そのエリアで人々は高い電気代払うほか、この地域に燃料を持ってくるのにも多額の費用がかかる

同じ会社は寒い天気でも動く電気自動車を再びテストした。

高い電力価格は人々が新しいエネルギーに目を向け始めている。

Check these out.

Fair enough. I think this probably is caricature to some extent, but I do think, at the end of the day, it is what he is arguing for.

In other words, something beyond argument itself. And yet, authoritarianism is bad.

This is the trick. Simply assume we all already share an understanding of "irrational attachment" and then proceed accordingly. Also, disparage "rhetoric," and then celebrate debate. This only works if everyone is already on the same page. Why can't everybody already have agreed with me on these fundamental points so that we can know debate these fundamental points.

by being out of it tho, the had some freedom

Interesting! how some epsecially those who had been repremarnded, were left with mental scars that, even if they were not being watched, made them feel that way.

no private life if they were a prisoner especially

no private life of enjoying books or personal improvment through non-fiction - this included the bible i think

shows how people would inform on one another

The girl is technically german (east german) - people still talk to her as if being 'german' is a thingl but its clear she doesn't think that - has the soviet union erased any sense of uniqueness? No longer private life of onesself, it was taken away.

In this specific examples we should make changes in the "Drug Approvals" facts and "drug status" should be a computed uneditable data. Simply we should plug in more data sources: - for example we could extract the China approval from press release - we could get additional EU approvals from other data sources

Could we avoid using the word "scrape"? These authoritative resources are ofte APIs. We could use "Ingest".

eLife Assessment

This manuscript identifies temperature-dependent alternative splicing of PIF4 in Arabidopsis thaliana and shows that heat stress promotes the accumulation of a short exon 5-skipping isoform that is predicted to encode a non-functional protein. This finding is important, and it provides an intriguing new layer of regulation for PIF4; however, the strength of the mechanistic conclusions is limited, and several key conclusions rely on indirect evidence. As a result, while the data robustly demonstrate heat-regulated alternative splicing of PIF4, the causal role of PIF4 isoforms' balance in shaping heat-induced developmental responses remains only partially supported and the strength of the evidence presented is incomplete. This work will be of interest to biologists working on alternative splicing.

Reviewer #1 (Public review):

This manuscript by Niño-González and collaborators shows that PIF4 undergoes alternative splicing in response to elevated temperature, generating distinct isoforms that may contribute to early seedling responses of Arabidopsis thaliana to heat stress (37 {degree sign}C). This work provides an intriguing perspective on how PIF activity may be modulated under stress conditions.

The authors report rapid heat-induced changes in seedling morphology, with cotyledon angle and hypocotyl length altered as early as 3 hours after transfer to 37 {degree sign}C. These responses correlate with a transient increase in PIF4 transcript levels, followed by a return to control values at later time points. Notably, heat induces preferential production of an exon 5-skipping isoform of PIF4. The resulting short protein variant (PIF4-S) lacks part of the bHLH domain and is therefore unlikely to be transcriptionally active.

To explore functional consequences, the authors expressed the exon 5 inclusion (functional) isoform, PIF4-L, in the pif4-101 mutant background. Some heat-induced phenotypes, such as protochlorophyllide accumulation and subsequent photobleaching, were reduced or absent in these lines. Interestingly, pif4-101 mutants themselves largely resemble WT plants for most heat-responsive traits, with the exception of hypocotyl length. PIF4-L expression specifically attenuates the cotyledon angle response to heat, without strongly affecting hypocotyl elongation.

An important point is that PIF4 itself is not essential for the observed heat responses, as pif4 mutants respond largely like wild-type plants. This implies that the phenotypes described are likely controlled by multiple PIFs acting redundantly. In this context, the generation of the PIF4-S isoform may represent one of several mechanisms by which heat stress reduces overall functional PIF levels, rather than a PIF4-specific regulatory switch.

Other caveats should be considered when interpreting the work. The functional relevance of the PIF4-S isoform under heat stress is not tested, as heat responses of these transgenic lines were not examined. Transcriptome analysis of heat-stressed WT, pif4-101 mutant, and PIF4-L-expressing plants revealed an enrichment of PIF-regulated genes, supporting a possible role for this family of transcription factors in the heat stress response. Notably, the heat responsiveness of the mutant and of the transgenic lines differs only marginally from that of WT plants. In addition, the study relies primarily on total transcript-level analyses, without quantitative assessment of individual PIF isoforms or direct measurement of PIF protein abundance. Given that other PIFs are also expressed and may be subject to alternative RNA processing, it needs to be determined whether PIF4-S alone could exert a dominant effect, counteracting all the other functional PIFs by itself, under heat stress. Hence, the proposed model is a plausible but still incomplete framework that requires further experimental validation and analysis.

Altogether, the results presented in this manuscript could also be interpreted as follows: multiple PIFs contribute to the observed phenotypes in response to heat, with overlapping (redundant) functions. Heat stress may reduce functional PIF levels through different mechanisms, one of which is the regulation of alternative splicing, as shown here for PIF4, leading to the production of non-functional proteins or protein variants that could act as negative competitors (such as PIF4-S). Restoring PIF levels to values of control conditions could therefore reverse heat-induced phenotypes, as observed in the PIF4-L expression lines.

Main concerns:

(1) The existence of a shorter isoform of PIF4 and PIF6 is relevant, and PIF4 could indeed play a role in the context of heat stress, as it does in thermomorphogenesis. In this sense, the interplay between PIF4-S and PIF4-L might be linked to plant morphological responses to heat; however, the present work requires further investigation to determine whether this is indeed the case. It is important to note that pif4 mutants behave similarly to WT plants, indicating that PIF4 is not necessary for the observed responses. These phenotypes are therefore most likely related to several PIFs rather than to one specific family member. The results obtained with the transgenic lines expressing PIF4-L or PIF4-S support this interpretation, as increasing a functional PIF (PIF4-L) reduces some phenotypes, while expressing a dominant-negative version mimics heat-induced phenotypes under control conditions. Thus, it is reasonable to interpret that under heat stress, functional PIF levels are reduced through multiple mechanisms, alternative splicing and PIF4-S generation being one of them in the case of PIF4, but likely with additional effects on other family members. This clearly requires further study.

(2) RT-qPCR quantification of total PIF4 transcripts, as well as the long and short isoforms under the tested conditions, is necessary. While we agree with the authors that PIF4-S could act as a dominant-negative factor, demonstrating this requires comparison of phenotypes under heat versus control conditions using the PIF4-S transgenic lines. Importantly, for the authors' hypothesis to be valid, PIF4-S must be able to outcompete other PIFs; therefore, accurate quantification of its expression levels across conditions is crucial. Combining the results shown in Figures 2A and Figure 2G suggests that the levels of the functional PIF4-L isoform are unchanged or even reduced after 3 h of heat treatment, as the increase in total PIF4 does not fully compensate for the diversion toward PIF4-S. Additionally, it would be equally relevant to quantify the expression of other PIFs (or at least those shown in Suppl. Fig. 6) to determine whether PIF4-S could exert such a strong effect even when expressed at relatively low levels. By "proper quantification", we refer specifically to functional protein-coding variants, as in the PIF4-L case. Supplemental Figure 6 shows that PIF3 and PIF5 appear unaffected by heat, while PIF1 expression is increased. However, JBrowse data for dark-grown seedlings indicate that PIF1 is subject to alternative transcription initiation, alternative splicing, and alternative polyadenylation at its 3′ end. A similar situation occurs for PIF3, at least at the 5′ end of the transcriptional unit. Therefore, alternative RNA processing mechanisms may play a key role in modulating functional PIF protein levels in response to heat. Without considering diverted isoforms of other PIFs, the interpretation becomes problematic, as PIF1 is upregulated by heat, and PIF4-S would therefore need to overcome its activity as well. This is particularly relevant given that the cotyledon angle phenotype at 37 {degree sign}C appears even stronger than in the pif1pif3pif5 triple mutant, if such a comparison is feasible.

(3) In addition, PP2A is a well-established housekeeping gene for normalization across different light regimes, as its expression is not affected by light. However, we are not convinced this holds true under heat stress conditions (see Li et al., Plant Cell 2019 Jul 29;31(10):2353-2369. doi:10.1105/tpc.19.00519).

(4) Furthermore, the mechanistic conclusions would be strengthened by directly assessing PIF protein levels, for example, by western blot analysis, to determine whether changes in transcript isoform abundance translate into corresponding changes in protein accumulation under heat stress.

(5) Importantly, the authors' interpretation that "PIF4-L.1 expresses the long isoform at levels similar to those of WT plants (Supplemental Figure 9A), ruling out the possibility that the suppression of heat-induced phenotypes (cotyledon opening and Pchlide accumulation) is due to elevated PIF4 expression levels" is not correct. The RT-qPCR assay quantifies all isoforms containing exon 6, which include both long and short variants with respect to exon 5 inclusion. Since WT plants at 37 {degree sign}C express both isoforms (L/S ≈ 60/40), the PIF4-L lines actually express 2-4-fold higher levels of the functional PIF4 isoform, based on the values shown in the figures.

(6) Figure 3B should include a statistical analysis, as it appears that PIF4-L expression does not significantly reduce photobleaching. Cotyledon angle is not affected by either the pif4 mutation or PIF4-L expression under 22 {degree sign}C conditions (Figure 3C). However, after 24 h at 37 {degree sign}C, there is a clear effect, with cotyledon angles closer to those observed in WT plants at 22 {degree sign}C. Regarding hypocotyl length, although statistical testing was not performed, it is evident that pif4-101 affects this parameter, while PIF4-L expression in this background does not substantially alter the mutant response.

Other comments:

(1) We do not believe that Figure 3E is an optimal way to demonstrate attenuation of transcriptional changes by PIF4-L expression in pif4 mutants. A heat map representation would likely be more direct and informative.<br /> The authors should consider expressing another functional PIF in the pif4 mutant background to determine whether the observed effects are specific to PIF4, as proposed, or whether they reflect a general PIF function.

(2) It would also be informative to examine the response under Light + 37 {degree sign}C conditions. Since PIF4 mRNA accumulation is induced by light, the authors should test whether plants incubated in light show a similar response to heat or whether it is attenuated. Potential cross-regulation between light and heat responses would be worth exploring.

(3) As the authors acknowledge in the introduction, most of our knowledge regarding PIFs in temperature signalling has focused on thermomorphogenesis. Therefore, we believe it is important to place these new findings (exon 5 skipping) within that framework, as they could help explain observations made under better-characterized conditions. In addition, would be interesting to see the phenotypes of the pifq mutant under heat stress. Even though this mutant line displays a heat-stress-like phenotype under control conditions, it may still respond to heat treatment. If so, this would indicate that PIFs are not fully determinative of this response.

(4) The authors should clearly state the genetic background of the PIF4-S expression lines, which appear to be in the pif4-101 background but are not explicitly described as such in the manuscript.

Reviewer #2 (Public review):

The manuscript "Alternative splicing of PIF4 regulates plant development under heat stress" by Niño-González et al. describes a heat-responsive alternative splicing (AS) event in PIF4 in Arabidopsis and its potential impact on seedling development. The authors observe that etiolated ings exposed to heat respond with a more photomorphogenic developmental behaviour, as reflected, for example, by increased cotyledon opening and reduced hypocotyl elongation. They propose that the AS event in PIF4 may contribute to this response, due to reduced formation of the full-length PIF4 protein and an increase in the shorter PIF4 protein with potentially dominant negative functions.

Expressing the individual variants in a pif4 mutant background was used to further examine their function. In the case of the full-length PIF4 variant, some of the heat-induced phenotypes were suppressed. For the lines overexpressing the shorter PIF4 variant, heat responses were not examined.

The authors describe an interesting phenotype and present an appealing model of how AS of PIF4, a well-known key regulator of developmental processes including light- and temperature responses, might be involved. However, I don't think that the authors provide strong evidence for their model, and the unaltered heat response of pif4 mutants argues against a major role of this gene and its AS event under these conditions. Regarding the heat responses, it remains open how distinct those are from thermomorphogenesis.

Weaknesses:

(1) In the manuscript, it is emphasized that previous studies on PIFs' role in temperature responses have mainly focused on thermomorphogenesis under high ambient temperature and not under hot temperatures causing heat stress. How do the authors know that the effects they are looking at are specific to hot temperatures and do not also occur at more moderate temperature increases? So, what would PIF4 splicing look like upon a shift from 22{degree sign}C to 28{degree sign}C (instead of 37{degree sign}C as used in the manuscript)?

(2) The potential role of PIF4 and its AS event in the heat response is the key point of this manuscript, as also reflected by the title. As summarized above, I don't see direct evidence for this and a functional characterization of the AS event is lacking. First, the pif4 mutant doesn't show an altered response, which argues against its requirement under these conditions, and in particular against the proposed model that a shortened version of PIF4 acts in a dominant negative manner. Second, the impact of AS on PIF4 protein levels remains open. Antibodies against PIF4 exist and have been used before, e.g. in Lee et al. (2021), Nat Comm, and Fan et al. (2025), Nat Comm - both studies address the role of PIF4 in thermomorphogenesis and should also be discussed in this manuscript. Detecting PIF4 proteins would allow testing if indeed both PIF4 protein variants are detectable and whether, upon heat stress, the longer variant decreases while the shorter variant increases. This could be expected based on transcript data; however, due to regulation at multiple steps, a correlation between transcript and protein levels might not exist. Third, the transgenic lines expressing either the short or long PIF4 variant do not really reflect the situation in the wild type and might be/are overexpression lines. Specifically, constructs for both variants lack the UTRs according to the description in the method section. Furthermore, is the short version expressed as GFP fusion, as I understood from the method description? The PIF4-L mutants have similar PIF levels as the WT (SFig. 9); however, this refers to total transcripts, which makes a difference in the wild type, in particular under heat stress. Comparing here only the PIF4-L levels would be more informative. Accordingly, the transgenic lines may overexpress PIF4-L compared to the wild type. All the PIF4-S lines show 4 to 5-fold overexpression (again for total transcripts) compared to WT. Including lines with lower overexpression levels would be needed for a direct comparison to the wild type. Moreover, immunoblot analysis of the PIF4 protein would be needed for a direct comparison between the wild type and the two types of mutants.

(3) Apart from the question of what level of (over)expression the transgenic lines have, several aspects of the phenotyping experiments are not in line with a simple model of PIF4 regulation or have not been addressed. Expressing the long PIF4 variant in the pif4 mutant background suppresses some of the heat-induced changes, but not the hypocotyl shortening, suggesting that the hypocotyl effect is not caused by a heat-induced lack of PIF4.

When expressing the short variant, the authors observe increased cotyledon opening in darkness, consistent with a suppression of skotomorphogenesis due to a negative function of PIF4-S, at least when it is overexpressed. For hypocotyl length, no consistent difference between wild type and PIF4-S lines was observed: seedlings grown for 3 d in darkness had identical lengths, for 4-d-old seedlings, the PIF4-S lines did not give consistent results: PIF4S.1 (which has highest transgene expression) had same length as wild type; a pronounced difference was only seen for PIF4-S.3, which is the line with lowest expression. Have the experiments been reproduced with independent seed badges? I'm also wondering why the authors haven't performed the heat stress experiments with these PIF4-S lines, as they did for the PIF4-L mutants. According to the authors' model, the PIF4-S lines might show an opposite response compared to the PIF4-L lines, i.e. an even more pronounced heat effect compared to the wild type.

(4) Why was the heat effect on AS of PIF6 not further analysed? Previous work showed the role of PIF6 in seed development and germination; in line with this, PIF6 expression is particularly high in embryos and seeds, but it is also expressed and alternatively spliced in other tissues and conditions, as shown in Figure 1 and SFigure 2. From the data in Figure 1, it looks like the AS pattern in heat might also be different from other conditions. So, it would be interesting to see how AS of PIF6 changes in the control and heat samples that the authors analysed for PIF4 AS, in particular, if this response is distinct for PIF4 versus PIF6.

(5) The presentation of the RNA-seq data is incomplete. According to the method section, WT, pif4-101, PIF4-L.1 and PIF4-L.2 seedlings upon 3 h heat/control treatment were analysed. Why are DE and DAS genes and comparisons of different genotypes not shown? The FC data displayed in Figure 2E and the overlap between heat-regulated genes (Fig. 3D; only in WT) and PIF regulation show only some aspects of the data.

Reviewer #3 (Public review):

Summary:

PIFs play a pivotal role not only in light and temperature signaling pathways, but in many other signaling pathways regulating plant development by modulating transcription of a large number of genes both directly and indirectly. Similarly, alternative splicing (AS) plays a critical role in shaping the splice isoforms of thousands of genes under different environmental conditions to regulate plant development. In fact, AS of PIF6 has been shown to be involved in seed development. PIF4 is a central transcription factor integrating light and temperature signaling pathways. However, AS of PIF4 has not been involved in any pathways. This story first describes how AS of PIF4 is regulated by heat stress, and this regulation is involved in heat stress signaling to regulate plant development. This is an important finding of general interest.

Strengths:

The authors first describe AS of PIF4 is regulated by heat stress, and this regulation is involved in heat stress signaling to regulate plant development.

Weaknesses:

There are many loose ends in this story that need to be tied up.

Major points:

(1) The authors are showing only the AS transcripts by PCR, but no protein data. