discutés

Est-ce que ce paragraphe n'aurait pas plus sa place après celui sur les étapes du travail ? Si on est pris, je pourrai participer à la rédaction de cette partie (surtout que les bibli ont pas toujours les infrastructures, selon les institutions, c'est parfois les presses, parfois les bibli, parfois la DSI... et ça renforce justement le côté "qui est le responsable" ?)

Il manque le "a"

beaucoup de par exemple, on peut enlever celui-ci qui en plus fait du bazar de parenthèses

Je pense qu'on peut juste s'arrêter à crucial, c'est crucial dans bcp de domaines

ça me semble très clair ! :)

pour que les presses universitaires soient en mesure de remplir leurs obligationS

Pour ma part, le 1er débrancher me semble très pertinent, et je proposerais "a disparu" ici

quels

confrontés, dans d'autres contextes, à des questions semblables aux nôtres.

d'institutions essentielles

décider ainsi du sort des œuvres.

pour éviter la répétition et jouer sur la métaphore : "jeter au pilon" ?

bisa juga tempelin CV interaktif yg dibuat pake Canva sih

caranya: 1. klik share 2. pilih embed 3. tempel di bio yg ada pada blog.uin-suka.ac.id

bisa juga cantumin blog personal atau medsos

gmn cara tahu kamu lebih lanjut atau cara reach kamu

sisi/versi lain dirimu, apa passionmu bisa ditaruh di sini

minat riset lainnya

area riset yang digeluti, usahakan lebih spesifik fokus riset

afiliasi aktif saat ini, bisa juga ditulis nama riset grup

foto, email, riwayat pendidikan hanya bisa diupdate via pak Aziz OKH

完成了

This section made me realize that storing personal data on social media is not just a technical question, but also an ethical one. For example, age can be stored as a number, but platforms still need to decide how precise it should be and how it might be used or misused. It also made me question whether some data, like exact address, really needs to be stored at all given the privacy risks.

In fact, I have always been puzzled about the collection of information such as "region" and "age". Is it really "necessary" for companies to collect such information? These pieces of information do not guarantee that the account is used by a real person - fake accounts can also randomly generate combinations of these pieces of information, but it will increase the risk of user information leakage

I found the discussion about representing gender as data especially thoughtful, because it shows how technical design decisions can have real social consequences. Treating gender as a simple binary might make data easier to process, but it can erase people’s identities and experiences. I also like the idea of combining preset options with an “other” field, since it balances inclusivity with the need for usable and consistent data.

to be changed to: https://api.razorpay.com/pg_router/v1/payments/pay_S3hhj7q9qpXKJ7/dcc_info

Can we remove this line for US?

Skill 和 skills

examples

Exercising is time-consuming, but it is essential for a healthy lifestyle ...

epidemiology focuses on preventing these diseases from occurring.

Nem kell a CIB-es anyagba

Töröljük ezt a részt teljesen

Ezt tegyük fel a Meeting notification sorhoz nem kell minden egyes Seev.001 sorba

Ez kerüljön fel Meeting notification cancellation szöveghez zárójelben

töröljük ezt a részt

3-4 days business days.

if any

This needs to be a physical address. No virtual addresses or P.O. Boxes allowed

Ez már szerepel az Értesítések küldése blokkban FAnni ez a komplett rész

EZt tegyük át légyszi . A vizsgálatok blokk alá a DTT tábla fölé

Data constraint plays a really important role in many social media platforms as they help set realistic boundaries when a person fills out their personal information. This also helps companies keep a record of what age the users usually are that uses their social media. Moreover, it all help prevent any possible trolls who will put random informations just to use the social media.

This is an important thing to consider when choosing how we store data and how we want to represent it. It is unrealistic to for example have someone who is 1000 years old. Another good case is if we only want to include a specific set of data but the user enters invalid characters which may distort our dataset.

Töröljük ezt a mondatot

Na szóval helyesen a mondat : Alletétkezelőtől, elszámolóháztól bejövő (MT567) szátuszüzenetek egy un. forgatótábla szerint kerülnek kiküldésre vagy megállításra ...

Ez ne link legyen FAnni csak annyi, hogy ,,,... egy forgatótábla szerint MT567 státusz üzenetek kerülnek kiküldésre vagy...

Dont understand this formula

The pixels that only have three color components have always made me really curious, as there is only green, blue,e and red, ed but once they are in a group,oup tcane to create a whole new different color that sometimes seems impossible. Additionally, one of the most intruiging thing about these grids is that with only these three colors, they can create the color white.

It’s cool to see how images are really just grids of pixels with RGB values, and even something like microRGB fits into that same idea of breaking color down into tiny components. Thinking about images this way makes them feel a lot less mysterious and more like something you can actually work with in code.

It’s interesting to think about how sound is really just a list of numbers that tell a speaker how to move, moment by moment, to recreate a noise or a voice. Once you see it that way, audio feels a lot less abstract and more like something you can store, edit, and mess with just like any other data.

I notice this sort of glitch sometimes when I'm using the app BeReal. When the notification goes off for everyone at the same time (no matter what time zone you are), the way the content is displayed to you is based entirely on what country you're currently in. Example- I have a friend who was visiting South Korea, and when the notification went off for us to take a photo through the app, it indicated that hers was 17 hours late. Interesting how there's a lack of solutions when technology has advanced so fast.

I find it so intriguing that, by simply posting a photo or tweet, a platform can gather immense amounts of data from the user. This type of data (metadata) is typically accessible to those who know their way around a computer, and one can assume how dangerous it can be when given to the wrong people.

As an international student, I have experience with this. Many social media platforms display the posting time based on the time zone of the viewer, i.e. the device time zone, when the publisher posts. That is to say, if you view the release time of the same tweet in different time zones, you will find that their release time has changed

I recently worked on a project using sound. All of the sound was stored as numbers like described here. The whole process of measuring physical sound waves is very interesting to learn about.

Instruction Processing Status Advice

Ezt a seev.31 alá tegyük be

ezeknél az eseményeknél nem épülnek tranzakciók

Kerüljön törlésre a fázis a CIB_nál

Ide nem kell a, b. csak sime bullet pontok legyenek

ezt törölhetjük

esemény létrehozása SWIFT üzenetből vagy kézzel történő rögzítése

Itt legyen még egy külön pont ezzel kezdődjön : MESSAGE FLOW: SWIFT társasági esemény üzenetek fogadása, megjelenítése külön felületen

Ez az ábra a másik ábra fölé kerüljön

opcionálisan az események...

társasági esemény üzenetek

Ide azt írjuk, opcionálisan az események ütemezett...

Platforms like Projectitude focus on providing ethical academic guidance that supports learning while maintaining originality and academic integrity.

Platforms like Projectitude focus on providing ethical academic guidance that supports learning while maintaining originality and academic integrity.

Very very nice - industry and leisurewere instricibly linked for the often working-class miners visiting Barry - their life was industry and so fittingly, their leisure (and pleasure) was watching their hardwork be shipped off across the globe from a beautiful vantage point on the beaches of Barry

Very interesting! Mitskell' arguement suggests how port and resort could co-exist peacefully, Croll goes further to argue that port and resort aided the growth of the other. In a prior chapter, Croll goes into depth with how the island's origins as a bathing resort brung attention to the area for industrialization, this was prohibited by the later owner Lord Windsor, however, upon his approval, the industrilisation of the area resulted in a boost for tourism which had suffered under Lord Windsor's ban!

Bathing machines were a significantly good sign - used for ladies to change and bath - it wasn't just a rough plae but a place where ladies could bathe and feel safe

idk need to waffle but brain cant lol

Highlights the leisure too - these were not tourists but residents. seaside resorts also became a place of genral recreation like the big parks in london - this is something mitskell doesn't highlight, but could be due to the resorts purpose moreso as a high-class resort - it still woulda had workers in the town tho

Very good - he highlights the weaknesses of primary sources available instead of making sweeping assumptions

Due to the ban, natural tourism of the beach was restricted - industrialisation helped revive this through the new 'industrial tourists', which then re-awakened the natural tourism again, people going against the visitor ban to barry island

new era where tourism became quite industry based. This is unlike mitskell's article yk?

Here we see a shift from a tourism based on natural beauty to industrial wonder - industrialisation here became a tourist attraction

Interesting use of primary source to back up his argument

Unlike Mitskell's article, Croll highlights how industrial elements could become a key part of the leisure industry through the advertisement of industrial ports as a tourist attraction, leisure and industrialisation having a strong and positive relationship in this example.

Naming a section of his work 'industrial tourists', Croll highlights a strog relationship between industrialisation and leisure through the new industrial workers who sought to utilise their new spending power and freetime through a trip to seaside resorts like Barr

Unlike Mitskell's choice of case study, Croll's decision of Barry has clear differences, it alludes greater to the impact of external factors like landowners on the relationship between industrialisation and tourism, with Lord Windsor, upon his purchase of Barry Island, Croll notes, banning visitors from the Island and prohibiting the becoming industrialisation of the Island. As such, Croll's choice of case study is interesting, and broadens the (complexities) of studying Welsh seaside resorts further. Through the case study, he suggests how landowners often decided the nature of the relationship between industrialisation and leisure, with the ammenities required for each at the whim of (blah)

Alludes to a differing relationship - industrialisation had caused the creation of large settlements of workers, who, with the railway, could now access (and afford) to engage in leisure activities in Barry

Industrialisation had hampered and then helped tourism, altho now largely for a more different class. Swansea seemed to remain more for the upperclass aided by industrialisation. This was unlike Barry which saw it's clientel shift more towards the woring classes.

Mitskell's point never really saw this break and overtake of industry, relationship was less frictional i think

• mitskell's case study doesn't seem to have this

confliction between industrialisation and leisure

Evidence of friction - the tourists didn't always like the industrialisation. Barry was especially known for it's lack of urbanisation and natural beuaty

Recommending Mitskell's article in a footnote at th eend of his introduction, Croll takes a similar view to his 2011 predeccessor, the example of Barry further proo that 'tourism could flourish alongside a commercial port', (do thing from option thing).

Written nine years after Mitskell's article, Croll places his work neatly within the historiography of the Welsh seaside, highlighting Borsay and Walton's thesis of the (expand girly). Like Mitskell, Borsay is a key historian of reference, and as such, it is unsurprising that both article's appear to highlight a less frictional relationship between industry and leisure through their different case studys.

eLife Assessment

This work significantly advances our understanding of chromatin organization within regions of repetitive sequences in the parasitic protozoan Trypanosoma brucei. Using cutting edge interdisciplinary tools, the authors provide compelling evidence for two discrete types of repetitive DNA element-associated proteins- one set involved in essential centromere function; and, the other involved in glycoprotein antigenic variation via homologous recombination. Thus, these fundamental findings have implications for this parasite's biology, and for therapeutic targeting in kinetoplastid diseases. This work will be exciting to those in the centromere/mitosis and parasite immunity fields.

[Editors' note: this paper was reviewed by Review Commons.]

Reviewer #1 (Public review):

Summary:

Carloni et al. comprehensively analyze which proteins bind repetitive genomic elements in Trypanosoma brucei. For this, they perform mass spectrometry on custom-designed, tagged programmable DNA-binding proteins. After extensively verifying their programmable DNA-binding proteins (using bioinformatic analysis to infer target sites, microscopy to measure localization, ChIP-seq to identify binding sites), they present, among others, two major findings: 1) 14 of the 25 known T. brucei kinetochore proteins are enriched at 177bp repeats. As T. brucei's 177bp repeat-containing intermediate-sized and mini-chromosomes lack centromere repeats but are stable over mitosis, Carloni et al. use their data to hypothesize that a 'rudimentary' kinetochore assembles at the 177bp repeats of these chromosomes to segregate them. 2) 70bp repeats are enriched with the Replication Protein A complex, which, notably, is required for homologous recombination. Homologous recombination is the pathway used for recombination-based antigenic variation of the 70bp-repeat-adjacent variant surface glycoproteins.

Strengths and Weaknesses:

The manuscript was previously reviewed through Review Commons. As noted there, the experiments are well controlled, the claims are well supported, and the methods are clearly described. The conclusions are convincing. All concerns I raised have been addressed except one (minor point #8):

"The way the authors mapped the ChIP-seq data is potentially problematic when analyzing the same repeat type in different genomic regions. Reads with multiple equally good mapping positions were assigned randomly. This is fine when analyzing repeats by type, independent of genomic position, which is what the authors do to reach their main conclusions. However, several figures (Fig. 3B, Fig. 4B, Fig. 5B, Fig. 7) show the same repeat type at specific genomic locations." Due to the random assignment, all of these regions merely show the average signal for the given repeat. I find it misleading that this average is plotted out at "specific" genomic regions.<br /> Initially, I suggested a workaround, but the authors clarified why the workaround was not feasible, and their explanation is reasonable to me. That said, the figures still show a signal at positions where they can't be sure it actually exists. If this cannot be corrected analytically, it should at least be noted in the figure legends, Results, or Discussion.

Importantly, the authors' conclusions do not hinge on this point; they are appropriately cautious, and their interpretations remain valid regardless.

Significance:

This work is of high significance for chromosome/centromere biology, parasitology, and the study of antigenic variation. For chromosome/centromere biology, the conceptual advancement of different types of kinetochores for different chromosomes is a novelty, as far as I know. It would certainly be interesting to apply this study as a technical blueprint for other organisms with mini-chromosomes or chromosomes without known centromeric repeats. I can imagine a broad range of labs studying other organisms with comparable chromosomes to take note of and build on this study. For parasitology and the study of antigenic variation, it is crucial to know how intermediate- and mini-chromosomes are stable through cell division, as these chromosomes harbor a large portion of the antigenic repertoire. Moreover, this study also found a novel link between the homologous repair pathway and variant surface glycoproteins, via the 70bp repeats. How and at which stages during the process, 70bp repeats are involved in antigenic variation is an unresolved, and very actively studied, question in the field. Of course, apart from the basic biological research audience, insights into antigenic variation always have the potential for clinical implications, as T. brucei causes sleeping sickness in humans and nagana in cattle. Due to antigenic variation, T. brucei infections can be chronic.

Comments on revised version:

All my recommendations have been addressed.

Reviewer #2 (Public review):

The Trypanosoma brucei genome, like that of other eukaryotes, contains diverse repetitive elements. Yet, the chromatin-associated proteome of these regions remains largely unexplored. This study represents a very important conceptual and technical advancement by employing synthetic TALE DNA-binding proteins fused to YFP to selectively capture proteins associated with specific repetitive sequences in T. brucei chromatin. The data presented here are convincing, supported by appropriate controls and a well-validated methodology, aligned with current state-of-the-art approaches.

The authors used synthetic TALE DNA binding proteins, tagged with YFP, which were designed to target five specific repeat elements in T. brucei genome, including centromere and telomeres-associated repeats and those of a transposon element. This is in order to identify specific proteins that bind to these repetitive sequences in T. brucei chromatin. Validation of the approach was done using a TALE protein designed to target the telomere repeat (TelR-TALE) that detected many of the proteins that were previously implicated with telomeric functions. A TALE protein designed to target the 70 bp repeats that reside adjacent to the VSG genes (70R-TALE) detected proteins that function in DNA repair and a protein designed to target the 177 bp repeat arrays (177R-TALE) identified kinetochore proteins associated T. brucei mega base chromosomes, as well as in intermediate and mini-chromosomes, which imply that kinetochore assembly and segregation mechanisms are similar in all T. brucei chromosomes.

This study represents a significant conceptual and technical advancement. To the best of our knowledge, it is the first report of employing TALE-YFP for affinity-based detection of protein complexes bound to repetitive genomic sequences in T. brucei. This approach enhances our understanding the organization in these important regions of the trypanosomal chromatin and provides the foundation for investigating the functional roles of associated proteins in parasite biology. These findings will be of particular interest to researchers studying the molecular biology of kinetoplastid parasites and other unicellular organisms, as well as to scientists investigating the roles of repetitive genomic elements in chromatin structure and their functional role in higher eukaryotes.

Importantly, any essential or unique interacting partners identified using the approach employed here, could serve as a potential target for therapeutic intervention in severe tropical diseases cause by kinetoplastids.

Author response:

Point-by-point description of the revisions:

Reviewer #1 (Evidence, reproducibility and clarity):

Summary

In this article, the authors used the synthetic TALE DNA binding proteins, tagged with YFP, which were designed to target five specific repeat elements in Trypanosoma brucei genome, including centromere and telomeres-associated repeats and those of a transposon element. This is in order to detect and identified, using YFP-pulldown, specific proteins that bind to these repetitive sequences in T. brucei chromatin. Validation of the approach was done using a TALE protein designed to target the telomere repeat (TelR-TALE) that detected many of the proteins that were previously implicated with telomeric functions. A TALE protein designed to target the 70 bp repeats that reside adjacent to the VSG genes (70R-TALE) detected proteins that function in DNA repair and the protein designed to target the 177 bp repeat arrays (177R-TALE) identified kinetochore proteins associated T. brucei mega base chromosomes, as well as in intermediate and mini-chromosomes, which imply that kinetochore assembly and segregation mechanisms are similar in all T. brucei chromosome.

Major comments:

Are the key conclusions convincing?

The authors reported that they have successfully used TALE-based affinity selection of proteinassociated with repetitive sequences in the T. brucei genome. They claimed that this study has provided new information regarding the relevance of the repetitive region in the genome to chromosome integrity, telomere biology, chromosomal segregation and immune evasion strategies. These conclusions are based on high-quality research, and it is, basically, merits publication, provided that some major concerns, raised below, will be addressed before acceptance for publication.

(1) The authors used TALE-YFP approach to examine the proteome associated with five different repetitive regions of the T. brucei genome and confirmed the binding of TALE-YFP with Chip-seq analyses. Ultimately, they got the list of proteins that bound to synthetic proteins, by affinity purification and LS-MS analysis and concluded that these proteins bind to different repetitive regions of the genome. There are two control proteins, one is TRF-YFP and the other KKT2-YFP, used to confirm the interactions. However, there are no experiment that confirms that the analysis gives some insight into the role of any putative or new protein in telomere biology, VSG gene regulation or chromosomal segregation. The proteins, which have already been reported by other studies, are mentioned. Although the author discovered many proteins in these repetitive regions, their role is yet unknown. It is recommended to take one or more of the new putative proteins from the repetitive elements and show whether or not they (1) bind directly to the specific repetitive sequence (e.g., by EMSA); (2) it is recommended that the authors will knockdown of one or a small sample of the new discovered proteins, which may shed light on their function at the repetitive region, as a proof of concept.

