high RC), it effectively does not "see" the competitors and then grows like it would be the sole species in the community, i.e. like in the partial density monoculture.

Overall, I am not very convinced by the proposed method.

(1) The proposed method seems not very systematic but rather "ad hoc". It also is much less a partitioning method than the AP method because the other term is simply the difference. It would be good if the authors investigated the mathematical form of this remainder and explored its properties.. when does complementarity occur? Would it capture complementarity and facilitation?

(2) The justification for the calculation of MG and RC does not seem to follow the very strict assumptions of what competition (in the absence of complementarity) is. See my specific comments above.

(3) Overall, the manuscript is hard to read. This is in part a problem of terminology and presentation, and it would be good to use more systematic terms for "response patterns" and "biological mechanisms".

Examples:<br /> - on line 30, the authors write that CE is used to measure "positive" interactions and SE to measure "competitive interactions", and later name "positive" and "negative" interactions "mechanisms of species interactions". Here the authors first use "positive interaction" as any type of effect that results in a community-level biomass gain, but then they use "interaction" with reference to specific biological mechanisms (e.g. one species might attract a parasite that infests another species, which in turn may cause further changes that modify the growth of the first and other species).

- on line 70, the authors state that "positive interaction" increases productivity relative to the null expectation, but it is clear that an interaction can have "negative" consequences for one interaction partner and "positive" ones for the other. Therefore, "positive" and "negative" interactions, when defined in this way, cannot be directly linked to "resource partitioning" and "facilitation", and "species interference" as the authors do. Also, these categories of mechanisms are still simple. For example, how do biotic interactions with enemies classify, see above?

- line 145: "Under the null hypothesis, species in the mixture are assumed to be competitively equivalent (i.e., absence of interspecific interactions)". This is wrong. The assumption is that there are interspecific interactions, but that these are the same as the intraspecific ones. Weirdly, what follows is a description of the AP method, which does not belong here. This paragraph would better be moved to the introduction where the AP method is mentioned. Or omitted, since it is basically a repetition of the original Loreau & Hector paper.

Other points:

- line 66: community productivity, not ecosystem productivity.<br /> - line 68: community average responses are with respect to relative yields - this is important!<br /> - line 64: what are "species effects of species interactions" ?<br /> - line 90: here "competitive" and "productive" are mixed up, and it is important to state that "suffers more" refers to relative changes, not yield changes.<br /> - line 92: "positive effect of competitive dominance": I don't understand what is meant here.

Reviewer #3 (Public Review):

Summary:

This manuscript by Tao et al. reports on an effort to better specify the underlying interactions driving the effects of biodiversity on productivity in biodiversity experiments. The authors are especially concerned with the potential for competitive interactions to drive positive biodiversity-ecosystem functioning relationships by driving down the biomass of subdominant species. The authors suggest a new partitioning schema that utilizes a suite of partial density treatments to capture so-called competitive ability. While I agree with the authors that understanding the underlying drivers of biodiversity-ecosystem functioning relationships is valuable - I am unsure of the added value of this specific approach for several reasons.

Strengths:

I can find a lot of value in endeavouring to improve our understanding of how biodiversity-ecosystem functioning relationships arise. I agree with the authors that competition is not well integrated into the complementarity and selection effect and interrogating this is important.

Weaknesses:

(1) The authors start the introduction very narrowly and do not make clear why it is so important to understand the underlying mechanisms driving biodiversity-ecosystem functioning relationships until the end of the discussion.

(2) The authors criticize the existing framework for only incorporating positive interactions but this is an oversimplification of the existing framework in several ways:<br /> a. The existing partitioning scheme incorporates resource partitioning which is an effect of competition.<br /> b. The authors neglect the potential that negative feedback from species-specific pests and pathogens can also drive positive BEF and complementarity effects but is not a positive interaction, necessarily. This is discussed in Schnitzer et al. 2011, Maron et al. 2011, Hendriks et al. 2013, Barry et al. 2019, etc.<br /> c. Hector and Loreau (and many of the other citations listed) do not limit competition to SE because resource partitioning is a byproduct of competition.

(3) It is unclear how this new measure relates to the selection effect, in particular. I would suggest that the authors add a conceptual figure that shows some scenarios in which this metric would give a different answer than the traditional additive partition. The example that the authors use where a dominant species increases in biomass and the amount that it increases in biomass is greater than the amount of loss from it outcompeting a subdominant species is a general example often used for a selection effect when exactly would you see a difference between the two? :<br /> a. Just a note - I do think you should see a difference between the two if the species suffers from strong intraspecific competition and has therefore low monoculture biomass but this would tend to also be a very low-density monoculture in practice so there would potentially be little difference between a low density and high-density monoculture because the individuals in a high-density monoculture would die anyway. So I am not sure that in practice you would really see this difference even if partial density plots were incorporated.

(4) One of the tricky things about these endeavors is that they often pull on theory from two different subfields and use similar terminology to refer to different things. For example - in competition theory, facilitation often refers to a positive relative interaction index (this seems to be how the authors are interpreting this) while in the BEF world facilitation often refers to a set of concrete physical mechanisms like microclimate amelioration. The truth is that both of these subfields use net effects. The relative interaction index is also a net outcome as is the complementarity effect even if it is only a piece of the net biodiversity effect. Trying to combine these two subfields to come up with a new partitioning mechanism requires interrogating the underlying assumptions of both subfields which I do not see in this paper.

(5) The partial density treatment does not isolate competition in the way that the authors indicate. All of the interactions that the authors discuss are density-dependent including the mechanism that is not discussed (negative feedback from species-specific pests and pathogens). These partial density treatment effects therefore cannot simply be equated to competition as the authors indicate.:<br /> a. Additionally - the authors use mixture biomass as a stand-in for competitive ability in some cases but mixture biomass could also be determined by the degree to which a plant is facilitated in the mixture (for example).

(6) I found the literature citation to be a bit loose. For example, the authors state that the additive partition is used to separate positive interactions from competition (lines 70-76) and cite many papers but several of these (e.g. Barry et al. 2019) explicitly do not say this.

(7) The natural take-home message from this study is that it would be valuable for biodiversity experiments to include partial density treatments but I have a hard time seeing this as a valuable addition to the field for two reasons:<br /> a. In practice - adding in partial density treatments would not be feasible for the vast majority of experiments which are already often unfeasibly large to maintain.<br /> b. The density effect would likely only be valuable during the establishment phase of the experiment because species that are strongly limited by intraspecific competition will die in the full-density plots resulting in low-density monocultures. You can see this in many biodiversity experiments after the first years. Even though they are seeded (or rarely planted) at a certain density, the density after several years in many monocultures is quite low.

Reviewer #4 (Public Review):

Summary:

This manuscript claims to provide a new null hypothesis for testing the effects of biodiversity on ecosystem functioning. It reports that the strength of biodiversity effects changes when this different null hypothesis is used. This main result is rather inevitable. That is, one expects a different answer when using a different approach. The question then becomes whether the manuscript's null hypothesis is both new and an improvement on the null hypothesis that has been in use in recent decades.

Strengths:

In general, I appreciate studies like this that question whether we have been doing it all wrong and I encourage consideration of new approaches.

Weaknesses:

Despite many sweeping critiques of previous studies and bold claims of novelty made throughout the manuscript, I was unable to find new insights. The manuscript fails to place the study in the context of the long history of literature on competition and biodiversity and ecosystem functioning. The Introduction claims the new approach will address deficiencies of previous approaches, but after reading further I see no evidence that it addresses the limitations of previous approaches noted in the Introduction. Furthermore, the manuscript does not reproducibly describe the methods used to produce the results (e.g., in Table 1) and relies on simulations, claiming experimental data are not available when many experiments have already tested these ideas and not found support for them. Finally, it is unclear to me whether rejecting the 'new' null hypothesis presented in the manuscript would be of interest to ecologists, agronomists, conservationists, or others. I will elaborate on each of these points below.

The critiques of biodiversity experiments and existing additive partitioning methods are overstated, as is the extent to which this new approach addresses its limitations. For example, the critique that current biodiversity experiments cannot reveal the effects of species interactions (e.g., lines 37-39) isn't generally true, but it could be true if stated more specifically. That is, this statement is incorrect as written because comparisons of mixtures, where there are interspecific and intraspecific interactions, with monocultures, where there are only intraspecific interactions, certainly provide information about the effects of species interactions (interspecific interactions). These biodiversity experiments and existing additive partitioning approaches have limits, of course, for identifying the specific types of interactions (e.g., whether mediated by exploitative resource competition, apparent competition, or other types of interactions). However, the approach proposed in this manuscript gets no closer to identifying these specific mechanisms of species interactions. It has no ability to distinguish between resource and apparent competition, for example. Thus, the motivation and framing of the manuscript do not match what it provides. I believe the entire Introduction would need to be rewritten to clarify what gap in knowledge this proposed approach is addressing and what would be gained by filling this knowledge gap.

I recommend that the Introduction instead clarify how this study builds on and goes beyond many decades of literature considering how competition and biodiversity effects depend on density. This large literature is insufficiently addressed in this manuscript. This fails to give credit to previous studies considering these ideas and makes it unclear how this manuscript goes beyond the many previous related studies. For example, see papers and books written by de Wit, Harper, Vandermeer, Connolly, Schmid, and many others. Also, note that many biodiversity experiments have crossed diversity treatments with a density treatment and found no significant effects of density or interactions between density and diversity (e.g., Finn et al. 2013 Journal of Applied Ecology). Thus, claiming that these considerations of density are novel, without giving credit to the enormous number of previous studies considering this, is insufficient.

Replacement series designs emerged as a consensus for biodiversity experiments because they directly test a relevant null hypothesis. This is not to say that there are no other interesting null hypotheses or study designs, but one must acknowledge that many designs and analyses of biodiversity experiments have already been considered. For example, Schmid et al. reviewed these designs and analyses two decades ago (2002, chapter 6 in Loreau et al. 2002 OUP book) and the overwhelming consensus in recent decades has been to use a replacement series and test the corresponding null hypothesis.

It is unclear to me whether rejecting the 'new' null hypothesis presented in the manuscript would be of interest to ecologists, agronomists, conservationists, or others. Most biodiversity experiments and additive partitions have tested and quantified diversity effects against the null hypothesis that there is no difference between intraspecific and interspecific interactions. If there was no less competition and no more facilitation in mixtures than in monocultures, then there would be no positive diversity effects. Rejecting this null hypothesis is relevant when considering coexistence in ecology, overyielding in agronomy, and the consequences of biodiversity loss in conservation (e.g., Vandermeer 1981 Bioscience, Loreau 2010 Princeton Monograph). This manuscript proposes a different null hypothesis and it is not yet clear to me how it would be relevant to any of these ongoing discussions of changes in biodiversity.

The claim that all previous methods 'are not capable of quantifying changes in ecosystem productivity by species interactions and species or community level' is incorrect. As noted above, all approaches that compare mixtures, where there are interspecific interactions, to monocultures, where there are no species interactions, do this to some extent. By overstating the limitations of previous approaches, the manuscript fails to clearly identify what unique contribution it is offering, and how this builds on and goes beyond previous work.

The manuscript relies on simulations because it claims that current experiments are unable to test this, given that they have replacement series designs (lines 128-131). There are, however, dozens of experiments where the replacement series was repeated at multiple densities, which would allow a direct test of these ideas. In fact, these ideas have already been tested in these experiments and density effects were found to be nonsignificant (e.g., Finn et al. 2013).

It seems that the authors are primarily interested in trees planted at a fixed density, with no opportunity for changes in density, and thus only changes in the size of individuals (e.g., Fig. 1). In natural and experimental systems, realized density differs from the initial planted density, and survivorship of seedlings can depend on both intraspecific and interspecific interactions. Thus, the constrained conditions under which these ideas are explored in this manuscript seem narrow and far from the more complex reality where density is not fixed.

Additional detailed comments:

It is unclear to me which 'effects' are referred to on line 36. For example, are these diversity effects or just effects of competition? What is the response variable?

The usefulness of the approach is overstated on line 52. All partitioning approaches, including the new one proposed here, give the net result of many types of species interactions and thus cannot 'disentangle underlying mechanisms of species interactions.'

The weaknesses of previous approaches are overstated throughout the manuscript, including in lines 60-61. All approaches provide some, but not all insights. Sweeping statements that previous approaches are not effective, without clarifying what they can and can't do, is unhelpful and incorrect. Also, these statements imply that the approach proposed here addresses the limitations of these previous approaches. I don't yet see how it does so.

The definitions given for the CE and SE on line 71 are incorrect. Competition affects both terms and CE can be negative or have nothing to do with positive interactions, as noted in many of the papers cited.

The proposed approach does not address the limitations noted on lines 73 and 74.

The definition of positive interactions in lines 77 and 78 seems inconsistent with much of the literature, which instead focuses on facilitation or mutualism, rather than competition when describing positive interactions.

Throughout the manuscript, competition is often used interchangeably with resource competition (e.g., line 82) and complementarity is often attributed to resource partitioning (e.g., line 77). This ignores apparent competition and partitioning enemy-free niche space, which has been found to contribute to biodiversity effects in many studies.

In what sense are competitive interactions positive for competitive species (lines 82-83)? By definition, competition is an interaction that has a negative effect. Do you mean that interspecific competition is less than intraspecific competition? I am having a very difficult time following the logic.

Results are asserted on lines 93-95, but I cannot find the methods that produced these results. I am unable to evaluate the work without a repeatable description of the methods.

The description of the null hypothesis in the common additive partitioning approach on lines 145-146 is incorrect. In the null case, it does not assume that there are no interspecific interactions, but rather that interspecific and intraspecific interactions are equivalent.

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eLife assessment

Here the authors present a useful extension of their previous method to cluster neuronal activity into cell assemblies (groups of neurons with correlated activity). The authors provide solid evidence that this method can identify temporal dynamics of neuronal clusters in sample simulated data, and they show how this method can be applied to whole-brain zebrafish data.

Reviewer #1 (Public Review):

Summary:

Understanding large-scale neural activity remains a formidable challenge in neuroscience. While several methods have been proposed to discover the assemblies from such large-scale recordings, most previous studies do not explicitly model the temporal dynamics. This study is an attempt to uncover the temporal dynamics of assemblies using a tool that has been established in other domains.

The authors previously introduced the compositional Restricted Boltzmann Machine (cRBM) to identify neuron assemblies in zebrafish brain activity. Building upon this, they now employ the Recurrent Temporal Restricted Boltzmann Machine (RTRBM) to elucidate the temporal dynamics within these assemblies. By introducing recurrent connections between hidden units, RTRBM could retrieve neural assemblies and their temporal dynamics from simulated and zebrafish brain data.

Strengths:

The RTRBM has been previously used in other domains. Training in the model has been already established. This study is an application of such a model to neuroscience. Overall, the paper is well-structured and the methodology is robust, the analysis is solid to support the authors' claim.

Weaknesses:

The overall degree of advance is very limited. The performance improvement by RTRBM compared to their cRBM is marginal, and insights into assembly dynamics are limited.

(1) The biological insights from this method are constrained. Though the aim is to unravel neural ensemble dynamics, the paper lacks in-depth discussion on how this method enhances our understanding of zebrafish neural dynamics. For example, the dynamics of assemblies can be analyzed using various tools such as dimensionality reduction methods once we have identified them using cRBM. What information can we gain by knowing the effective recurrent connection between them? It would be more convincing to show this in real data.

(2) Despite the increased complexity of RTRBM over cRBM, performance improvement is minimal. Accuracy enhancements, less than 1% in synthetic and zebrafish data, are underwhelming (Figure 2G and Figure 4B). Predictive performance evaluation on real neural activity would enhance model assessment. Including predicted and measured neural activity traces could aid readers in evaluating model efficacy.

Reviewer #2 (Public Review):

Summary:

In this work, the authors propose an extension to some of the last author's previous work, where a compositional restricted Boltzmann machine was considered as a generative model of neuron-assembly interaction. They augment this model by recurrent connections between the Boltzmann machine's hidden units, which allow them to explicitly account for temporal dynamics of the assembly activity. Since their model formulation does not allow the training towards a compositional phase (as in the previous model), they employ a transfer learning approach according to which they initialise their model with a weight matrix that was pre-trained using the earlier model so as to essentially start the actually training in a compositional phase. Finally, they test this model on synthetic and actual data of whole-brain light-sheet-microscopy recordings of spontaneous activity from the brain of larval zebrafish.

Strengths:

This work introduces a new model for neural assembly activity. Importantly, being able to capture temporal assembly dynamics is an interesting feature that goes beyond many existing models. While this work clearly focuses on the method (or the model) itself, it opens up an avenue for experimental research where it will be interesting to see if one can obtain any biologically meaningful insights considering these temporal dynamics when one is able to, for instance, relate them to development or behaviour.

Weaknesses:

For most of the work, the authors present their RTRBM model as an improvement over the earlier cRBM model. Yet, when considering synthetic data, they actually seem to compare with a "standard" RBM model. This seems odd considering the overall narrative, and it is not clear why they chose to do that. Also, in that case, was the RTRBM model initialised with the cRBM weight matrix?

A few claims made throughout the work are slightly too enthusiastic and not really supported by the data shown. For instance, when the authors refer to the clusters shown in Figure 3D as "spatially localized", this seems like a stretch, specifically in view of clusters 1, 3, and 4. Moreover, when they describe the predictive performance of their model as "close to optimal" when the down-sampling factor coincided with the interaction time scale, it seems a bit exaggerated given that it was more or less as close to the upper bound as it was to the lower bound.

When discussing the data statistics, the authors quote correlation values in the main text. However, these do not match the correlation values in the figure to which they seem to belong. Now, it seems that in the main text, they consider the Pearson correlation, whereas in the corresponding figure, it is the Spearman correlation. This is very confusing, and it is not really clear as to why the authors chose to do so.

Finally, when discussing the fact that the RTRBM model outperforms the cRBM model, the authors state it does so for different moments and in different numbers of cases (fish). It would be very interesting to know whether these are the same fish or always different fish.

Reviewer #3 (Public Review):

With ever-growing datasets, it becomes more challenging to extract useful information from such a large amount of data. For that, developing better dimensionality reduction/clustering methods can be very important to make sense of analyzed data. This is especially true for neuroscience where new experimental advances allow the recording of an unprecedented number of neurons. Here the authors make a step to help with neuronal analyses by proposing a new method to identify groups of neurons with similar activity dynamics. I did not notice any obvious problems with data analyses here, however, the presented manuscript has a few weaknesses:

(1) Because this manuscript is written as an extension of previous work by the same authors (van der Plas et al., eLife, 2023), thus to fully understand this paper it is required to read first the previous paper, as authors often refer to their previous work for details. Similarly, to understand the functional significance of identified here neuronal assemblies, it is needed to go to look at the previous paper.

(2) The problem of discovering clusters in data with temporal dynamics is not unique to neuroscience. Therefore, the authors should also discuss other previously proposed methods and how they compare to the presented here RTRBM method. Similarly, there are other methods using neural networks for discovering clusters (assemblies) (e.g. t-SNE: van der Maaten & Hinton 2008, Hippocluster: Chalmers et al. 2023, etc), which should be discussed to give better background information for the readers.

(3) The above point to better describe other methods is especially important because the performance of the presented here method is not that much better than previous work. For example, RTRBM outperforms the cRBM only on ~4 out of 8 fish datasets. Moreover, as the authors nicely described in the Limitations section this method currently can only work on a single time scale and clusters have to be estimated first with the previous cRBM method. Thus, having an overview of other methods which could be used for similar analyses would be helpful.

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