The main request from Referee 1 is for individual evaluation of protein-DNA interaction for a few candidates identified in our TALE-YFP affinity purifications, particularly using EMSA to identify binding to the DNA repeats used for the TALE selection. In our opinion, such an approach would not actually provide the validation anticipated by the reviewer. The power of TALE-YFP affinity selection is that it enriches for protein complexes that associate with the chromatin that coats the target DNA repetitive elements rather than only identifying individual proteins or components of a complex that directly bind to DNA assembled in chromatin.

The referee suggests we express recombinant proteins and perform EMSA for selected candidates, but many of the identified proteins are unlikely to directly bind to DNA – they are more likely to associate with a combination of features present in DNA and/or chromatin (e.g. specific histone variants or histone post-translational modifications). Of course, a positive result would provide some validation but only IF the tested protein can bind DNA in isolation – thus, a negative result would be uninformative.

In fact, our finding that KKT proteins are enriched using the 177R-TALE (minichromosome repeat sequence) identifies components of the trypanosome kinetochore known (KKT2) or predicted (KKT3) to directly bind DNA (Marciano et al., 2021; PMID: 34081090), and likewise the TelR-TALE identifies the TRF component that is known to directly associate with telomeric (TTAGGG)n repeats (Reis et al 2018; PMID: 29385523). This provides reassurance on the specificity of the selection, as does the lack of cross selectivity between different TALEs used (see later point 3 below). The enrichment of the respective DNA repeats quantitated in Figure 2B (originally Figure S1) also provides strong evidence for TALE selectivity.

It is very likely that most of the components enriched on the repetitive elements targeted by our TALE-YFP proteins do not bind repetitive DNA directly. The TRF telomere binding protein is an exception – but it is the only obvious DNA binding protein amongst the many proteins identified as being enriched in our TelR-TALE-YFP and TRF-YFP affinity selections.

The referee also suggests that follow up experiments using knockdown of the identified proteins found to be enriched on repetitive DNA elements would be informative. In our opinion, this manuscript presents the development of a new methodology previously not applied to trypanosomes, and referee 2 highlights the value of this methodological development which will be relevant for a large community of kinetoplastid researchers. In-depth follow-up analyses would be beyond the scope of this current study but of course will be pursued in future. To be meaningful such knockdown analyses would need to be comprehensive in terms of their phenotypic characterisation (e.g. quantitative effects on chromosome biology and cell cycle progression, rates and mechanism of recombination underlying antigenic variation, etc) – simple RNAi knockdowns would provide information on fitness but little more. This information is already publicly available from genome-wide RNAi screens (www.tritrypDB.org), with further information on protein location available from the genome-wide protein localisation resource (Tryptag.org). Hence basic information is available on all targets selected by the TALEs after RNAi knock down but in-depth follow-up functional analysis of several proteins would require specific targeted assays beyond the scope of this study.

(2) NonR-TALE-YFP does not have a binding site in the genome, but YFP protein should still be expressed by T. brucei clones with NLS. The authors have to explain why there is no signal detected in the nucleus, while a prominent signal was detected near kDNA (see Fig.2). Why is the expression of YFP in NonR-TALE almost not shown compared to other TALE clones?

The NonR-TALE-YFP immunolocalisation signal indeed is apparently located close to the kDNA and away from the nucleus. We are not sure why this is so, but the construct is sequence validated and correct. However, we note that artefactual localisation of proteins fused to a globular eGFP tag, compared to a short linear epitope V5 tag, near to the kinetoplast has been previously reported (Pyrih et al, 2023; PMID: 37669165).

The expression of NonR-TALE-YFP is shown in Supplementary Fig. S2 in comparison to other TALE proteins. Although it is evident that NonR-TALE-YFP is expressed at lower levels than other TALEs (the different TALEs have different expression levels), it is likely that in each case the TALE proteins would be in relative excess.

It is possible that the absence of a target sequence for the NonR-TALE-YFP in the nucleus affects its stability and cellular location. Understanding these differences is tangential to the aim of this study.

However, importantly, NonR-TALE-YFP is not the only control for used for specificity in our affinity purifications. Instead, the lack of cross-selection of the same proteins by different TALEs (e.g. TelR-TALE-YFP, 177R-TALE-YFP) and the lack of enrichment of any proteins of interest by the well expressed ingiR-TALE-YFP or 147R-TALE-YFP proteins each provide strong evidence for the specificity of the selection using TALEs, as does the enrichment of similar protein sets following affinity purification of the TelR-TALE-YFP and TRF-YFP proteins which both bind telomeric (TTAGGG)n repeats. Moreover, control affinity purifications to assess background were performed using cells that completely lack an expressed YFP protein which further support specificity (Figure 6).

We have added text to highlight these important points in the revised manuscript:

Page 8:

“However, the expression level of NonR-TALE-YFP was lower than other TALE-YFP proteins; this may relate to the lack of DNA binding sites for NonR-TALE-YFP in the nucleus.”

Page 8:

“NonR-TALE-YFP displayed a diffuse nuclear and cytoplasmic signal; unexpectedly the cytoplasmic signal appeared to be in the vicinity the kDNA of the kinetoplast (mitochrondria). We note that artefactual localisation of some proteins fused to an eGFP tag has previously been observed in T. brucei (Pyrih et al, 2023).”

Page 10:

Moreover, a similar set of enriched proteins was identified in TelR-TALE-YFP affinity purifications whether compared with cells expressing no YFP fusion protein (No-YFP), the NonR-TALE-YFP or the ingiR-TALE-YFP as controls (Fig. S7B, S8A; Tables S3, S4). Thus, the most enriched proteins are specific to TelR-TALE-YFP-associated chromatin rather than to the TALE-YFP synthetic protein module or other chromatin.

(3) As a proof of concept, the author showed that the TALE method determined the same interacting partners enrichment in TelR-TALE as compared to TRF-YFP. And they show the same interacting partners for other TALE proteins, whether compared with WT cells or with the NonR-TALE parasites. It may be because NonR-TALE parasites have almost no (or very little) YFP expression (see Fig. S3) as compared to other TALE clones and the TRF-YFP clone. To address this concern, there should be a control included, with proper YFP expression.

See response to point 2, but we reiterate that the ingi-TALE -YFP and 147R-TALE-YFP proteins are well expressed (western original Fig. S3 now Fig. S2) but few proteins are detected as being enriched or correspond to those enriched in TelR-TALE-YFP or TRF-YFP affinity purifications (see Fig. S9). Therefore, the ingi-TALE -YFP and 147R-TALE-YFP proteins provide good additional negative controls for specificity as requested. To further reassure the referee we have also included additional volcano plots which compare TelR-TALE-YFP, 70R-TALE-YFP or 177R-TALE-YFP to the ingiR-TALE-YFP affinity selection (new Figure S8). As with No-YFP or NonR-TALE-YFP controls, the use of ingiR-TALE-YFP as a negative control demonstrates that known telomere associated proteins are enriched in TelR-TALE-YFP affinity purification, RPA subunits enriched with 70R-TALE-YFP and Kinetochore KKT poroteins enriched with 177RTALE-YFP. These analyses demonstrate specificity in the proteins enriched following affinity purification of our different TALE-YFPs and provide support to strengthen our original findings.

We now refer to use of No-YFP, NonR-TALE-YFP, and ingiR-TALE -YFP as controls for comparison to TelR-TALE-YFP, 70R-TALE-YFP or 177R-TALE-YFP in several places:

Page10:

“Moreover, a similar set of enriched proteins was identified in TelR-TALE-YFP affinity purifications whether compared with cells expressing no YFP fusion protein (No-YFP), the NonR-TALE-YFP or the ingiR-TALE-YFP as controls (Fig. S7B, S8A; Tables S3, S4).”

Page 11:

“Thus, the nuclear ingiR-TALE-YFP provides an additional chromatin-associated negative control for affinity purifications with the TelR-TALE-YFP, 70R-TALE-YFP and 177R-TALE-YFP proteins (Fig. S8).”

“Proteins identified as being enriched with 70R-TALE-YFP (Figure 6D) were similar in comparisons with either the No-YFP, NonR-TALE-YFP or ingiR-TALE-YFP as negative controls.”

Top Page 12:

“The same kinetochore proteins were enriched regardless of whether the 177R-TALE proteomics data was compared with No-YFP, NonR-TALE or ingiR-TALE-YFP controls.”

Discussion Page 13:

“Regardless, the 147R-TALE and ingiR-TALE proteins were well expressed in T. brucei cells, but their affinity selection did not significantly enrich for any relevant proteins. Thus, 147R-TALE and ingiR-TALE provide reassurance for the overall specificity for proteins enriched TelR-TALE, 70R-TALE and 177R-TALE affinity purifications.”

(4) After the artificial expression of repetitive sequence binding five-TALE proteins, the question is if there is any competition for the TALE proteins with the corresponding endogenous proteins? Is there any effect on parasite survival or health, compared to the control after the expression of these five TALEs YFP protein? It is recommended to add parasite growth curves, for all the TALE proteins expressing cultures.

Growth curves for cells expressing TelR-TALE-YFP, 177R-TALE-YFP and ingiR-TALE-YFP are now included (New Fig S3A). No deficit in growth was evident while passaging 70R-TALE-YFP, 147R-TALE-YFP, NonR-TALE-YFP cell lines (indeed they grew slightly better than controls).

The following text has been added page 8:

“Cell lines expressing representative TALE-YFP proteins displayed no fitness deficit (Fig. S3A).”

(5) Since the experiments were performed using whole-cell extracts without prior nuclear fractionation, the authors should consider the possibility that some identified proteins may have originated from compartments other than the nucleus. Specifically, the detection of certain binding proteins might reflect sequence homology (or partial homology) between mitochondrial DNA (maxicircles and minicircles) and repetitive regions in the nuclear genome. Additionally, the lack of subcellular separation raises the concern that cytoplasmic proteins could have been co-purified due to whole cell lysis, making it challenging to discern whether the observed proteome truly represents the nuclear interactome.

In our experimental design, we confirmed bioinformatically that the repeat sequences targeted were not represented elsewhere in the nuclear or mitochondrial genome (kDNA). The absence of subcellular fractionation could result in some cytoplasmic protein selection, but this is unlikely since each TALE targets a specific DNA sequence but is otherwise identical such that cross-selection of the same contaminating protein set would be anticipated if there was significant non-specific binding. We have previously successfully affinity selected 15 chromatin modifiers and identified associated proteins without major issues concerning cytoplasmic protein contamination (Staneva et al 2021 and 2022; PMID: 34407985 and 36169304). Of course, the possibility that some proteins are contaminants will need to be borne in mind in any future follow-up analysis of proteins of interest that we identified as being enriched on specific types of repetitive element in T. brucei. Proteins that are also detected in negative control, or negative affinity selections such as No-YFP, NoR-YFP, IngiR-TALE or 147R-TALE must be disregarded.

(6) Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

As mentioned earlier, the author claimed that this study has provided new information concerning telomere biology, chromosomal segregation mechanisms, and immune evasion strategies. But there are no experiments that provides a role for any unknown or known protein in these processes. Thus, it is suggested to select one or two proteins of choice from the list and validate their direct binding to repetitive region(s), and their role in that region of interaction.

As highlighted in response to point 1 the suggested validation and follow up experiments may well not be informative and are beyond the scope of the methodological development presented in this manuscript. Referee 2 describes the study in its current form as “a significant conceptual and technical advancement” and “This approach enhances our understanding of chromatin organization in these regions and provides a foundation for investigating the functional roles of associated proteins in parasite biology.”

The Referee’s phrase ‘validate their direct binding to repetitive region(s)’ here may also mean to test if any of the additional proteins that we identified as being enriched with a specific TALE protein actually display enrichment over the repeat regions when examined by an orthogonal method. A key unexpected finding was that kinetochore proteins including KKT2 are enriched in our affinity purifications of the 177R-TALE-YFP that targets 177bp repeats (Figure 6F). By conducting ChIP-seq for the kinetochore specific protein KKT2 using YFP-KKT2 we confirmed that KKT2 is indeed enriched on 177bp repeat DNA but not flanking DNA (Figure 7). Moreover, several known telomere-associated proteins are detected in our affinity selections of TelRTALE-YFP (Figure 6B, FigS6; see also Reis et al, 2018 Nuc. Acids Res. PMID: 29385523; Weisert et al, 2024 Sci. Reports PMID: 39681615).

Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

The answer for this question depends on what the authors want to present as the achievements of the present study. If the achievement of the paper was is the creation of a new tool for discovering new proteins, associated with the repeat regions, I recommend that they add a proof for direct interactions between a sample the newly discovered proteins and the relevant repeats, as a proof of concept discussed above, However, if the authors like to claim that the study achieved new functional insights for these interactions they will have to expand the study, as mentioned above, to support the proof of concept.

See our response to point 1 and the point we labelled ‘6’ above.

Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

I think that they are realistic. If the authors decided to check the capacity of a small sample of proteins (which was unknown before as a repetitive region binding proteins) to interacts directly with the repeated sequence, it will substantially add of the study (e.g., by EMSA; estimated time: 1 months). If the authors will decide to check the also the function of one of at least one such a newly detected proteins (e.g., by KD), I estimate the will take 3-6 months.

As highlighted previously the proposed EMSA experiment may well be uninformative for protein complex components identified in our study or for isolated proteins that directly bind DNA in the context of a complex and chromatin. RNAi knockdown data and cell location data (as well as developmental expression and orthology data) is already available through tritrypDB.org and trtyptag.org

Are the data and the methods presented in such a way that they can be reproduced? Yes

Are the experiments adequately replicated, and statistical analysis adequate?

The authors did not mention replicates. There is no statistical analysis mentioned.

The figure legends indicate that all volcano plots of TALE affinity selections were derived from three biological replicates. Cutoffs used for significance: P < 0.05 (Student's t-test).

For ChiP-seq two biological replicates were analysed for each cell line expressing the specific YFP tagged protein of interest (TALE or KKT2). This is now stated in the relevant figure legends – apologies for this oversight. The resulting data are available for scrutiny at GEO: GSE295698.

Minor comments:

Specific experimental issues that are easily addressable.

The following suggestions can be incorporated:

(1) Page 18, in the material method section author mentioned four drugs: Blasticidine, Phleomycin and G418, and hygromycin. It is recommended to mention the purpose of using these selective drugs for the parasite. If clonal selection has been done, then it should also be mentioned.

We erroneously added information on several drugs used for selection in our labaoratory. In fact all TALE-YFP construct carry the Bleomycin resistance genes which we select for using Phleomycin. Also, clones were derived by limiting dilution immediately after transfection. We have amended the text accordingly:

Page 17/18:

“Cell cultures were maintained below 3 x 106 cells/ml. Pleomycin 2.5 µg/ml was used to select transformants containing the TALE construct BleoR gene.”

“Electroporated bloodstream cells were added to 30 ml HMI-9 medium and two 10-fold serial dilutions were performed in order to isolate clonal Pleomycin resistant populations from the transfection. 1 ml of transfected cells were plated per well on 24-well plates (1 plate per serial dilution) and incubated at 37°C and 5% CO2 for a minimum of 6 h before adding 1 ml media containing 2X concentration Pleomycin (5 µg/ml) per well.”

(2) In the method section the authors mentioned that there is only one site for binding of NonR-TALE in the parasite genome. But in Fig. 1C, the authors showed zero binding site. So, there is one binding site for NonR-TALE-YFP in the genome or zero?

We thank the reviewer for pointing out this discrepancy. We have checked the latest Tb427v12 genome assembly for predicted NonR-TALE binding sites and there are no exact matches. We have corrected the text accordingly.

Page 7:

“A control NonR-TALE protein was also designed which was predicted to have no target sequence in the T. brucei genome.”

Page 17:

“A control NonR-TALE predicted to have no recognised target in the T. brucei geneome was designed as follows: BLAST searches were used to identify exact matches in the TREU927 reference genome. Candidate sequences with one or more match were discarded.”

(3) The authors used two different anti-GFP antibodies, one from Roche and the other from Thermo Fisher. Why were two different antibodies used for the same protein?

We have found that only some anti-GFP antibodies are effective for affinity selection of associated proteins, whereas others are better suited for immunolocalisation. The respective suppliers’ antibodies were optimised for each application.

(4) Page 6: in the introduction, the authors give the number of total VSG genes as 2,634. Is it known how many of them are pseudogenes?

This value corresponds to the number reported by Consentino et al. 2021 (PMID: 34541528) for subtelomeric VSGs, which is similar to the value reported by Muller et al 2018 (PMID: 30333624) (2486), both in the same strain of trypanosomes as used by us. Based on the earlier analysis by Cross et al (PMID: 24992042), 80% of the identified VSGs in their study (2584) are pseudogenes. This approximates to the estimation by Consentino of 346/2634 (13%) being fully functional VSG genes at subtelomeres, or 17% when considering VSGs at all genomic locations (433/2872).

(5) I found several typos throughout the manuscript.

Thank you for raising this, we have read through the manuscipt several times and hopefully corrected all outstanding typos.

(6) Fig. 1C: Table: below TOTAL 2nd line: the number should be 1838 (rather than 1828)

Corrected- thank you.

- Are prior studies referenced appropriately? Yes

- Are the text and figures clear and accurate? Yes

- Do you have suggestions that would help the authors improve the presentation of their data and conclusions? Suggested above

Reviewer #1 (Significance):

Describe the nature and significance of the advance (e.g., conceptual, technical, clinical) for the field:

This study represents a significant conceptual and technical advancement by employing a synthetic TALE DNA-binding protein tagged with YFP to selectively identify proteins associated with five distinct repetitive regions of T. brucei chromatin. To the best of my knowledge, it is the first report to utilize TALE-YFP for affinity-based isolation of protein complexes bound to repetitive genomic sequences in T. brucei. This approach enhances our understanding of chromatin organization in these regions and provides a foundation for investigating the functional roles of associated proteins in parasite biology. Importantly, any essential or unique interacting partners identified could serve as potential targets for therapeutic intervention.

- Place the work in the context of the existing literature (provide references, where appropriate). I agree with the information that has already described in the submitted manuscript, regarding its potential addition of the data resulted and the technology established to the study of VSGs expression, kinetochore mechanism and telomere biology.

- State what audience might be interested in and influenced by the reported findings. These findings will be of particular interest to researchers studying the molecular biology of kinetoplastid parasites and other unicellular organisms, as well as scientists investigating chromatin structure and the functional roles of repetitive genomic elements in higher eukaryotes.

- (1) Define your field of expertise with a few keywords to help the authors contextualize your point of view. Protein-DNA interactions/ chromatin/ DNA replication/ Trypanosomes

- (2) Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. None

Reviewer #2 (Evidence, reproducibility and clarity):

Summary

Carloni et al. comprehensively analyze which proteins bind repetitive genomic elements in Trypanosoma brucei. For this, they perform mass spectrometry on custom-designed, tagged programmable DNA-binding proteins. After extensively verifying their programmable DNA-binding proteins (using bioinformatic analysis to infer target sites, microscopy to measure localization, ChIP-seq to identify binding sites), they present, among others, two major findings: 1) 14 of the 25 known T. brucei kinetochore proteins are enriched at 177bp repeats. As T. brucei's 177bp repeatcontaining intermediate-sized and mini-chromosomes lack centromere repeats but are stable over mitosis, Carloni et al. use their data to hypothesize that a 'rudimentary' kinetochore assembles at the 177bp repeats of these chromosomes to segregate them. 2) 70bp repeats are enriched with the Replication Protein A complex, which, notably, is required for homologous recombination. Homologous recombination is the pathway used for recombination-based antigenic variation of the 70bp-repeat-adjacent variant surface glycoproteins.

Major Comments

None. The experiments are well-controlled, claims well-supported, and methods clearly described. Conclusions are convincing.

Thank you for these positive comments.

Minor Comments

(1) Fig. 2 - I couldn't find an uncropped version showing multiple cells. If it exists, it should be linked in the legend or main text; Otherwise, this should be added to the supplement.

The images presented represent reproducible analyses, and independently verified by two of the authors. Although wider field of view images do not provide the resolution to be informative on cell location, as requested we have provided uncropped images in new Fig. S4 for all the cell lines shown in Figure 2A.

In addition, we have included as supplementary images (Fig. S3B) additional images of TelRTALE-YFP, 177R-TALE-YFP and ingiR-TALE YFP localisation to provide additional support their observed locations presented in Figure 1. The set of cells and images presented in Figure 2A and in Fig S3B were prepared and obtained by a different authors, independently and reproducibly validating the location of the tagged protein.

(2) I think Suppl. Fig. 1 is very valuable, as it is a quantification and summary of the ChIP-seq data. I think the authors could consider making this a panel of a main figure. For the main figure, I think the plot could be trimmed down to only show the background and the relevant repeat for each TALE protein, leaving out the non-target repeats. (This relates to minor comment 6.) Also, I believe, it was not explained how background enrichment was calculated.

We are grateful for the reviewer’s positive view of original Fig. S1 and appreciate the suggestion. We have now moved these analysis to part B of main Figure 2 in the revised manuscript – now Figure 2B. We have also provided additional details in the Methods section on the approaches used to assess background enrichment.

Page 19:

“Background enrichment calculation

The genome was divided into 50 bp sliding windows, and each window was annotated based on overlapping genomic features, including CIR147, 177 bp repeats, 70 bp repeats, and telomeric (TTAGGG)n repeats. Windows that did not overlap with any of these annotated repeat elements were defined as "background" regions and used to establish the baseline ChIP-seq signal. Enrichment for each window was calculated using bamCompare, as log₂(IP/Input). To adjust for background signal amongst all samples, enrichment values for each sample were further normalized against the corresponding No-YFP ChIP-seq dataset.”

Note: While revising the manuscript we also noticed that the script had a nomalization error. We have therefore included a corrected version of these analyses as Figure 2B (old Fig. S1)

(3) Generally, I would plot enrichment on a log2 axis. This concerns several figures with ChIP-seq data.

Our ChIP-seq enrichment is calculated by bamCompare. The resulting enrichment values are indeed log2 (IP/Input). We have made this clear in the updated figures/legends.

(4) Fig. 4C - The violin plots are very hard to interpret, as the plots are very narrow compared to the line thickness, making it hard to judge the actual volume. For example, in Centromere 5, YFP-KKT2 is less enriched than 147R-TALE over most of the centromere with some peaks of much higher enrichment (as visible in panel B), however, in panel C, it is very hard to see this same information. I'm sure there is some way to present this better, either using a different type of plot or by improving the spacing of the existing plot.

We thank the reviewer for this suggestion; we have elected to provide a Split-Violin plot instead. This improves the presentation of the data for each centromere. The original violin plot in Figure 4C has been replaced with this Split-Violin plot (still Figure 4C).

(5) Fig. 6 - The panels are missing an x-axis label (although it is obvious from the plot what is displayed).

Maybe the "WT NO-YFP vs" part that is repeated in all the plot titles could be removed from the title and only be part of the x-axis label?

In fact, to save space the X axis was labelled inside each volcano plot but we neglected to indicate that values are a log2 scale indicating enrichment. This has been rectified – see Figure 6, and Fig. S7, S8 and S9.

(6) Fig. 7 - I would like to have a quantification for the examples shown here. In fact, such a quantification already exists in Suppl. Figure 1. I think the relevant plots of that quantification (YFPKKT2 over 177bp-repeats and centromere-repeats) with some control could be included in Fig. 7 as panel C. This opportunity could be used to show enrichment separated out for intermediate-sized, mini-, and megabase-chromosomes. (relates to minor comment 2 & 8)

The CIR147 sequence is found exclusively on megabase-sized chromosomes, while the 177 bp repeats are located on intermediate- and mini-sized chromosomes. Due to limitations in the current genome assembly, it is not possible to reliably classify all chromosomes into intermediate- or mini- sized categories based on their length. Therefore, original Supplementary Fig. S1 presented the YFP-KKT2 enrichment over CIR147 and 177 bp repeats as a representative comparison between megabase chromosomes and the remaining chromosomes (corrected version now presented as main Figure 2B). Additionally, to allow direct comparison of YFP-KKT2 enrichment on CIR147 and 177 bp repeats we have included a new plot in Figure 7C which shows the relative enrichment of YFP-KKT2 on these two repeat types.

We have added the following text , page 12:

“Taking into account the relative to the number of CIR147 and 177 bp repeats in the current T.brucei genome (Cosentino et al., 2021; Rabuffo et al., 2024), comparative analyses demonstrated that YFP-KKT2 is enriched on both CIR147 and 177 bp repeats (Figure 7C).”

(7) Suppl. Fig. 8 A - I believe there is a mistake here: KKT5 occurs twice in the plot, the one in the overlap region should be KKT1-4 instead, correct?

Thanks for spotting this. It has been corrected

(8) The way that the authors mapped ChIP-seq data is potentially problematic when analyzing the same repeat type in different regions of the genome. The authors assigned reads that had multiple equally good mapping positions to one of these mapping positions, randomly.

This is perfectly fine when analysing repeats by their type, independent of their position on the genome, which is what the authors did for the main conclusions of the work.

However, several figures show the same type of repeat at different positions in the genome. Here, the authors risk that enrichment in one region of the genome 'spills' over to all other regions with the same sequence. Particularly, where they show YFP-KKT2 enrichment over intermediate- and mini-chromosomes (Fig. 7) due to the spillover, one cannot be sure to have found KKT2 in both regions.

Instead, the authors could analyze only uniquely mapping reads / read-pairs where at least one mate is uniquely mapping. I realize that with this strict filtering, data will be much more sparse. Hence, I would suggest keeping the original plots and adding one more quantification where the enrichment over the whole region (e.g., all 177bp repeats on intermediate-/mini-chromosomes) is plotted using the unique reads (this could even be supplementary). This also applies to Fig. 4 B & C.

We thank the reviewer for their thoughtful comments. Repetitive sequences are indeed challenging to analyze accurately, particularly in the context of short read ChIP-seq data. In our study, we aimed to address YFP-KKT2 enrichment not only over CIR147 repeats but also on 177 bp repeats, using both ChIP-seq and proteomics using synthetic TALE proteins targeted to the different repeat types. We appreciate the referees suggestion to consider uniquely mapped reads, however, in the updated genome assembly, the 177 bp repeats are frequently immediately followed by long stretches of 70 bp repeats which can span several kilobases. The size and repetitive nature of these regions exceeds the resolution limits of ChIP-seq. It is therefore difficult to precisely quantify enrichment across all chromosomes.

Additionally, the repeat sequences are highly similar, and relying solely on uniquely mapped reads would result in the exclusion of most reads originating from these regions, significantly underestimating the relative signals. To address this, we used Bowtie2 with settings that allow multi-mapping, assigning reads randomly among equivalent mapping positions, but ensuring each read is counted only once. This approach is designed to evenly distribute signal across all repetitive regions and preserve a meaningful average.

Single molecule methods such as DiMeLo (Altemose et al. 2022; PMID: 35396487) will need to be developed for T. brucei to allow more accurate and chromosome specific mapping of kinetochore or telomere protein occupancy at repeat-unique sequence boundaries on individual chromosomes.

Reviewer #2 (Significance):

This work is of high significance for chromosome/centromere biology, parasitology, and the study of antigenic variation. For chromosome/centromere biology, the conceptual advancement of different types of kinetochores for different chromosomes is a novelty, as far as I know. It would certainly be interesting to apply this study as a technical blueprint for other organisms with minichromosomes or chromosomes without known centromeric repeats. I can imagine a broad range of labs studying other organisms with comparable chromosomes to take note of and build on this study. For parasitology and the study of antigenic variation, it is crucial to know how intermediate- and mini-chromosomes are stable through cell division, as these chromosomes harbor a large portion of the antigenic repertoire. Moreover, this study also found a novel link between the homologous repair pathway and variant surface glycoproteins, via the 70bp repeats. How and at which stages during the process, 70bp repeats are involved in antigenic variation is an unresolved, and very actively studied, question in the field. Of course, apart from the basic biological research audience, insights into antigenic variation always have the potential for clinical implications, as T. brucei causes sleeping sickness in humans and nagana in cattle. Due to antigenic variation, T. brucei infections can be chronic.

Thank you for supporting the novelty and broad interest of our manuscript

My field of expertise / Point of view:

I'm a computer scientist by training and am now a postdoctoral bioinformatician in a molecular parasitology laboratory. The laboratory is working on antigenic variation in T. brucei. The focus of my work is on analyzing sequencing data (such as ChIP-seq data) and algorithmically improving bioinformatic tools.

eLife Assessment

This important study examines the role of map3k1, a MAP3K family member that has both kinase and ubiquitin ligase domains, in the differentiation of progenitors in the flatworm Planaria. The convincing analyses demonstrate that map3k1 acts within progenitors to restrict their premature differentiation and to prevent formation of teratomas. This work would be of interest to researchers in the fields of regeneration, developmental biology, and aging.

Reviewer #1 (Public review):

Summary:

The authors assess the role of map3k1 in adult Planaria through whole body RNAi for various periods of time. The authors' prior work has shown that neoblasts (stem cells that can regenerate the entire body) for various tissues are intermingled in the body. Neoblasts divide to produce progenitors that migrate within a "target zone" to the "differentiated target tissues" where they differentiate into a specific cell type. Here the authors show that map3k1-i animals have ectopic eyes that form along the "normal" migration path of eye progenitors, ectopic neurons and glands along the AP axis and pharynx in ectopic anterior positions. The rest of the study shows that positional information is largely unaffected by loss of map3k1. However, loss of map3k1 leads to premature differentiated of progenitors along their normal migratory route. They also show that "long-term" whole body depletion of map3k1 results in mis-specified organs and teratomas. In short, this study convincingly demonstrates that in planaria, map3k1 maintains progenitor cells in an undifferentiated state, preventing premature fate commitment until they encounter the appropriate signals, either positional cues within a designated region or contact-dependent inputs from surrounding tissues.

Strengths:

(1) The study has appropriate controls, sample sizes and statistics.

(2) The work is high-quality.

(3) The conclusions are supported by the data.

(4) Planaria is a good system to analyze the function of map3k1, which exists in mammals but not other invertebrates.

Weaknesses:

None noted.

Reviewer #2 (Public review):

Summary:

The flatworm planarian Schmidtea mediterranea is an excellent model for understanding cell fate specification during tissue regeneration and adult tissue maintenance. Planarian stem cells, known as neoblasts, are continuously deployed to support cellular turnover and repair tissues damaged or lost due to injury. This reparative process requires great precision to recognize the location, timing, and cellular fate of a defined number of neoblast progeny. Understanding the molecular mechanisms driving this process could have important implications for regenerative medicine and enhance our understanding of how form and function are maintained in long-lived organisms such as humans. Unfortunately, the molecular basis guiding cell fate and differentiation remains poorly understood.

In this manuscript, Canales et al. identified the role of the map3k1 gene in mediating the differentiation of progenitor cells at the proper target tissue. The map3k1 function in planarians appears evolutionarily conserved as it has been implicated in regulating cell proliferation, differentiation, and cell death in mammals. The results show that the downregulation of map3k1 with RNAi leads to spatial patterning defects in different tissue types, including the eye, pharynx, and the nervous system. Intriguingly, long-term map3k1-RNAi resulted in ectopic outgrowths consistent with teratomas in planarians. The findings suggest that map3k1 mediates signaling, regulating the timing and location of cellular progenitors to maintain correct patterning during adult tissue maintenance.

Strengths:

The authors provide an entry point to understanding molecular mechanisms regulating progenitor cell differentiation and patterning during adult tissue maintenance.

The diverse set of approaches and methods applied to characterize map3k1 function strengthens the case for conserved evolutionary mechanisms in a selected number of tissue types. The creativity using transplantation experiments is commendable, and the findings with the teratoma phenotype are intriguing and worth characterizing.

Weaknesses:

The authors have satisfactorily addressed our previous concerns.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors assess the role of map3k1 in adult Planaria through whole body RNAi for various periods of time. The authors' prior work has shown that neoblasts (stem cells that can regenerate the entire body) for various tissues are intermingled in the body. Neoblasts divide to produce progenitors that migrate within a "target zone" to the "differentiated target tissues" where they differentiate into a specific cell type. Here the authors show that map3k1-i animals have ectopic eyes that form along the "normal" migration path of eye progenitors (Fig. 1), ectopic neurons and glands along the AP axis (Fig. 2) and pharynx in ectopic anterior positions (Fig. 3). The rest of the study show that positional information is largely unaffected by loss of map3k1 (Fig. 4,5). However, loss of map3k1 leads to premature differentiated of progenitors along their normal migratory route (Fig. 6). They also show that an ill-defined "long-term" whole body depletion of map3k1 results in mis-specified organs and teratomas.

Strengths:

(1) The study has appropriate controls, sample sizes and statistics.

(2) The work appears to be high-quality.

(3) The conclusions are supported by the data.

(4) Planaria is a good system to analyze the function of map3k1, which exists in mammals but not in other invertebrates.

Weaknesses:

(1) The paper is largely descriptive with no mechanistic insights. 

The mechanistic insights we aim to address are primarily at the cellular systems level – how adult progenitor cells produce pattern. Specifically, we uncovered strong evidence that regulation of differentiation is an active process occurring in migratory progenitors and that this regulation is a major component of pattern formation during the adult processes of tissue turnover and regeneration. The map3k1 phenotype provided a tool used to reveal the existence of this regulation, and to understand the patterning abnormalities prevented by this regulatory mechanism. We updated the text in several places to make clearer some of this emphasis. For example, in the Discussion: "We suggest that differentiation is restricted during migratory targeting as an essential component of pattern formation, with the map3k1 RNAi phenotype indicating the existence and purpose of this element of patterning." 

Naturally, identifying a particular molecule involved in this process is of interest for understanding molecular mechanism; this would allow for comparison to other cellular systems in other organisms and would focus future molecular inquiry. Future molecular studies into the mechanism of Map3k1 regulation and its downstream signaling will be fascinating as next steps towards understanding the process at the molecular level more deeply. We also added some discussion considering the types of upstream activation cues that could potentially be associated with Map3k1 regulation to suppress differentiation. 

(2) Given the severe phenotypes of long-term depletion of map3k1, it is important that this exact timepoint is provided in the methods, figures, figure legends and results. 

We removed the use of the term “long-term” and instead added timepoints used to all figure legends. We also added a summary of timepoints used in the methods section and included RNAi timepoint labels in figures where a phenotype progression over time is relevant to interpretation. For timecourses, we also added suitable time information to text in the results. 

(3) Figure 1C, the ectopic eyes are difficult to see, please add arrows. 

To improve visualization, we replaced the example animal in the original Figure 1C with one that has a stronger phenotype, including arrows pointing to every ectopic event. Additionally, we included magnified images of optic cup cells and photoreceptor neurons in the trunk and tail region. This is now Figure 1B.

(4) line 217 - why does the n=2/12 animals not match the values in Figure 3B, which is 11/12 and 12/12. The numbers don't add up. Please correct/explain. 

In Figure 3B in the submitted version (3/18 had cells in the tail) had more animals scored (6 animals from a replicate experiment where 1/6 showed the cells in the tail) than the total scored (2/12 had cells in the tail) in the text, which did not have the animals from the replicate added during writing. The results are the same, just different sample sizes were noted in those locations and we fixed this issue. In the updated Figure 3, the order of presentation has shifted (e.g., prior 3B is now in 3C and Figure 3_figure supplement 1). We made sure to include numbers to all figure panels. 

(5) Figure panels do not match what is written in the results section. There is no Figure 6E. Please correct.

Thank you for catching this. We have gone through figures and text after editing to make sure that text callouts are appropriately matched to the figures. 

Reviewer #2 (Public review):

Summary:

The flatworm planarian Schmidtea mediterranea is an excellent model for understanding cell fate specification during tissue regeneration and adult tissue maintenance. Planarian stem cells, known as neoblasts, are continuously deployed to support cellular turnover and repair tissues damaged or lost due to injury. This reparative process requires great precision to recognize the location, timing, and cellular fate of a defined number of neoblast progeny. Understanding the molecular mechanisms driving this process could have important implications for regenerative medicine and enhance our understanding of how form and function are maintained in long-lived organisms such as humans. Unfortunately, the molecular basis guiding cell fate and differentiation remains poorly understood.

In this manuscript, Canales et al. identified the role of the map3k1 gene in mediating the differentiation of progenitor cells at the proper target tissue. The map3k1 function in planarians appears evolutionarily conserved as it has been implicated in regulating cell proliferation, differentiation, and cell death in mammals. The results show that the downregulation of map3k1 with RNAi leads to spatial patterning defects in different tissue types, including the eye, pharynx, and the nervous system. Intriguingly, long-term map3k1-RNAi resulted in ectopic outgrowths consistent with teratomas in planarians. The findings suggest that map3k1 mediates signaling, regulating the timing and location of cellular progenitors to maintain correct patterning during adult tissue maintenance.

Strengths:

The authors provide an entry point to understanding molecular mechanisms regulating progenitor cell differentiation and patterning during adult tissue maintenance.

The diverse set of approaches and methods applied to characterize map3k1 function strengthens the case for conserved evolutionary mechanisms in a selected number of tissue types. The creativity using transplantation experiments is commendable, and the findings with the teratoma phenotype are intriguing and worth characterizing.

Thank you to the reviewer for the positive feedback

Weaknesses:

The article presents a provocative idea related to the importance of positional control for organs and cells, which is at least in part regulated by map3k1. Nonetheless, the role of map3k1 or its potential interaction with regulators of the anterior-posterior, mediolateral axes, and PCGs is somewhat superficial. The authors could elaborate or even speculate more in the discussion section and the different scenarios incorporating these axial modulators into the map3k1 model presented in Figure 8 

First, to strengthen the support for our finding that positional information is largely unaffected in map3k1 RNAi animals, we added data regarding the expression of additional relevant position control genes (PCGs) –ndl-4, ptk7, sp5, and wnt11-1 – to the PCG panel in Figure 5. The expression domain of ndl-4, an FGF receptor-like protein family member that contributes to head patterning and anterior pole maintenance, was normal in map3k1 RNAi. wnt11-1, a PCG with expression concentrated in the posterior end of the animal and with expression dependent on general Wnt activity, was also normal in map3k1 RNAi animals. ptk7, RNAi of which can result in supernumerary pharynges, also showed normal expression in map3k1 RNAi animals. Finally, sp5, a Wnt-activated gene with expression in the tail, also showed normal expression in map3k1 RNAi animals. 

Second, to further support the conclusion that cells are not suitably responding to positional information after map3k1 RNAi, which we argue normally dictates where differentiation should occur, we added examples of differentiated cell types that are ectopically positioned within an atypical PCG expression domain for that cell type (Figure 5C). This underscores that following map3k1 RNAi the PCG expression domains do not change, but the pattern of differentiated cell types relative to these domains does shift. We also added data showing that regenerating tails had a proper wntP-2 gradient, but an anterior regenerating pharynx appeared outside of this wntP-2⁺ zone and inside of an ndl-5⁺ zone (Figure 5- figure supplement 1E). We added some discussion of these new data in the Figure 5 results section. We also noted, regarding independent recent map3k1 work (Lo, 2025), some evidence exists that a minor posterior shift in ndl-5 expression can occur after map3k1 RNAi.

Next, we added a new element to the model figure (Figure 8B) depicting that PCG expression domains remain normal after map3k1 RNAi, with ectopic differentiation occurring in an incorrect positional information environment. We refer to this new panel in the discussion: "We suggest that map3k1 is not required for the spatial distribution of progenitor-extrinsic differentiation-promoting cues themselves, but for progenitors to be restricted from differentiating until these cues are received (Figure 8B)."; we then follow this statement with a summary in the Discussion of six pieces of evidence that support this model.

Finally, we added some additional text to the discussion section about candidate mechanisms by which extrinsic cues could potentially regulate Map3k1, pointing to potential future inquiry directions. We suggest that map3k1 is not involved in regulating PCG activity domains themselves, but instead acts as a brake on differentiation within migratory progenitors through active signaling. This brake is then lifted when the progenitors hit their correct PCG-based migratory target, or when they hit their target tissue. How that occurs mechanistically is unknown. One scenario is that each progenitor is tuned to respond to a particular PCG-regulated environment (such as a particular ECM or signaling environment) to generate a molecular change that inactivates Map3K1 signaling, such as by inactivating or disengaging an RTK signal. Alternatively, the migratory process in progenitors could engage the Map3K1 signal, enabling signal cessation with arrival at a target location. When Map3K1 is active it could result in a transcriptional state that prevents full expression of differentiated factors required for maturation, tissue incorporation, and cessation of migration. These considerations are now added to the discussion.

The article can be improved by addressing inconsistencies and adding details to the results, including the main figures and supplements. This represents one of the most significant weaknesses of this otherwise intriguing manuscript. Below are some examples of a few figures, but the authors are expected to pay close attention to the remaining figures in the paper.

Details associated with the number of animals per experiment, statistical methods used, and detailed descriptions of figures appear inconsistent or lacking in almost all figures. In some instances, the percentage of animals affected by the phenotype is shown without detailing the number of animals in the experiment or the number of repeats. Figures and their legends throughout the paper lack details on what is represented and sometimes are mislabeled or unrelated. 

We endeavored to ensure that these noted details are present throughout the legends and figures for all figure panels.

Specifically, the arrows in Figure 1A are different colors. Still, no reasoning is given for this, and in the exact figure, the top side (1A) shows the percentages and the number of animals below. 

The only reason for the different colored arrows was for visibility purposes. To avoid confusion, we now use white arrows for all FISH images in figure 1, and where ever else possible. We also replaced the percentages with n numbers in the bottom left corner of the live images in Figure 1A. 

Conversely, in Figures 1B, C, and D, no details on the number of animals or percentages are shown, nor an explanation of why opsin was used in Figure 1A but not 1B. 

The original Figure 1B represented a few different examples of ectopic eye/eye cell patterns in the map3k1 RNAi animals to demonstrate the variable and disorganized nature of the phenotype. To address this, we added further explanation in the legend. We also merged 1A and 1B for simplicity of interpretation. opsin was used in Figure 1A to label cell bodies of photoreceptors. anti-Arrestin was used in the example FISH images to see if these cells were interconnected via projections, which we now clarify in the legend and in the text. 

Is Figure 1B missing an image for the respective control? Figure 1C needs details regarding what the two smaller boxes underneath are. 

The control for Figure 1B was in Figure 1A; the merger of Figures 1A/B should address this. Boxes in Figure 1C were labelled with numbers corresponding to the image above them.

Figure 1C could use an AP labeling map in 10 days (e.g., AP6 has one optic cup present). Figure 1C and F counts do not match. 

We added a cartoon to 1C to show the region imaged. Note that the 36d trunk image (now Fig. 1B) has now been replaced with a full animal image and magnified boxes that show locations of example ectopic cells. That cell in 1C was categorized as in AP5. Note that additional animals were analyzed and data added to the distribution (now Fig. 1D). 

In Figure 1C, we do not know the number of animals tested, controls used, the scale bar sizes in the first two images, nor the degree of magnification used despite the pharynx region appearing magnified in the second image.  Figure 1C is also shown out of chronological order; 36 days post RNAi is shown before 10 days post RNAi. Moreover, the legends for Figures 1C and 1D are swapped.

We have endeavored to ensure sample numbers, control images, and appropriate scale bar notation in legends are present for all images. Figure 1C has now been split into two panels: Figure 1B and Figure 1C. It does not follow a chronological order in presentation for the following logic flow: we uncover and describe the phenotype; then, with knowledge of the defect, we walk back to see how early the phenotype starts after RNAi and what the pattern of ectopic cell distribution is when the phenotype starts to emerge (using the knowledge of which cells are affected from the overt phenotype described in 1A/B). 

Additionally, Figure 1F and many other figures throughout the paper lack overall statistical considerations. Furthermore, Figure 1F has three components, but only one is labeled. Labeling each of them individually and describing them in the corresponding figure legend may be more appropriate.

The main point of the graphs in 1F (now 1D) was the overt overall pattern difference with the wild-type, which never has ectopic eye cells in the midbody or tail, and that the ectopic eye cells progress throughout the entire AP axis. However, we concur that a statistical test is a reasonable thing to show here and that is now included in the legend. The 3 components (in Figure 1F, now Figure 1D) where kept together with one figure label (D) for simplicity, but were rearranged (top and bottom) with a cartoon to the side and with modified labeling for extra clarity. 

Figure 2C shows images of gene expression for two genes, but the counts are shown for only one in Figure 2D. It is challenging to follow the author's conclusions without apparent reasoning and by only displaying quantitative considerations for one case but not the other. These inconsistencies are also observed in different figures. 

In Figure 2C, FISH images of cintillo+ and dd_17258+ neurons are shown to display the specificity of this effect to some neurons and not others. Because cintillo+ cells did not expand at all (n=24/24 animals), the counts for them would all be zero values. We only counted data for dd_17258 cells because it was the neuron that expanded compared to the control animals. We have now added a note in the legend explaining this.

In Figure 2D, 24/24 animals were reported to show the phenotype, but only eight were counted (is there a reason for this?).

8 animals were used to quantitatively characterize the spread of cells along the AP axis, as it was deemed an adequate sample size to capture the change in distribution of 17258+ cells from being head restricted to being present throughout the body. Through multiple cohorts of animals in replicates, a total of 24/24 examined animals showed this expansion phenotype. Double FISH experiments were additionally carried out using dd_17258 and various PCGs; these data are now included in Figure 5C, and these animals were added to the total counts regarding quantitative analysis of the phenotype in Figure 2D. 

In Figure 2E, the expression for three genes is shown, with some displaying anterior and posterior regions while others only show the anterior picture. Is there a particular reason for this? 

The original first panel in Figure 2E showed an example of a non-expanding gland cell type, dd_9223, which is very restricted to the head in both control and map3k1 RNAi animals. Because we did not observe a phenotype for this cell type (no cells in all control and map3k1 RNAi animal tails), we only included tail images of cell types that showed an abnormal phenotype with clear expanded to the posterior (dd_8476 and dd_7131). However, we have now included tail images of dd_9223 cells and added data for dd_9223 to the graph in Figure 2E. 

Also, in Figure 2F, the counts are shown for only the posterior region of two genes out of the three displayed in Figure 2E. It is unclear why the authors do not show counts for the anterior areas considered in Figure 2E. Furthermore, the legend for Figure 2D is missing, and the legend for 2F is mislabeled as a description for Figure 2D.

We now include tail images for dd_9223 in Figure 2E to show that there are no ectopic cells in tails. We did not originally include counts of dd_9223 because there was no phenotype observed. dd_7131 and dd_8476 cell types appeared in the posterior of even control animals at a low frequency, unlike dd_9223 cells. However, we did now add counts for dd_9223 tail regions in the graph. We did not count the anterior regions of the animal because our goal was to show data for the visible phenotype (ectopic cells in the tail) not only with an example image, but also by showing the number of cells in the tail with a graph and statistical test. Legends have been updated with correct details.

Supplement Figure 1 B reports data up to 6 weeks, but no text in the manuscript or supplement mentions any experiment going up to 6 weeks. There are no statistics for data in Supplement Figure 1E. Any significance between groups is unclear.

More details about the RNAi feeding schedules have been added in the methods section. All RNAi timepoints are now specified specifically in the legends. The Figure 1F and Figure 1- figure supplement 1E (additional data: ovo⁺; smedwi-1^- cell counts) and legends now mention the statistical tests performed and annotations (not significant *ns) or p values have been added to the graphs. For simplicity, we decided to include all smedwi-1+ counts together rather than splitting them into low and high smedwi-1+ cells, because we weren't really making any claims about low and high cells. 

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

It would be good to acknowledge in the discussion the recent paper from the Petersen lab on map3k1, published in PLoS Genet 2025, especially if the results differ between the two labs.

We added reference/discussion regarding the recent PLoS Genetics Lo, 2025 map3k1 paper at several suitable points in the manuscript.

Reviewer #2 (Recommendations for the authors):

Please pay close attention to the description of experimental details and the consistency throughout the paper. It seems like the reader has to assume or come across information that is not readily available from the text or the legends in the paper. This is an interesting paper with intriguing findings. However, the version presented here appears rushed or put together on the flight.

Thank you for your thorough feedback. We have endeavored to ensure all appropriate details are present in figures and/or figure legends.

Simplifying data may frequently be convenient when creating a widely-applicable program, but it involves leaving at least one group or perspective out. Because of this, simplification of data often contains inherent bias and developers should be aware of this.

In the modern age, it is important to understand what truly counts as a “bot” considering the intricacies of automation and the philosophical question of autonomous AI. Can we consider bots as a valid representation of general public consensus as they become more prevalent?

make this not bold

People typically don’t expect to glean much useful information from bots. In my case, at least, I would typically block or ignore them. Additionally, it’s often easy to identify a bit, but in the age of AI these lines are becoming more blurred.

eLife Assessment

This important study employs a closed-loop, theta-phase-specific optogenetic manipulation of medial septal parvalbumin-expressing neurons in rats and reports that disrupting theta-timescale coordination impairs performance of challenging aspects of spatial behaviors, while sparing hippocampal replay and spatial coding in hippocampal place cells. The findings are expected to advance theoretical understanding of learning and memory operations and to provide practical implications for the application of similar optogenetic approaches. The experiments were viewed as technically rigorous, but the strength of evidence provided in the current version of the manuscript was viewed as incomplete, mostly due to limited analyses and the descriptions of some of the experimental protocols.

Reviewer #1 (Public review):

Summary:

This manuscript by Joshi and colleagues demonstrates that the precise theta-phase timing of spikes is causal for CA1 hippocampal theta sequences during locomotion on a linear track and is necessary for learning the cognitively demanding outbound component of a hippocampus-dependent alternation task (W-maze), independently of replay during immobility. To reach these conclusions, the authors developed a theta-phase-specific, closed-loop manipulation that used optogenetic activation of medial septal parvalbumin (PV) interneurons at the ascending phase of theta during locomotion. This protocol preserved immobility periods, allowing a clean and elegant dissociation from SWR-associated replay.

The manuscript is well written and was a pleasure to read. The work described is of high quality and introduces several notable advances to the field:

(a) It extends prior studies that manipulated theta oscillations by examining precise temporal structure (specifically theta sequences) rather than only LFP features.

(b) The closed-loop manipulation enabled dissociation between deficits in theta sequences during a behavioural task and SWR-associated replay activity.

(c) As controls, the authors included rats with suboptimal viral transduction or optic-fibre placement, and, within subjects, both stimulation-on (stim-on) and stimulation-off (stim-off) trials. Notably, sequence disruption persisted into stim-off periods within the same session.

Overall, this is a strong manuscript that will provide valuable insights to the field. I have only minor comments:

(1) As the authors note, it is striking that both behavioural performance and spike patterns are altered during stim-off trials. They propose that "disruption of theta sequences during the initial experience in an environment is sufficient to have lasting effects," implying that rapid, experience-dependent plasticity is driven by sequential firing. Does this imply that if rats were previously trained on the task, subsequent stim-on and stim-off trials would yield different outcomes, with stim-off trials showing improved performance and intact theta sequences? For example, if the sequence of one-third stim-on, one-third stim-off, one-third stim-on were inverted to off-on-off, would theta sequences be expected to emerge, disappear, and potentially re-emerge? While I am not asking for additional experiments, I think the discussion could be extended in this aspect.

Alternatively, could the number of stim-off trials (one third of the total) be insufficient to support learning/induce plasticity? In the controls, ~50-100 trials appear necessary to achieve high performance.

(2) In line with the point above, the authors characterise the behavioural changes induced by MS optogenetic stimulation specifically as a "learning deficit," as rats failed to improve across 300 trials in an initially novel environment (W-maze). While they present this as complementary to prior demonstrations of impaired performance on previously learned tasks (Zutshi et al., 2018; Quirk et al., 2021; Etter et al., 2023; Petersen et al., 2020), an alternative interpretation is a working-memory deficit. This would produce the same behavioural pattern, with reference memory (the less cognitively demanding trials) remaining intact despite stimulation and concomitant changes in theta sequences. This interpretation would also be consistent with work in certain disease models, where reduced synaptic plasticity and working-memory deficits co-occur with preserved place coding despite impaired theta sequences (e.g., Viana da Silva et al., 2024; Donahue et al., 2025).

(3) It was not immediately clear whether SWR-associated activity was derived from the interleaved ~15-min rest sessions in a rest box, or from periods of immobility or reward consumption in the maze (aSWR, as in Jadhav et al 2012). Regardless, it would be informative to compare aSWR events within the maze to rest-box SWRs that may occur during more prolonged slow-wave episodes (even if not full sleep). This contrasts with Liu et al. (2024), who analysed replay during ~1.5-h sleep sessions.

Reviewer #2 (Public review):

Summary:

The authors of this study developed a closed-loop optogenetic stimulation system with high temporal precision in rats to examine the effect of medial septum (MS) stimulation on the disruption of hippocampal activity at both behavioral and compressed time scales. They found that this manipulation preserved hippocampus single-cell-level spatial coding but affected theta sequences and performance during a spatial alternation task. The performance deficits were observed during the more cognitively demanding component of the task and even persisted after the stimulation was turned off. However, the effects of this disruption were confined to locomotor periods and did not impact waking rest replay, even during the early phase of stimulation-on. Their conclusion is consistent with previous findings from the Pastalkova lab, where MS disruption (using different methods) affected theta sequences and task performance but spared replay (Wang et al., 2015; Wang et al., 2016). However, it differs from a recent study in which optogenetic disruption of EC inputs during running affected both theta sequences and replay (Liu et al., 2023).

Strengths:

The experiments were well designed and controlled, and the results were generally well presented.

Weaknesses:

Major concerns are primarily technical but also conceptual. To further increase the impact of this study by contrasting findings from different disruptions, it is necessary to better align the analysis and detection methods.

Major concerns:

(1) To show that MS disruption does not affect spatial tuning, the authors computed the KL divergence of tuning curves between stimulation-on and stimulation-off conditions. I have two main questions about this analysis:

(1.1) The authors seem to impose stringent inclusion criteria requiring a large number of spikes and a strong concentration of tuning curves. These criteria may have selected strongly spatially tuned cells, which are typically more stable and potentially less vulnerable to perturbations. Based on the Figure 2 caption, it seems that fewer than 10% of cells were included in the KL divergence analysis, which is lower than the usual proportion of place cells reported in the literature. What is the rationale for using such strict inclusion criteria? What happens to the cells that are not as strongly tuned but are still identified as significant place cells?

(1.2) The KL divergence was computed between stimulation-on and stimulation-off conditions within the same animal group. However, the authors also showed that MS stimulation had lasting effects on theta sequences and performance even during stimulation-off periods. Would that lasting effect also influence spatial tuning? Based on these questions, the authors should perform additional analyses that directly measure spatial tuning quality and compare results across control and experimental groups - for example, spatial information of spikes (Skaggs et al., 1996), tuning stability, field length, and decoding error during running.

(2) The authors compared their results with those from Liu et al. (2023) and proposed that the different outcomes could be explained by different sites of disruption. However, the detection and quantification methods for theta sequences and replay differ substantially between the two studies, emphasizing different aspects of the phenomenon. I am not suggesting that either method is superior, but providing additional analyses using aligned detection methods would better support the authors' interpretations and benefit the field by enabling clearer comparisons across studies. In the current analysis, the power spectrum of the decoded ahead/behind distance only indicates that there is a rhythmic pattern, without specifying the decoding features at different theta phases. Moreover, the continuous non-local representations during ripples could include stationary representations of a location or zigzag representations that do not exhibit a linear sequential trace. Given that, the authors should show averaged decoding results corrected by the animal's actual position within theta cycles and compute a quadrant ratio. For replay analysis, they could use a linear fit (as in Liu et al., 2023) and report the proportion of significant replay events.

(3) The finding that theta sequences and performance were impaired even during stimulation-off periods is particularly interesting and warrants deeper exploration. In the Discussion, the authors claim that this may arise from "the rapid plasticity engaged during early learning." However, this explanation does not fully account for the observation. Previous studies have shown that theta sequences can develop very rapidly (Feng et al., Foster lab, 2015; Zhou et al., Dragoi lab, 2025). If the authors hypothesize that rapid plasticity during early stimulation-on disrupts the theta sequence, then the plasticity window must also be short and terminate during the subsequent stimulation-off period. Otherwise, why can't animals redevelop theta sequences during stimulation-off? The authors should conduct additional analyses during the stimulation-off periods of the W-maze task. For example:

(3.1) What is the spike-theta phase relationship? Do the phases return to normal or remain altered as during stimulation-on?

(3.2) Is there a significant place-field remapping from stimulation-on to stimulation-off? (Supplementary Figure 3F includes only a small subset of cells; what if population vector correlations are computed across all cells, or Bayesian decoding of stimulation-on spikes is performed using stimulation-off tuning curves?)

(3.3) The authors should also discuss why the stimulation-off epochs were not sufficient to support learning, and if the stimulation-off place cell sequences could have supported replay.

(4) Citations and/or discussion of key studies relevant to the current work are missing: Wang et al. in Pastalkova lab 2015-2016 studies for disruption of theta sequence (but not place cell sequence) disrupting learning but not replay, Drieu et al. in Zugaro lab 2018 study on disruption of theta sequence affecting sleep replay, Farooq and Dragoi 2019 for association between a lack of theta sequence and presence of waking rest replay during postnatal development, etc. The authors should discuss what the conceptually new findings in the current study are, given the findings of the previous literature above.

(5) The assessment of theta sequence is not state-of-the-art:

(5.1) Detecting the peak of cross-correlograms between neurons (CCG) relates to behavioral timescale CCG, not the theta sequence one; for the theta sequence, the closest to zero local peak should be used instead.

(5.2) How were other methods of detecting theta sequences performing on the stimulation-on/stimulation-off data: Bayesian decoding, firing sequences?

(5.3) How was phase precession during stimulation-on/stimulation-off?

(6) It would be important to calculate additional variables in the replay part of the study to compare the quality of replay across the 2 groups:

(6.1) Proportion of significant replay events out of the detected multiunit events.

(6.2) The average extent of trajectory depicted by the significant replay events in the targeted compared to the control, stimulation-on/stimulation-off.

Reviewer #3 (Public review):

Joshi et al. present an elegant and technically rigorous study examining how the temporal structure of hippocampal spiking during locomotion contributes to spatial learning. Using a closed-loop, theta phase-specific optogenetic manipulation of medial septal parvalbumin-expressing neurons in rats, the authors demonstrate that disrupting theta-timescale coordination impairs performance on the cognitively demanding component outbound trajectory of a spatial alternation task, while sparing hippocampal replay, place coding, and the simpler inbound learning. The work aims to dissociate the role of theta-associated temporal organization during navigation from sharp-wave ripple-associated replay during subsequent rest periods, providing a mechanistic link between theta sequences and learning. The findings have important implications for models of septo-hippocampal coordination and the functional segregation between online (theta) and offline (SWR) network states. That said, there are a few conceptual and methodological issues that need to be addressed.

One concern is the overall novelty of this work; the dissociation between online temporal sequence and offline replay events following memory deficits has previously been shown by Wang et al., 2016 elife. While the authors discuss Lui et al., 2023, which demonstrates MEC activation of inhibitory neurons at gamma frequencies during locomotion disrupts theta sequences, subsequent replay and learning (line 65-66), they do not reference Wang et al., 2016 who performed a very similar study with MS pharmacological inactivation, and report large decreases in theta power, attenuated theta frequencies together with behavioural deficits but SWR replay persisted. Given strong similarities in the manipulation and findings, this study should be discussed.

Along the same lines, it should be noted that Brandon et al. (2014, Neuron) demonstrated that hippocampal place codes can still form in novel environments despite MS inactivation and loss of theta, indicating that spatial representations can emerge without intact septal drive. Referencing this study would strengthen the discussion of how temporal coordination, rather than spatial coding per se, underlies the learning deficits observed here.

The conclusion that disrupting "theta microstructure" impairs learning relies on the assumption that the observed behavioral deficits arise from altered temporal coding from within hippocampal CA1 only. However, optogenetic modulation of medial septal PV neurons influences multiple downstream regions (entorhinal cortex, retrosplenial cortex) via widespread GABAergic projections. While the authors do touch on this, their discussion should expand to include the network-level consequences of entorhinal grid-cell disruption and how this could affect temporal coding both online and offline.

The finding that replay content, rate, and duration are unchanged is critical to the paper's claim of dissociation. However, the analysis is restricted to immobility on the track. Given evidence for distinct awake vs. sleep replay, confirming that off-track rest and post-session sleep replays are similarly unaffected would confirm the conclusions of the paper. If these data are unavailable, the limitation should be acknowledged explicitly. Moreover, statistical power for detecting subtle differences in replay organization or spatial bias should be added to the supplement (n of events per animal, variability across sessions).

The exact protocol for optogenetic stimulation is a bit confusing. For the task, the first and final third (66%) of trials were disrupted and were only stimulated when away from the reward well and only when the animal was moving. What proportion of time within "stimulated" trials remained unstimulated? Why were only 66% of trials stimulated?

Author response:

We thank all reviewers for their overall assessment, thoughtful comments, and suggestions. We are working to address each reviewer’s comment in detail. In this provisional response, we provide clarifications regarding our experimental approach and the novelty of our work, and include additional analyses that we have performed since the submission of the manuscript. We are also happy to report that we have now shared the raw data, intermediate analysis files, and the complete repository to facilitate replication of the analysis and figures.

Code repo: github.com/LorenFrankLab/ms_stim_analysis

Data repo: dandiarchive.org/dandiset/001634

Docker containers (see GitHub repo for use instructions):

Database: https://hub.docker.com/r/samuelbray32/spyglass-db-ms_stim_analysis

Python notebooks: https://hub.docker.com/r/samuelbray32/spyglass-hub-ms_stim_analysis

(1) Novelty and contrast with earlier manipulations:

We thank the reviewers for suggesting that we explicitly contrast our results with prior pharmacological (Wang et al., 2016; Wang et al., 2015; Koenig et al., 2011; Brandon et al., 2014), systemic (Robbe & Buzsaki 2009; Petersen and Buzsáki 2020), and behavioral (Drieu et al., 2018) manipulations that also assessed some of the physiological features we evaluated. We will add a discussion of these studies, which will help us emphasize both the insights and discrepancies observed using these prior approaches. We will also more clearly explain the the novelty and importance of our specific approach for temporally and physiologically precise manipulation. Specifically, our approach (closed-loop theta-phase stimulation during locomotion) provides a level of physiological specificity that made it possible to dissociate theta-state dynamics from other hippocampal processes. This in turn allowed us to address a question that has remained unresolved across prior studies: Are hippocampal spatial sequences during locomotion (i.e., theta sequences) necessary to learn a novel hippocampal-dependent task?

(2) Additional analysis on SWRs during rest:

since submitting the manuscript, we have conducted additional analysis on the rate and length of SWRs in the rest box and found that their rate and length are also indistinguishable between targeted and control animals (effect of manipulation between control and targeted animals; rSWR rate: p=0.45; rSWR length: p=0.94, mixed effect model). We also find evidence for sequential neural representations in the rest box, when the encoding was performed in the behavioral arena. Example trajectories are shown below. These results are consistent with our observations on SWRs rate, length, and content in the behavioral arena. Additionally, we are in the process of evaluating and quantifying the results of decoding the rSWRs and will include those in the next version of the manuscript.

Author response image 1.

Sequential replay events observed in the rest box

(3) Theta sequence measurement in the absence of theta:

In the next version of the manuscript, we will explicitly explain why our manipulation makes it is more appropriate to measure sequential hippocampal representations during locomotion (i.e., theta sequences) without using theta oscillation or an epoch-averaged relatively large sliding window as a reference. The key insight here is that our manipulation suppresses theta and thus makes it difficult or impossible to accurately identify theta phase. We understand that theta-phase based approaches were used in prior work; however, these prior analyses may have confounded the absence of hippocampal theta sequences during locomotion by the inability to detect theta oscillatory phase reliably. We will show that our method of using clusterless Bayesian decoding in which we estimate the decoded position at every 2ms timestep is indeed able to capture endogenous hippocampal sequences even without imposing any requirements of aligning to theta oscillations, thus providing an unbiased estimate of the rhythmicity of hippocampal spatial representations.

(4) Additional analysis on place cell stability and tuning:

We thank the reviewer for this question. For the KL divergence analysis, we have imposed a spike-count criterion (100 spikes for each interval type —stimulation-off, stimulation-on, and the stimulus sub-interval) and a coverage criterion (50% HPD of the units’ spatial firing distribution was contained within 40cm on the linear track and 100cm on the w-track). These criteria were chosen to ensure that spatial tuning curves were sufficiently well sampled and localized to allow reliable estimation of KL divergence, which is particularly sensitive to noise arising from low spike counts or diffuse firing. Based on the reviewer’s suggestion, we have relaxed the unit inclusion criteria for KL divergence by relaxing the criteria for number of spikes and spatial coverage criterion to include more weakly tuned place cells and replicated our results (p=.146). Further, we have also evaluated the stability of place field order between stimulation-on and stimulation-off conditions using more standard methods (as in Wang et. al., 2015; spearman correlation of place field order, control vs targeted, p = .920, t-test). These results are consistent with our observations about place field stability during stimulation-off and stimulation-on conditions (Fig. 2F).

Author response image 2.

Spearman correlation of place field order during stimulation-on and stimulation-off conditions.

複雜的 səˈfistəˌkādəd 形容詞 复雜的 involuted, mazy, sophisticated 詭辯的 choplogical, sophistic, sophisticated 尖端 sophisticated

反轉

被拒絕 Bèi jùjué 動詞 駁斥 deny, disprove, rebut 抵賴 deny, disavow 否 deny, negate 否定 negative 否認 repudiate 拒絕 exclude, negative, nix 賴 rely 謝絕 turn down

被告

Synthèse : Enfants Violents à l'École - Entre Aide et Répression

Résumé Exécutif

Ce document de synthèse analyse les tensions et les débats entourant la gestion de la violence chez les jeunes enfants au sein du système scolaire et de la société française.

Il ressort que l'école se trouve démunie face à des comportements extrêmes, conduisant à la création de structures expérimentales comme "r'école" pour éviter la déscolarisation.

Parallèlement, une tendance croissante à la médicalisation des troubles du comportement, incarnée par le diagnostic d'hyperactivité et la prescription de Ritaline, suscite une vive controverse.

Des experts dénoncent l'influence du lobbying pharmaceutique et une simplification qui ignore les causes profondes de la souffrance de l'enfant.

Cette approche s'inscrit dans un contexte de "psychose médiatique" qui exagère le phénomène de la violence infantile, contredit par la réalité judiciaire qui atteste de la rareté des cas criminels chez les très jeunes.

L'analyse des cas individuels révèle que la violence est souvent le symptôme d'une souffrance psychique profonde, liée à des contextes familiaux difficiles (ruptures, violence parentale) et socio-économiques précaires.

Face à des réponses répressives ou médicamenteuses, des initiatives de prévention de proximité, comme l'association "Mission Possible", démontrent qu'un accompagnement axé sur l'écoute et le soutien aux familles est non seulement plus humain, mais aussi considérablement moins coûteux et plus efficace à long terme pour la société.

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1. Le Défi de l'École Face aux Comportements Extrêmes

Le système éducatif est confronté à une difficulté croissante pour gérer les comportements violents et ingérables de certains très jeunes élèves.

Les enseignants et les directions d'école expriment un sentiment d'impuissance et de manque de formation, menant à des situations d'échec et à l'exclusion des enfants concernés comme ultime recours.

• Le Cas d'Ethan Paul et Hamadi (6 ans) : Ces deux élèves de CP ont été exclus de leur école pour "comportement violent et ingérable".

Ethan Paul aurait tenté d'étrangler un camarade, conduisant des parents à porter plainte. Sa maîtresse reconnaît son échec :

"Je n'ai pas réussi à ce qu'ils puissent être intégrés en classe et faire les apprentissages de la place de façon satisfaisante." Elle décrit un enfant en "très très très très grande souffrance".

• La Structure "r'école" : Mise en place en janvier 2009 par le rectorat de Paris, cette structure unique en France accueille pour trois mois des enfants exclus.

◦ Objectif : Éviter la déscolarisation en offrant une "surveillance éducative renforcée" et en réapprenant aux enfants les règles de la vie en groupe.  

◦ Fonctionnement : Les enfants sont pris en charge par un personnel mixte (enseignante, auxiliaire de vie scolaire), mais seule l'enseignante est spécifiquement formée. Les crises de violence y sont fréquentes et difficiles à gérer pour le personnel.   

◦ Limites : Bien que présentée comme le "seul espoir", cette solution est coûteuse et soulève le risque de créer des filières alternatives pour enfants "difficiles", comme le souligne Jean-Louis Barateau, initiateur du projet : "Ça pourrait être dangereux si on multipliait des r'écoles au point d'avoir finalement des écoles alternatives."

2. La Médicalisation de la Violence Infantile : Une Solution Controversée

Face à l'inquiétude grandissante, une approche tendant à considérer les troubles du comportement comme des pathologies médicales à traiter a émergé, non sans susciter de vives critiques.

2.1. Le Rapport de l'INSERM et la Récupération Politique

• Le Rapport (2006) : Consacré aux "troubles des conduites", ce rapport d'experts visait à dépister les "facteurs de risque" et de "vulnérabilité" chez l'enfant.

• La Controverse : Le rapport a servi de caution scientifique à un projet de loi sur la délinquance des mineurs, porté par Nicolas Sarkozy, alors ministre de l'Intérieur.

Celui-ci affirmait : "Plutôt on n'intervient plus mieux on a de chances d'éviter le drame d'un enfant qui évolue vers la délinquance."

• La Réponse de la Société Civile : Des experts ayant participé au rapport ont précisé n'avoir "jamais écrit un rapport sur la prévention de la délinquance".

En réaction, la pétition "Pas de zéro de conduite pour les enfants de 3 ans" a recueilli près de 200 000 signatures, dénonçant le risque de transformer des comportements normaux (morsures, colères) en symptômes d'un trouble mental à rééduquer.

2.2. L'Hyperactivité et la Ritaline : Remède ou Simplification ?

Le diagnostic de "l'hyperactivité avec déficit de l'attention" (TDAH) et son traitement par la Ritaline, une amphétamine, sont au cœur du débat.

• La Défense du Traitement : La pédopsychiatre Marie-France Le Heuzey justifie son usage pour "améliorer la qualité de vie et d'améliorer le quotidien de ces enfants", soulignant la souffrance liée au rejet social et familial.

Pour elle, si le médicament permet à l'enfant de ne plus être puni et aux parents de moins se disputer, "on a aussi fait du bien largement à l'enfant".

• La Critique de la Sur-médicalisation :

◦ Rareté de la pathologie : Selon le Pr Bernard Golse (Hôpital Necker), l'hyperactivité "vraie" est très rare (1 à 2 cas pour 1000), loin des 5 à 10% avancés par certains. Il dénonce "l'effet direct du lobbying pharmaceutique qui veut élargir coûte que coûte la prescription de médicaments".   

◦ Création de la demande : Philippe Pignarre, ancien cadre de l'industrie pharmaceutique, explique la stratégie marketing : "L'industrie pharmaceutique travaille à créer à la fois l'offre et la demande... On va la créer la demande en disant aux gens... ce que vous saviez pas, c'est qu'il a un trouble mental et qu'on peut soigner ce trouble mental."   

◦ Traitement des symptômes, pas des causes : La Ritaline, surnommée "pilule de l'obéissance", agit sur les symptômes mais ne traite pas les causes sous-jacentes de la souffrance.

2.3. L'Expérience Vécue : Le Cas d'Aymeric

Aymeric, 16 ans, a été traité à la Ritaline pendant des années.

Son témoignage illustre l'ambivalence du traitement : "C'était bien mais c'est pas bien. Pourquoi c'était bien ? Parce que ça me calmait d'un côté. Mais... c'était bien pour eux, mais pour moi c'était pas bien parce que là je mangeais plus... j'étais tout le temps fatigué."

3. Psychose Médiatique et Réalité Judiciaire

La perception publique de la violence infantile est fortement influencée par un traitement médiatique qui tend à se focaliser sur des faits divers extrêmes, créant une forme de psychose collective.

• L'Affaire d'Uckange (2009) : Un Emballement Révélateur :

◦ Un garçon de 5 ans est accusé d'avoir poignardé sa sœur de 8 ans, prétendument sous l'influence de jeux vidéo.

L'affaire est largement médiatisée, et la thèse de l'enfant coupable est acceptée par les médias, la police et la justice.   

◦ Quelques jours plus tard, la mère avoue être l'auteure du coup de couteau. L'affaire démontre la rapidité avec laquelle "les médias ont véhiculé un peu trop vite le scénario de l'enfant criminel".

• La Perspective des Magistrats : La juge pour enfants Marie-Pierre Hourcade affirme que les affaires de violence au pénal impliquant de très jeunes enfants sont très rares.

Le critère de la responsabilité pénale est le discernement, qui apparaît vers 7-8 ans. "En aucune façon le parquet ne nous saisit pour des situations de violences commises par des très jeunes enfants."

4. Derrière la Violence : Souffrance Psychique et Contexte Familial

L'analyse approfondie des cas révèle que les comportements violents sont presque toujours l'expression d'une souffrance profonde, souvent enracinée dans des histoires familiales et sociales complexes.

• L'Expression de la Souffrance : Le Cas de Sami (13 ans) :

◦ Retiré à 8 ans d'un contexte familial violent, Sami a été ballotté de foyer en foyer. Sa violence est une manifestation de sa tristesse face aux ruptures affectives répétées.  

◦ Le Dr Roger Teboul, psychiatre, explique : "Bien souvent, quand vous parlez de la violence, vous parlez de la tristesse... Le seul truc qui permet de tenir, c'est d'être en colère."

L'objectif de son service est de permettre à ces jeunes d'exprimer leur tristesse pour ne plus avoir à l'agir par la violence.

• L'Impact du Contexte Socio-économique : Le Cas de Florian (7 ans) :

◦ Florian vit dans un quartier précaire d'Amiens. Sa mère élève seule 5 enfants avec le RMI. L'État s'est largement désengagé du quartier.  

◦ Cet environnement de précarité rend l'éducation extrêmement difficile. La mère de Florian exprime sa peur que son fils devienne délinquant si elle n'est pas soutenue.

5. Stratégies d'Intervention : La Prévention comme Alternative

Face aux approches répressives ou médicales, les initiatives de prévention axées sur l'accompagnement et le soutien des familles démontrent leur pertinence humaine et économique.

• "Mission Possible" : Un Modèle de Prévention de Proximité :

◦ Créée par le juge des enfants Claude Baud, cette association à Amiens accueille librement des familles sans obligation judiciaire.  

◦ Elle offre un soutien aux parents, souvent démunis et en rupture avec les services sociaux, sans les juger ni les culpabiliser.   

◦ Elle apprend aux enfants les règles de vie en société par le dialogue et un cadre clair, en cherchant à comprendre le sens de leurs comportements plutôt qu'à les étiqueter.

• L'Analyse d'un Juge : Coût et Efficacité : Claude Baud souligne les avantages de la prévention :

◦ Moins Stigmatisant : "Un parcours judiciaire pour un enfant est dix fois plus stigmatisant qu'un parcours de prévention."   

◦ Moins Coûteux : Il établit une comparaison financière éloquente :      

▪ Prévention (Mission Possible) : 8 € par jour et par enfant.     

▪ Placement Éducatif : 200 à 400 € par jour.    

▪ Détention en section mineurs : 700 à 1000 € par jour.

La conclusion est claire : investir dans des moyens humains, de l'écoute et du personnel bien formé pour aider les familles et les enfants en souffrance est infiniment moins coûteux que de devoir gérer, quelques années plus tard, les conséquences de cette souffrance non traitée.

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Cher Tony Gheeraert,

Merci pour cet article, que j'ai vraiment apprécié. Parce que c'est une chronique, elle pourrait être publiée telle quelle. j'ai fait un certain nombre de commentaires, que vous être libre d'utiliser ou non, qui ne s'insèrent pas toujours dans la logique de l'article (reprendre la théorie marxiste de la création de valeurs et essayer de l'appliquer à la situation présente de l'IA) et que vous êtes bien évidemment libre de reprendre ou non.

Reprenons les différents critères d'évaluation :

Pertinence de la réflexion

Cet article propose une relecture marxiste de l'économie de l'IA générative, de la bulle spéculative qu'elle forme, etc. Même pour un non-marxiste, cette relecture est intéressante: dans mon expérience de chercheur non-marxiste, les écrits marxistes (orthodoxes ou hétérodoxes) m'ont toujours forcé à regarder des situations avec un autre regard et, donc, à varier mes analyses.

Deux exemple: le livre de Gavin Mueller qui essaye de réconcilier luddisme et marxisme (Mueller Gavin, Breaking things at work: the Luddites are right about why you hate your job, London New York, Verso, 2021.) J'avoue que je me contrefiche un peu de la compatibilité du luddisme avec le marxisme (sauf par intérêt historique). Mais cela a poussé Mueller à analyser la nature du mouvement du logiciel libre comme forme de luddisme technophile, idée que je repends depuis à mon tompte.

Second exemple, très loin de ce qui nous préoccupe ici: ce sont notamment des analyses marxistes hétérodoxes qui ont poussé les historiens et historiennes à regarder de plus près les politiques financière, budgétaires des dictatures fascistes et nazies, dès la fin des années 1940.

Le seul problème de cette démarche de relecture marxiste est qu'elle peut être souvent frustrante pour le lecteur. Est-ce qu'il y a des moyens d'éviter cela, je n'en suis pas sûr.

Je reste cela dit peu convaincu par l'entrée en matière de l'article. Le texte de gemini est vraiment mauvais et caricatural (et en outre, j'aime bien avoir les prompts quand quelqu'un cite une IA).

Subjectivité et démarche

Je renvoie à la section précédente sur ce point, car la démarche, une relecture marxiste, y est bien commentée.

Contribution au champ disciplinaire

Il est indéniable que cet artcile participe à quelque chose d'important, démystifier l'IA générative. Ce que je regrette un peu, c'est le fait que l'auteur ne fait pas complètement le tri (de manière explicite, s'entend) entre certains mythes de l'IA et la réalité économique de l'IA. Je reconnais que c'est assez difficile, dans la mesure où certains éléments relèvent des deux, la question de la bulle spéculative en premier lieu. Mais on a parfois l'impression que lauteur succombe lui-même un peu au mythe de l'IA (ce que je ne juge pas, moi-même y succombant parfois aussi). Mais je ne peux qu'être d'accord avec la logique d'ensemble de l'article et notamment la section "Par-delà « le libre »".

Avis général sur la publication

Comme c'est une chronique, je dirais que l'article peut être publié sans modification. À l'auteur, ou à l'éditeurice, de voir s'il souhaite prendre en compte certaines de mes remarques.

Bien à vous.

oui! C'est aussi le raisonnement de Bender et Hannah dans The AI Con.

À titre personnel, je ne peux qu'approuver ces trois points. La question reste de savoir s'ils sont applicables, car: - quelles conditions économiques pour qu'ils soient applicables? Les grandes plateformes sont quand même assez douées pour étouffer ce type de modèles alternatifs, - quelles conditions sociales, disons, et institutionnelles?

Sur ce second point, ce que je veux dire, c'est qu'il faudrait notamment -- pour juste considérer le milieu universitaire -- que ce milieu s'empare de l'IA générative, pour être prête à proposer des modèles alternatifs de développement de l'IA générative (et au-delà). Or celles et ceux qui veulent s'en emparer et en faire quelque chose d'autre vont de plus en plus vers un rejet complet de l'IA générative (voir les deux prises de position sur la liste DH: https://groupes.renater.fr/sympa/arc/dh/2025-12/msg00047.html , https://www.lobrassard.net/carnet/2025-12-19-ia-agnotologie.html et https://atecopol.hypotheses.org/13082)

Le résultat est que beaucoup d'usage de l'IA dans le monde universitaire se fait à bas bruit (du côté des chercheurs et chercheuses en SHS, s'entend, pas du côté des institutions qui elles font grand bruit de beaucoup de bullshit). Bref, les conditions ne sont pas réunies pour un tel programme.

Il y a des travaux des Digital Humanities sur la frugalité informatique: https://dhq-static.digitalhumanities.org/pdf/000646.pdf

Et ce texte de Lauren Klein, qui reprend des éléments de son Data feminism (avec d'Ignazio) et essaye de l'appliquer à l'IA (et doute): https://zenodo.org/records/6567985

À nouveau, les travaux de Casilli et du diplab, mais pas uniquement d'eux d'ailleurs, seraient éclairants ici, je pense.

Sans vouloir faire de la pub, il y a les travaux de pleias, startup, qui a mis en place un corpus d'entraînement constitué de données du domaine public: https://huggingface.co/blog/Pclanglais/common-corpus

(le fait que deux des co-fondateurs, si mes souvenirs sont bons, de pleias viennent des SHS est probablement lié à leur recherche d'utiliser les communs pour l'IA).

Ce qui n'est pas totalement nouveau, en effet -- Graeber l'a montré, mais beaucoup d'économistes (je n'ai pas ici de références précises, pardon, sauf Robert Solow et le paradoxe de la productivité) ont mis en doute les gains de productivité proclamés de l'informatique dès les années 1980.

eLife Assessment

This is a useful study that investigates the role of the long non-coding RNA Dreg1 for the development, differentiation, or maintenance of group 2 ILC (ILC2). The authors generate Dreg1-/- mice and show a reduction of group 2 innate lymphoid cells (ILC2). However, the strength of evidence supporting the impact of Dreg1 on Gata3 expression, a transcription factor required for ILC2 cell fate decisions, and the cell-intrinsic requirement of Dreg1 for ILC2 remain incomplete. This study will be of interest to immunologists.

Reviewer #1 (Public review):

Summary:

This study examines the role of the long non-coding RNA Dreg1 in regulating Gata3 expression and ILC2 development. Using Dreg1-deficient mice, the authors show a selective loss of ILC2s but not T or NK cells, suggesting a lineage-specific requirement for Dreg1. By integrating public chromatin and TF-binding datasets, they propose a Tcf1-Dreg1-Gata3 regulatory axis. The topic is relevant for understanding epigenetic regulation of ILC differentiation.

Strengths:

(1) Clear in vivo evidence for a lineage-specific role of Dreg1.

(2) Comprehensive integration of genomic datasets.

(3) Cross-species comparison linking mouse and human regulatory regions.

Weaknesses:

(1) Mechanistic conclusions remain correlative, relying on public data.

(2) Lack of direct chromatin or transcriptional validation of Tcf1-mediated regulation.

(3) Human enhancer function is not experimentally confirmed.

(4) Insufficient methodological detail and limited mechanistic discussion.

Reviewer #2 (Public review):

The authors investigate the role of the long non-coding RNA Dreg1 for the development, differentiation, or maintenance of group 2 ILC (ILC2). Dreg1 is encoded close to the Gata3 locus, a transcription factor implicated in the differentiation of T cells and ILC, and in particular of type 2 immune cells (i.e., Th2 cells and ILC2). The center of the paper is the generation of a Dreg1-deficient mouse. While Dreg1-/- mice did not show any profound ab T or gd T cell, ILC1, ILC3, and NK cell phenotypes, ILC2 frequencies were reduced in various organs tested (small intestine, lung, visceral adipose tissue). In the bone marrow, immature ILC2 or ILC2 progenitors were reduced, whereas a common ILC progenitor was overrepresented, suggesting a differentiation block. Using ATAC-seq, the authors find that the promoter of Dreg1 is open in early lymphoid progenitors, and the acquisition of chromatin accessibility downstream correlates with increased Dreg1 expression in ILC2 progenitors. Examining publicly available Tcf1 CUT&Run data, they find that Tcf1 was specifically bound to the accessible sites of the Dreg1 locus in early innate lymphoid progenitors. Finally, the syntenic region in the human genome contains two non-coding RNA genes with an expression pattern resembling mouse Dreg1.

The topic of the manuscript is interesting. However, there are various limitations that are summarized below.

(1) The authors generated a new mouse model. The strategy should be better described, including the genetic background of the initially microinjected material. How many generations was the targeted offspring backcrossed to C57BL/6J?

(2) The data is obtained from mice in which the Dreg1 gene is deleted in all cells. A cell-intrinsic role of Dreg1 in ILC2 has not been demonstrated. It should be shown that Dreg1 is required in ILC2 and their progenitors.

(3) The data on how Dreg1 contributes to the differentiation and or maintenance of ILC2 is not addressed at a very definitive level. Does Dreg1 affect Gata3 expression, mRNA stability, or turnover in ILC2? Previous work of the authors indicated that knockdown of Dreg1 does not affect Gata3 expression (PMID: 32970351).

(4) How Dreg1 exactly affects ILC2 differentiation remains unclear.

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https://k51qzi5uqu5div1auuxm59ygav4p7gdg9z4e9iggtu6m43rmc3xw75mczx2b7x.ipns.dweb.link/%E2%99%96/hyperpost/~/indyweb/%F0%9F%93%93/20/26/14/setup-indy0wiki.pad.html

Synthèse sur la Maltraitance Infantile : Thèmes, Intervenants et Cas d'Étude

Synthèse Exécutive

Ce document de synthèse analyse les thèmes centraux, les dynamiques et les conséquences de la maltraitance infantile, en se basant sur une série d'études de cas et d'interventions d'experts.

L'analyse révèle que la maltraitance est un phénomène polymorphe, incluant la violence physique extrême, le syndrome du bébé secoué, les abus sexuels et les négligences graves.

Une conclusion alarmante émerge : dans la majorité des cas (neuf sur dix), les sévices sont infligés au sein même de la cellule familiale, transformant le lieu de sécurité supposé en principal foyer de danger.

Le silence des victimes, la complicité passive ou active de certains membres de la famille et l'aveuglement de l'entourage constituent des obstacles majeurs à la protection des enfants.

La chaîne d'intervention, bien que complexe, est clairement définie : elle commence par une alerte (via le 119 ou un signalement médical), se poursuit par une enquête policière (Brigade des Mineurs), aboutit à une réponse judiciaire (Procureur, Juge des enfants) et se conclut par une prise en charge spécialisée (placement, suivi psychologique).

Les séquelles de la maltraitance sont profondes et durables, affectant les victimes sur les plans physique, psychologique et comportemental.

Néanmoins, les témoignages de résilience, illustrés par des parcours de reconstruction personnelle et la recréation de liens affectifs, soulignent que la guérison, bien que longue et ardue, reste possible grâce à un soutien adéquat et continu.

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1. Les Multiples Visages de la Maltraitance

La maltraitance infantile se manifeste sous diverses formes, souvent cumulatives, allant des sévices physiques aux abus psychologiques et sexuels.

Violences Physiques et Sévices Graves

La violence physique est la forme la plus visible de la maltraitance. Les statistiques présentées sont alarmantes : chaque semaine en France, trois enfants meurent des suites de mauvais traitements.

• Le cas de Gaël : Adolescent de 15 ans, il a été victime de violences extrêmes de la part de son père pendant 21 mois, à l'âge de 8 ans. Ses témoignages décrivent un calvaire :

◦ Brûlures de cigarettes.  

◦ Coups portés avec divers objets : manche à balai, fourchette à poulet, bouteille de verre, assiette, pare-chocs de voiture.  

◦ Tentative de noyade dans la baignoire.  

◦ Humiliations extrêmes, comme être forcé à manger des excréments de chien.

Son père a été condamné à 14 ans de prison ferme.

Gaël utilise aujourd'hui la boxe à haut niveau pour "dégager la haine" et se reconstruire.

• Le cas de Dylan : Enfant de 4 ans décédé en 2003, son corps présentait d'innombrables traces de coups, de morsures et de brûlures de cigarettes, infligées par son beau-père.

Il était devenu son "souffre-douleur" depuis plusieurs mois.

Le Syndrome du Bébé Secoué

Une forme de violence spécifique aux nourrissons est mise en évidence : le syndrome du bébé secoué.

• Mécanisme : Le Dr Philippe Meyer explique qu'il ne s'agit pas d'un simple jeu, mais de "mouvements très répétitifs" et "extrêmement violents".

La tête du bébé, très lourde et mal soutenue par les muscles du cou, subit des accélérations et décélérations qui provoquent des hémorragies cérébrales (hématome sous-dural).

• Prévalence : L'hôpital Necker reçoit plus de 50 bébés par an présentant ces symptômes.

• Conséquences : Les séquelles peuvent être irréversibles, et un bébé secoué sur dix en meurt.

• Cas étudiés :

◦ Louis (6 jours) : Arrivé pour un hématome sous-dural, son cas est d'autant plus suspect que son frère est décédé cinq ans plus tôt dans des circonstances similaires, conduisant les médecins à faire un signalement au procureur.  

◦ Willy (3,5 mois) : Admis pour le même symptôme, son père avoue lui avoir porté un coup lors d'une dispute.

Il reconnaît son geste : "J'ai craqué [...] j'ai fait ces gestes là j'ai regretté".

Abus Sexuels

Les abus sexuels, souvent perpétrés par des proches, sont une autre facette de la maltraitance.

• Le cas d'Elena (7 ans) : La fillette se plaint d'avoir été touchée par Yvon, l'ami de sa grand-mère.

L'enquête de la Brigade des Mineurs révèle que l'agresseur présumé a déjà des antécédents pour "agression sexuelle sur mineur" en 1998. Confronté, il avoue les faits.

• Le cas d'Estelle : Violée de 2 à 12 ans par son grand-père maternel, elle n'a osé en parler que dix ans plus tard.

Son parcours illustre la difficulté de la révélation et le poids de la culpabilité et de la honte, qui se sont traduits par des conduites à risque (tentatives de suicide, drogue) à l'adolescence.

Négligences Graves et Violences Psychologiques

La maltraitance ne se limite pas aux actes de commission.

• Le placement d'un enfant de 8 ans : La Brigade des Mineurs intervient pour retirer un enfant de sa famille suite à des "graves négligences". L'enfant n'est pas scolarisé et les services sociaux n'ont plus accès à la famille.

• Le cas de Marie : Placée à 15 ans, elle a fui une famille où, au-delà des violences physiques, régnait une "violence psychologique permanente". Elle témoigne : "chaque fois je faisais quelque chose ma mère me disait que ça allait pas tout le temps tout le temps". Cette emprise psychologique l'a conduite à des pensées suicidaires.

2. L'Environnement Familial : Principal Foyer de Danger

Le documentaire souligne de manière récurrente que le danger provient le plus souvent de l'entourage immédiat de l'enfant.

La Responsabilité des Auteurs et le Silence Complice

Les auteurs des violences sont les parents, beaux-parents ou des proches. Le silence d'un des parents peut être assimilé à une forme de complicité.

• Le cas d'Adeline, mère de Dylan : Elle est jugée pour ne pas avoir dénoncé les violences infligées par son compagnon à son fils.

Elle a retiré Dylan de l'école pour cacher ses blessures. Son procès en appel aboutit à une peine alourdie à 20 ans de réclusion criminelle.

Pour l'avocat du père de Dylan, son comportement n'était pas un simple silence mais une "dissimulation" active des faits, court-circuitant toute aide possible.

L'Aveuglement et la Culpabilité de l'Entourage

L'entourage élargi peine souvent à percevoir ou à admettre la réalité de la maltraitance, ce qui engendre une profonde culpabilité a posteriori.

• L'entourage de Gaël : La mère de Gaël, Carole, a lutté seule pendant deux ans pour récupérer son fils, séquestré par son ex-mari.

Les grands-parents expriment leur regret : "Carole disait toujours mon enfant est en danger et nous autour d'elle, on le croyait pas [...] on ne peut pas imaginer qu'on s'est rendu compte de rien." Ils avouent même avoir pensé qu'elle "amplifiait la chose".

• L'indifférence du voisinage : Gaël raconte avoir dormi en slip sur le toit du garage, visible par des centaines de personnes, y compris les parents et enfants de l'école voisine. "Personne a jugé bon de signaler qu'il y avait un souci, c'est inadmissible."

3. La Chaîne d'Intervention : Du Signalement à la Protection

Le processus de prise en charge d'un enfant en danger implique une succession d'acteurs institutionnels.

| Étape | Acteurs Clés | Actions et Observations | | --- | --- | --- | | L'Alerte | Ligne 119, entourage, écoles, médecins | Le service du 119 reçoit plus de 4000 appels par jour. L'alerte est le point de départ crucial qui déclenche l'intervention. | | Le Diagnostic Médical | Médecins hospitaliers (pédiatres, réanimateurs) | Ils sont en première ligne pour détecter les signes physiques (hématomes, fractures). Leur rôle est de soigner mais aussi de signaler les suspicions aux autorités judiciaires, comme dans le cas de Louis. | | L'Enquête Policière | Brigade de Protection des Mineurs | Les policiers mènent des auditions et des interrogatoires pour établir les faits. Leur travail consiste à démêler le vrai du faux face aux dénégations initiales des parents (cas du bébé secoué) ou à obtenir les aveux (cas d'Elena). | | La Réponse Judiciaire | Procureur de la République, Juge des enfants | Le procureur décide des suites à donner (mise en examen, contrôle judiciaire, procès). Le juge des enfants prend les mesures de protection nécessaires (enquête sociale, placement) et évalue la sécurité de l'enfant dans son milieu familial (cas d'Elena et de Marie). | | Le Placement et le Soin | Foyers, pouponnières, éducateurs spécialisés, pédopsychiatres | Lorsque le danger est avéré, les enfants sont retirés de leur famille et placés dans des structures spécialisées. Le placement est souvent un traumatisme, comme le montre l'intervention forcée pour l'enfant de 8 ans. Le soin vise à "réparer" les traumatismes (cas de Roxane et Charlotte à la pouponnière). |

4. Les Séquelles et le Chemin de la Reconstruction

Les conséquences de la maltraitance sont profondes et nécessitent un travail de reconstruction de longue haleine.

Traumatismes Physiques et Psychologiques

• Séquelles physiques : Gaël conserve de multiples cicatrices de ses blessures.

• Séquelles psychologiques : Me Brun Meyrin, l'avocate de Gaël, souligne : "Il a surtout des séquelles morales dont on se demande bien comment elles pourraient ne pas avoir de conséquences dans son futur."

La psychologue Martine Nisse explique que la communication paradoxale dans les familles maltraitantes ("c'est pour ton bien que je te frappe") rend les enfants "difficiles à comprendre".

• Comportements post-traumatiques : Les enfants placés en pouponnière manifestent des troubles du comportement :

Roxane, exposée à la violence, développe de l'agressivité et des difficultés relationnelles ; Charlotte, bébé secoué, a appris à "éviter la relation" en se protégeant du contact physique.

La Thérapie comme Voie de Guérison

Le suivi psychologique est essentiel pour surmonter le traumatisme.

• Le cas d'Estelle : Après quatre ans de thérapie, elle a pu mettre des mots sur l'inceste subi et déconstruire le sentiment de culpabilité. "Là j'ai compris vraiment que j'y étais pour rien [...] la honte elle reste mais elle s'estompe."

• L'importance de la parole : L'éducateur de Marie souligne que "le fait qu'il y ait une intervention du commissariat [...] n'a pas réglé les problèmes". Il a fallu deux ans et demi pour qu'elle arrive progressivement à "prendre en main sa vie".

La Résilience et la Reconstruction des Liens

Malgré la gravité des faits, des parcours de résilience sont possibles.

• Gaël : La boxe lui sert d'exutoire et il retisse un lien fort avec sa mère, Carole. Il parvient à formuler : "Grâce à ma mère, je suis là."

• Marie : Bien qu'inquiète de sa majorité, elle demande à la juge de continuer à la protéger, montrant sa volonté de se construire un avenir stable.

• Cindy, mère de Roxane : En désintoxication et séparée de son conjoint violent, elle s'engage dans un processus pour recréer un lien avec ses enfants et espère pouvoir un jour les récupérer.

5. Citations Clés

• Gaël, victime de violences paternelles : "C'est pour pouvoir me défendre, c'est pour pouvoir dégager la haine que j'ai sur lui."

• Père de Willy, auteur de secouement : "J'ai craqué [...] je pardonne pas parce qu'on fait pas ça à un bébé mais je sais que ça peut arriver à n'importe qui."

• Grand-père de Gaël, sur sa culpabilité : "Carole disait toujours mon enfant est en danger et nous autour d'elle, on le croyait pas [...] je m'imaginais jamais ce qui se passe."

• Maître Bejo, avocat du père de Dylan : "On n'est pas dans le silence, on est dans un comportement actif de dissimulation des faits et c'est ce comportement actif qui a court-circuité toutes les velléités d'intervention."

• Dr Renier, pionnier sur le syndrome du bébé secoué : "Ce qui fait la différence entre un bien-traitant pour un bébé et un non bien-traitant [...] c'est la maîtrise et la maîtrise elle est indispensable en toutes circonstances."

• Françoise Achard, médecin scolaire, aux enseignants : "On sait que tout le monde peut être maltraitant, c'est-à-dire que ces parents qui avaient l'air bien sympathiques, et ben ça veut pas dire pour autant qu'ils soient pas maltraitants dans l'intimité de leur maison."

• Martine Nisse, psychologue : "Je crois que les principaux sévices c'est la famille, c'est le principal danger pour l'enfant."

• Carole, mère de Gaël : "On essaie de récupérer mais on récupérera jamais ces années, c'est des années qui vont nous manquer toujours."

what is embedded mean?

Note d'information : La Stratégie d'Expansion du Groupe Emeis (ex-Orpea) dans le Secteur de la Psychiatrie en France

Synthèse Exécutive

Cette note d'information analyse la stratégie d'expansion du groupe privé lucratif Emeis (anciennement Orpea) dans le secteur de la psychiatrie en France.

Elle s'appuie sur une enquête journalistique qui met en lumière comment le groupe, marqué par le scandale de ses EHPAD, capitalise sur la crise profonde de la psychiatrie publique pour s'implanter sur ce marché jugé très rentable.

L'analyse révèle une situation de crise systémique dans le secteur public : un sous-financement chronique, un manque criant de personnel (seulement 600 pédopsychiatres en France), des infrastructures vétustes et une explosion de la demande de soins, notamment chez les jeunes depuis la crise du Covid (+77 % d'épisodes dépressifs chez les 18-24 ans).

Dans ce contexte, Emeis déploie une stratégie agressive pour s'imposer, illustrée par un projet de clinique de 80 lits près de Strasbourg.

Cette implantation, menée via sa filiale Clinea, s'est initialement appuyée sur une alliance "étonnante" avec un concurrent, Clinipsy.

L'enquête suggère que cette alliance aurait pu servir de "cheval de Troie" pour Emeis, lui permettant d'obtenir des autorisations administratives que le groupe, sous le nom d'Orpea, s'était vu refuser à plusieurs reprises depuis 2007.

Les principales préoccupations soulevées sont le risque d'affaiblissement de l'hôpital public par le débauchage de son personnel, une complémentarité illusoire où le privé se concentrerait sur les cas les plus rentables en laissant les plus complexes au public, et un modèle économique basé sur la rentabilité qui pourrait se faire au détriment de la qualité des soins par la réduction des effectifs.

Enfin, le document souligne l'opacité de l'Agence Régionale de Santé (ARS) Grand Est, qui a refusé de communiquer des documents essentiels sur ce projet malgré les importants financements publics engagés.

1. Le Contexte : Une Psychiatrie Publique en Crise Profonde

La psychiatrie en France est décrite comme étant "malade" et "abandonnée par les pouvoirs publics".

Ce secteur est devenu le "parent pauvre de la santé", confronté à un manque critique de moyens alors que les besoins de soins explosent.

• Explosion de la demande : La crise du Covid et les confinements ont provoqué une forte augmentation des pathologies mentales.

◦ +77 % d'épisodes dépressifs chez les 18-24 ans.    ◦ +133 % d'hospitalisations pour tentative de suicide ou automutilation.

• Manque de moyens structurel : Le secteur public souffre d'un sous-investissement chronique.

◦ Une politique ambulatoire non financée : Depuis les années 1980, une politique de fermeture de lits a été menée au profit de soins ambulatoires (hors de l'hôpital).

Cependant, les moyens financiers n'ont pas suivi pour développer ces structures alternatives comme les Centres Médico-Psychologiques (CMP).  

◦ Pénurie de personnel : La France compte environ 600 pédopsychiatres, laissant des départements entiers sans spécialiste.  

◦ Diminution des capacités : L'hôpital public a perdu près de 7 000 places de prise en charge psychiatrique à temps complet en 15 ans.

• Vétusté des infrastructures : L'état des bâtiments publics est alarmant.

À Strasbourg, le secteur de la pédopsychiatrie des Hôpitaux Universitaires est logé dans des "bâtiments complètement vétustes" et des "préfabriqués".

L'Inspection générale des affaires sociales (Igas) a signalé des risques d'incendie et demandé un déménagement en urgence, qui n'a été annoncé que 15 ans plus tard.

"On a alerté qu'on allait droit dans le mur et le mur aujourd'hui on se le prend en pleine face." - Un soignant, cité dans le documentaire de Laurence Deur.

2. L'Émergence d'un Nouvel Eldorado : Le Secteur Privé Lucratif

La défaillance du système public crée une opportunité majeure pour les groupes privés à but lucratif, qui considèrent la psychiatrie comme un "marché très rentable".

• Un secteur profitable : Selon un rapport récent du Sénat, la psychiatrie est l'un des secteurs de la santé les plus rentables, avec des marges estimées entre 5 % et 8 %.

L'investissement principal étant l'humain, la réduction du personnel est le principal levier pour augmenter les profits.

• Une croissance rapide : La part du secteur privé lucratif dans l'offre de soins psychiatriques a considérablement augmenté :

◦ 1975 : 11 % des lits.    ◦ Aujourd'hui : Plus de 30 % des lits.

• Un parallèle avec les EHPAD : La situation actuelle en psychiatrie est comparée à la privatisation du secteur des EHPAD dans les années 1980.

Face à des établissements publics vieillissants et coûteux à rénover, l'État avait ouvert la porte au privé qui promettait de "faire moins cher, plus vite".

• Le rôle des ARS : Les Agences Régionales de Santé, autrefois réticentes à ouvrir la psychiatrie au privé, sont aujourd'hui plus enclines à le faire.

Face à l'incapacité du public à répondre à la demande immense, elles autorisent l'ouverture de cliniques et d'hôpitaux de jour privés.

3. Étude de Cas : La Stratégie d'Implantation d'Emeis à Strasbourg

L'enquête se concentre sur un projet de clinique psychiatrique privée de 80 lits à Schiltigheim, près de Strasbourg, porté par le groupe Emeis (ex-Orpea), rebaptisé pour faire oublier le scandale révélé par le livre Les Fossoyeurs. Ce projet est jugé "démesuré" et "anachronique" par les acteurs locaux.

Une Alliance Stratégique Inédite

Le projet est né d'une alliance "étonnante" entre deux concurrents :

1. Clinea : La filiale sanitaire d'Emeis/Orpea.

2. Clinipsy : Un acteur plus petit, déjà connu pour une enquête du Parquet National Financier (PNF) concernant des autorisations obtenues en région Rhône-Alpes par d'anciens fonctionnaires de l'ARS locale, ensuite embauchés par des filiales du groupe.

Cette collaboration entre concurrents directs est jugée inhabituelle, comparable à "si Intermarché et Leclerc montaient un supermarché ensemble".

L'Hypothèse du "Cheval de Troie"

L'enquête soulève l'hypothèse que cette alliance aurait servi de stratégie à Emeis pour contourner des obstacles réglementaires.

• Dissimulation : Emeis se serait "dissimulé un petit peu" derrière le nom de Clinipsy, un groupe plus petit avec une "moins mauvaise image" auprès des ARS, pour obtenir plus facilement les autorisations.

• Historique des refus : Des documents montrent qu'Orpea tentait d'ouvrir une clinique psychiatrique dans la région depuis au moins 2007 et avait essuyé au moins deux refus de la part de l'agence régionale (alors ARH).

Depuis, Clinipsy s'est désengagé du projet de clinique de 80 lits pour se concentrer sur des hôpitaux de jour, des structures moins coûteuses et "extrêmement rentables", laissant le champ libre à Emeis pour le projet principal.

"La question [...] se pose de savoir si Clinipsy a été un petit peu le cheval de Troie d'Orpea dans cette affaire." - Laurence Deur, journaliste.

4. Les Risques Systémiques de la Privatisation

L'arrivée massive d'acteurs privés lucratifs comme Emeis dans la psychiatrie fait peser plusieurs risques majeurs sur l'équilibre global du système de santé mentale.

Le "Pillage" des Ressources Humaines du Public

La principale inquiétude est que les nouvelles cliniques privées, en offrant de meilleures conditions de travail ou de rémunération, ne débauchent le personnel médical et soignant déjà en sous-effectif dans le secteur public.

• Un exemple concret : Un courrier de 2022 révèle qu'une clinique privée près de Nancy a débauché cinq médecins de l'hôpital public local, fragilisant ce dernier.

• L'inquiétude de la Mairie de Strasbourg : La maire, Jeanne Barségan, craint que le projet de 80 lits n'aggrave la pénurie de psychiatres et ne "vide" l'hôpital public de ses forces.

Une Complémentarité Illusoire : Le "Triage" des Patients

L'offre privée est souvent présentée comme "complémentaire" du public. Cependant, l'analyse montre qu'elle ne remplit pas les mêmes missions.

• Évitement des cas complexes : Le privé évite généralement les missions les plus lourdes et les moins rentables, comme l'hospitalisation sous contrainte, qui nécessite plus de personnel et de temps.

• Gestion des urgences "à la carte" : Dans le projet d'Emeis, la prise en charge des urgences se ferait "de gré à gré", sans obligation contraignante. Le médecin du privé peut accepter ou refuser un patient envoyé par le public.

• La conclusion : "Tout ce qui est complexe reste dans l'hôpital public", tandis que le privé se positionne sur des missions "plus faciles à assurer".

Le Modèle Économique : Profit vs. Qualité des Soins

Emeis est une entreprise cotée en bourse qui doit générer du profit pour ses actionnaires.

• Le levier du personnel : En psychiatrie, où "l'investissement, c'est l'humain", la principale méthode pour augmenter la rentabilité est de réduire les effectifs.

• Conflits sociaux : Plusieurs conflits sociaux ont éclaté dans des cliniques psychiatriques d'Emeis (Thionville, Nord, Isère) où le personnel dénonçait un manque d'effectifs et une réorganisation du travail impactant la qualité des soins.

Une grève de trois semaines a eu lieu à Seyssins, un événement "extrêmement rare dans le privé".

"Une entreprise est là pour faire du profit alors que l'hôpital public on lui demande pas d'être profitable, on lui demande d'être à l'équilibre." - Laurence Deur, journaliste.

5. Le Rôle et l'Opacité des Autorités de Régulation

L'enquête met en cause le manque de transparence de l'Agence Régionale de Santé (ARS) Grand Est.

• Rétention d'information : La journaliste a été "baladée pendant un mois et demi" sans obtenir de réponse ni les documents demandés concernant le projet de clinique Emeis.

L'ARS a fini par envoyer un document public générique qui ne correspondait pas à la demande.

• Recours à la CADA : Il a fallu saisir la Commission d'Accès aux Documents Administratifs (CADA) pour obtenir une partie des informations.

• Enjeux financiers publics : Cette opacité est jugée problématique car le projet engage d'importants fonds publics.

Les autorisations délivrées "valent des millions d'euros" et le groupe peut prétendre à une "dotation d'amorçage" de l'État pour financer son démarrage.

Cette situation soulève des questions sur le contrôle et la régulation de l'expansion du secteur privé lucratif, financée en partie par de l'argent public, dans un domaine aussi sensible que la santé mentale.

Synthèse du rapport : Protection de l’enfance et maltraitances — État des lieux 2025

Résumé Exécutif

Ce document de synthèse présente les principales conclusions du rapport "Protection de l’enfance et maltraitances — État des lieux 2025", publié par l’Observatoire national de la protection de l’enfance (ONPE).

L'analyse des données, arrêtées au 31 décembre 2023, révèle plusieurs tendances structurelles profondes qui redéfinissent le paysage de la protection de l'enfance en France.

Au 31 décembre 2023, 364 200 prestations et mesures étaient en cours pour les mineurs et 33 400 pour les jeunes majeurs, des chiffres en augmentation significative sur la dernière décennie.

Les dynamiques clés sont les suivantes :

1. Une croissance globale et continue : Le nombre total d'interventions pour les mineurs a augmenté de 22 % entre 2013 et 2023.

Le taux de prise en charge pour 1 000 mineurs a quant à lui progressé de 27 % sur la même période, passant de 20,3 ‰ à 25,8 ‰, une hausse accentuée par la baisse démographique de cette tranche d'âge.

2. Un basculement structurel vers l'accueil : Pour la première fois depuis le début du suivi, l'accueil (placement hors du domicile familial) est devenu majoritaire, représentant 52,2 % des interventions pour les mineurs.

Cette inversion de tendance, amorcée en 2018, marque un changement profond par rapport au suivi en milieu ouvert (à domicile).

3. La prédominance de l'hébergement en établissement : Une seconde inversion de tendance est observée dans les modalités d'accueil.

L'hébergement en établissement (41 %) dépasse désormais l'accueil familial traditionnel chez les assistants familiaux (36 %), qui voit sa part diminuer de manière continue.

4. Une judiciarisation accrue des mesures : La part des interventions décidées par un juge ne cesse de croître, atteignant 82,4 % de l'ensemble des mesures pour mineurs en 2023, contre 78,6 % en 2013.

Cette tendance est particulièrement marquée pour les mesures d'accueil (92,1 %).

5. L'impact majeur des mineurs non accompagnés (MNA) : La forte augmentation du nombre de MNA pris en charge (46 200 mineurs et jeunes majeurs fin 2023) influence profondément les statistiques globales, notamment la hausse des accueils, la prédominance masculine chez les adolescents et l'augmentation des saisines judiciaires.

6. Des disparités territoriales persistantes et croissantes : Des écarts considérables subsistent entre les départements, que ce soit pour les taux de prise en charge globaux, les taux de judiciarisation ou les modalités d'intervention. Ces disparités tendent à se creuser au fil du temps.

7. Une attention renforcée aux jeunes majeurs : Bien qu'en légère baisse depuis un pic en 2021, le soutien aux 18-20 ans a fortement augmenté sur dix ans (+53 %).

Le taux de poursuite de l'accompagnement après la majorité a atteint 52 % en 2023, retrouvant son niveau d'avant 2013, signe d'une politique active contre les "sorties sèches".

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1. Vue d'ensemble de la prise en charge en protection de l'enfance

1.1. Augmentation continue des interventions

Le nombre d'interventions en protection de l'enfance pour les mineurs (0-17 ans) a connu une croissance soutenue sur la dernière décennie.

• Nombre total d'interventions : Au 31 décembre 2023, 364 200 prestations administratives et mesures judiciaires étaient en cours, soit une augmentation de 22 % par rapport à 2013 (297 500).

• Taux de prise en charge : Le taux d'intervention pour 1 000 mineurs est passé de 20,3 ‰ en 2013 à 25,8 ‰ en 2023, une augmentation de 27 %.

Cette hausse est plus rapide que celle des effectifs en raison d'une diminution de 4 % de la population des moins de 18 ans sur la même période.

• Estimation du nombre de mineurs : En croisant diverses sources (DREES, Olinpe), le nombre de mineurs uniques suivis est estimé à environ 351 500 au 31 décembre 2023.

| Année | Nombre de prestations et mesures | Taux pour 1 000 mineurs | | --- | --- | --- | | 2013 | 297 500 | 20 ‰ | | 2017 | 342 900 | 23 ‰ | | 2020 | 338 600 | 24 ‰ | | 2022 | 347 100 | 24 ‰ | | 2023 | 364 200 | 26 ‰ |

Source : DREES, DPJJ, Insee, calculs ONPE

La croissance a été particulièrement marquée entre 2022 et 2023, avec une hausse de 5 %.

Cette dynamique fait écho à l'augmentation des saisines des juges des enfants observée après la crise sanitaire.

1.2. Disparités territoriales croissantes

Les écarts de prise en charge entre les départements non seulement persistent mais se sont accentués entre 2013 et 2023.

• Écart des taux : Au 31 décembre 2023, le taux de prise en charge des mineurs variait de 13,5 ‰ (Yvelines) à 48,1 ‰ (Nièvre). En 2013, l'écart était moins prononcé, allant de 10,9 ‰ à 37 ‰.

• Tendances géographiques :

◦ Les taux les plus faibles se concentrent majoritairement en Île-de-France et en Auvergne-Rhône-Alpes.  

◦ Les taux les plus élevés sont observés dans des territoires souvent moins densément peuplés.

• Évolutions hétérogènes : Entre 2013 et 2023, le taux de prise en charge a augmenté dans 98 départements, mais avec des variations extrêmes, allant de -7 % (Hauts-de-Seine, Loiret) à +101 % (Lozère).

2. La dynamique des types d'intervention

2.1. Une judiciarisation accrue

La prise en charge des mineurs est majoritairement décidée par l'autorité judiciaire, et cette tendance se renforce.

• Part des mesures judiciaires : Au 31 décembre 2023, 82,4 % des 364 200 interventions résultaient d'une décision judiciaire, contre 78,6 % en 2013.

• Répartition par type d'intervention :

◦ Accueil : 92,1 % des mesures sont judiciaires.    ◦ Milieu ouvert : 71,7 % des mesures sont judiciaires.

• Disparités départementales : Le "taux de judiciarisation" varie fortement, de 66,9 % (Morbihan) à 94,9 % (Seine-Saint-Denis).

Les départements des Hauts-de-France et du Grand-Est affichent des taux particulièrement élevés.

2.2. Le basculement vers l'accueil au détriment du milieu ouvert

Un changement structurel majeur s'est opéré : le nombre de placements d'enfants (accueil) dépasse désormais le nombre d'interventions à domicile (milieu ouvert).

• Inversion de la tendance : En 2023, les mesures d'accueil s'élèvent à près de 190 300 (52,2 % du total), tandis que les mesures en milieu ouvert sont de 174 000 (47,8 %). Le point de bascule s'est produit en 2018.

• Croissance différentielle (2013-2023) :

◦ Le taux de mineurs accueillis a augmenté de 40 % (passant de 9,7 ‰ à 13,5 ‰).    ◦ Le taux de mineurs suivis en milieu ouvert a augmenté de 16 % (passant de 10,7 ‰ à 12,3 ‰).

• Facteurs explicatifs : Cette évolution est notamment liée à la forte augmentation des accueils de mineurs non accompagnés (MNA) et au développement de nouvelles mesures comme le "placement à domicile", comptabilisées comme de l'accueil.

3. L'accueil des mineurs : modalités et profils

3.1. Évolution des modes d'hébergement : l'établissement devance la famille d'accueil

Pour la deuxième année consécutive, l'accueil en établissement est la modalité la plus fréquente, devant l'accueil familial traditionnel.

| Mode d'hébergement | Part en 2013 | Part en 2023 | Évolution en effectifs (2013-2023) | | --- | --- | --- | --- | | Établissement | 38 % | 41 % | +50 % | | Famille d'accueil | 52 % | 36 % | \-4 % (depuis 2019) | | Hébergement autonome | 4 % | 6 % | +143 % | | Autres modes d'hébergement | 6 % | 17 % | +321 % |

Source : DREES, calculs ONPE. Champ : Mineurs et jeunes majeurs confiés à l'ASE.

• La catégorie "Autres modes d'hébergement" inclut les placements chez un tiers digne de confiance, en internat, l'accueil durable et bénévole, etc. Son explosion est un facteur clé de la restructuration du secteur.

• Cette tendance coïncide avec une baisse de 11 % du nombre d'assistants familiaux employés par les départements entre 2016 et 2023.

3.2. Le placement direct : le recours croissant au "tiers digne de confiance"

Le placement direct, décidé par un juge sans passer par une mesure de confiement à l'ASE, évolue également.

• Au 31 décembre 2023, 17 100 enfants bénéficiaient d'un placement direct.

• La part des placements chez un "tiers digne de confiance" a fortement augmenté, passant de 69 % en 2013 à 86 % en 2023.

• Cette évolution est directement liée à la loi du 7 février 2022, qui systématise la recherche d'un membre de la famille ou d'un proche pour accueillir l'enfant.

3.3. Profils démographiques des enfants accueillis

• Prédominance masculine : Les garçons représentent 59 % des mineurs accueillis (hors placement direct).

Ce déséquilibre s'accentue avec l'âge, atteignant 69 % chez les 16-17 ans, principalement en raison de la population de MNA (à plus de 90 % masculine).

• Répartition par âge : Entre 2015 et 2023, la croissance des accueils a été la plus forte aux âges extrêmes : +38 % pour les moins de 6 ans et +50 % pour les 16-17 ans.

• Profil en placement direct : La population en placement direct est très différente, avec un équilibre quasi parfait entre les sexes (50,2 % de filles) et une part plus importante de 11-15 ans (39 % contre 34 %).

4. La situation spécifique des jeunes majeurs (18-20 ans)

4.1. Tendances générales et disparités

L'accompagnement des jeunes majeurs a connu une croissance massive, bien qu'en léger recul depuis 2021.

• Effectifs : 33 400 jeunes majeurs étaient pris en charge fin 2023, soit une augmentation de 53 % depuis 2013. Le pic a été atteint en 2021 avec 35 100 jeunes.

• Nature de l'intervention : La prise en charge est quasi exclusivement administrative (99,8 %) et consiste très majoritairement en un accueil (92,2 %).

• Taux de prise en charge : Le taux national est de 13,6 ‰, mais les disparités départementales sont extrêmes, allant de 1,6 ‰ (Hautes-Alpes) à 28,5 ‰ (Allier).

4.2. La poursuite de l'accompagnement après 18 ans

Un nouvel indicateur, le "taux de poursuite en Accueil Provisoire Jeunes Majeurs (APJM)", mesure la probabilité pour un jeune confié à 17 ans de continuer à être hébergé après sa majorité.

• Après une chute à un niveau plancher de 37 % en 2018, ce taux a connu une remontée spectaculaire pour atteindre 52 % en 2023.

• Cette hausse s'explique par les mesures liées à la crise sanitaire puis par la Stratégie nationale de prévention et de protection de l’enfance, qui a fait de la lutte contre les "sorties sèches" un objectif prioritaire.

5. Facteurs d'influence et dynamiques transversales

5.1. L'impact des mineurs non accompagnés (MNA)

Les MNA constituent une part croissante et influente de la population protégée.

• Effectifs : Au 31 décembre 2023, 46 200 mineurs et jeunes majeurs MNA étaient pris en charge, une hausse de 17 % en un an.

• Répartition : 65 % sont mineurs et 35 % sont de jeunes majeurs. Cette proportion de jeunes majeurs a diminué depuis son pic à 50 % en 2021.

• Influence statistique : Les MNA contribuent significativement à la hausse du nombre d'accueils, à la surreprésentation des garçons de 16-17 ans et à l'augmentation des saisines judiciaires.

5.2. L'augmentation des saisines des juges des enfants

L'activité judiciaire en assistance éducative est en forte croissance.

• En 2023, les juges des enfants ont été saisis pour 124 117 nouveaux mineurs, un chiffre en hausse de 10 % par rapport à 2022 et de 50 % depuis 2013.

• Le rapport note une corrélation entre la courbe des saisines judiciaires et celle des évaluations de minorité pour les MNA, suggérant un lien de cause à effet partiel.

DOI: 10.4110/in.2023.23.e1

Resource: (BD Biosciences Cat# 564806, RRID:AB_2738963)

Curator: @sofiakhan13

SciCrunch record: RRID:AB_2738963

What is this?

DOI: 10.4110/in.2023.23.e1

Resource: (BD Biosciences Cat# 555558, RRID:AB_395940)

Curator: @scibot

SciCrunch record: RRID:AB_395940

What is this?

DOI: 10.4110/in.2023.23.e1

Resource: (BD Biosciences Cat# 560744, RRID:AB_1727527)

Curator: @scibot

SciCrunch record: RRID:AB_1727527

What is this?

DOI: 10.4110/in.2023.23.e1

Resource: (BD Biosciences Cat# 555551, RRID:AB_395934)

Curator: @scibot

SciCrunch record: RRID:AB_395934

What is this?

DOI: 10.17912/micropub.biology.000461

Resource: RRID:BDSC_28202

Curator: @maulamb

SciCrunch record: RRID:BDSC_28202

What is this?

DOI: 10.17912/micropub.biology.000461

Resource: RRID:BDSC_25210

Curator: @maulamb

SciCrunch record: RRID:BDSC_25210

What is this?

DOI: 10.17912/micropub.biology.000461

Resource: RRID:BDSC_28198

Curator: @maulamb

SciCrunch record: RRID:BDSC_28198

What is this?

DOI: 10.17912/micropub.biology.000461

Resource: RRID:BDSC_28241

Curator: @maulamb

SciCrunch record: RRID:BDSC_28241

What is this?

DOI: 10.17912/micropub.biology.000461

Resource: RRID:BDSC_25191

Curator: @maulamb

SciCrunch record: RRID:BDSC_25191

What is this?

DOI: 10.17912/micropub.biology.000461

Resource: RRID:BDSC_25205

Curator: @maulamb

SciCrunch record: RRID:BDSC_25205

What is this?

DOI: 10.17912/micropub.biology.000461

Resource: RRID:BDSC_28189

Curator: @maulamb

SciCrunch record: RRID:BDSC_28189

What is this?

DOI: 10.17912/micropub.biology.000461

Resource: RRID:BDSC_28185

Curator: @maulamb

SciCrunch record: RRID:BDSC_28185

What is this?

DOI: 10.17912/micropub.biology.000461

Resource: RRID:BDSC_28184

Curator: @maulamb

SciCrunch record: RRID:BDSC_28184

What is this?

DOI: 10.17912/micropub.biology.000461

Resource: RRID:BDSC_28227

Curator: @maulamb

SciCrunch record: RRID:BDSC_28227

What is this?

DOI: 10.17912/micropub.biology.000461

Resource: RRID:BDSC_25200

Curator: @maulamb

SciCrunch record: RRID:BDSC_25200

What is this?

DOI: 10.17912/micropub.biology.000461

Resource: RRID:BDSC_83728

Curator: @maulamb

SciCrunch record: RRID:BDSC_83728

What is this?

DOI: 10.17912/micropub.biology.000461

Resource: RRID:BDSC_25198

Curator: @maulamb

SciCrunch record: RRID:BDSC_25198

What is this?

DOI: 10.17912/micropub.biology.000461

Resource: RRID:BDSC_28171

Curator: @maulamb

SciCrunch record: RRID:BDSC_28171

What is this?

DOI: 10.17912/micropub.biology.000461

Resource: RRID:BDSC_28148

Curator: @maulamb

SciCrunch record: RRID:BDSC_28148

What is this?

DOI: 10.17912/micropub.biology.000461

Resource: RRID:BDSC_28134

Curator: @maulamb

SciCrunch record: RRID:BDSC_28134

What is this?

DOI: 10.17912/micropub.biology.000461

Resource: RRID:BDSC_28123

Curator: @maulamb

SciCrunch record: RRID:BDSC_28123

What is this?

DOI: 10.17912/micropub.biology.000461

Resource: RRID:BDSC_28262

Curator: @maulamb

SciCrunch record: RRID:BDSC_28262

What is this?

DOI: 10.14218/JCTH.2022.00346

Resource: (RRID:CVCL_4972)

Curator: @sofiakhan13

SciCrunch record: RRID:CVCL_4972

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_26751

Curator: @maulamb

SciCrunch record: RRID:BDSC_26751

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_50662

Curator: @maulamb

SciCrunch record: RRID:BDSC_50662

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_65129

Curator: @maulamb

SciCrunch record: RRID:BDSC_65129

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_31902

Curator: @maulamb

SciCrunch record: RRID:BDSC_31902

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_53991

Curator: @maulamb

SciCrunch record: RRID:BDSC_53991

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_34993

Curator: @maulamb

SciCrunch record: RRID:BDSC_34993

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_50792

Curator: @maulamb

SciCrunch record: RRID:BDSC_50792

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_35644

Curator: @maulamb

SciCrunch record: RRID:BDSC_35644

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_62873

Curator: @maulamb

SciCrunch record: RRID:BDSC_62873

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_34067

Curator: @maulamb

SciCrunch record: RRID:BDSC_34067

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_35413

Curator: @maulamb

SciCrunch record: RRID:BDSC_35413

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_33638

Curator: @maulamb

SciCrunch record: RRID:BDSC_33638

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_40824

Curator: @maulamb

SciCrunch record: RRID:BDSC_40824

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_42637

Curator: @maulamb

SciCrunch record: RRID:BDSC_42637

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_64926

Curator: @maulamb

SciCrunch record: RRID:BDSC_64926

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_37519

Curator: @maulamb

SciCrunch record: RRID:BDSC_37519

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_32863

Curator: @maulamb

SciCrunch record: RRID:BDSC_32863

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_35812

Curator: @maulamb

SciCrunch record: RRID:BDSC_35812

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_38902

Curator: @maulamb

SciCrunch record: RRID:BDSC_38902

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_29337

Curator: @maulamb

SciCrunch record: RRID:BDSC_29337

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_36092

Curator: @maulamb

SciCrunch record: RRID:BDSC_36092

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_33761

Curator: @maulamb

SciCrunch record: RRID:BDSC_33761

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_26752

Curator: @maulamb

SciCrunch record: RRID:BDSC_26752

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_65132

Curator: @maulamb

SciCrunch record: RRID:BDSC_65132

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_33715

Curator: @maulamb

SciCrunch record: RRID:BDSC_33715

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_32486

Curator: @maulamb

SciCrunch record: RRID:BDSC_32486

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_41699

Curator: @maulamb

SciCrunch record: RRID:BDSC_41699

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_38199

Curator: @maulamb

SciCrunch record: RRID:BDSC_38199

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_55646

Curator: @maulamb

SciCrunch record: RRID:BDSC_55646

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_40849

Curator: @maulamb

SciCrunch record: RRID:BDSC_40849

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_26225

Curator: @maulamb

SciCrunch record: RRID:BDSC_26225

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_35645

Curator: @maulamb

SciCrunch record: RRID:BDSC_35645

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_36721

Curator: @maulamb

SciCrunch record: RRID:BDSC_36721

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_57718

Curator: @maulamb

SciCrunch record: RRID:BDSC_57718

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_51167

Curator: @maulamb

SciCrunch record: RRID:BDSC_51167

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_41682

Curator: @maulamb

SciCrunch record: RRID:BDSC_41682

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_33652

Curator: @maulamb

SciCrunch record: RRID:BDSC_33652

What is this?

DOI: 10.1371/journal.pgen.1009460

Resource: RRID:BDSC_54852

Curator: @maulamb

SciCrunch record: RRID:BDSC_54852

What is this?

DOI: 10.1158/2643-3230.BCD-25-0112

Resource: cutadapt (RRID:SCR_011841)

Curator: @evieth

SciCrunch record: RRID:SCR_011841

What is this?

DOI: 10.1158/2643-3230.BCD-25-0112

Resource: RRID:Addgene_43974

Curator: @evieth

SciCrunch record: RRID:Addgene_43974

What is this?

DOI: 10.1158/2159-8290.CD-25-0605

Resource: (Cell Signaling Technology Cat# 2920, RRID:AB_1147620)

Curator: @evieth

SciCrunch record: RRID:AB_1147620

What is this?

DOI: 10.1158/2159-8290.CD-25-0605

Resource: (Cell Signaling Technology Cat# 4267BF, RRID:AB_2895042)

Curator: @evieth

SciCrunch record: RRID:AB_2895042

What is this?

DOI: 10.1158/2159-8290.CD-25-0605

Resource: (Cell Signaling Technology Cat# 2242, RRID:AB_331015)

Curator: @evieth

SciCrunch record: RRID:AB_331015

What is this?

DOI: 10.3390/molecules31010003

Resource: RRID:Addgene_85042

Curator: @scibot

SciCrunch record: RRID:Addgene_85042

What is this?

DOI: 10.3390/ijms27010023

Resource: (RRID:CVCL_1922)

Curator: @scibot

SciCrunch record: RRID:CVCL_1922

What is this?

DOI: 10.3390/cancers18010132

Resource: (Thermo Fisher Scientific Cat# PA5-19462, RRID:AB_10981523)

Curator: @scibot

SciCrunch record: RRID:AB_10981523

What is this?

DOI: 10.3390/cancers18010132

Resource: (Thermo Fisher Scientific Cat# MA5-32654, RRID:AB_2809931)

Curator: @scibot

SciCrunch record: RRID:AB_2809931

What is this?

DOI: 10.3390/ani16010138

Resource: Microsoft Excel (RRID:SCR_016137)

Curator: @scibot

SciCrunch record: RRID:SCR_016137

What is this?

DOI: 10.15698/mic2025.12.863

Resource: Leica Application Suite X (RRID:SCR_013673)

Curator: @scibot

SciCrunch record: RRID:SCR_013673

What is this?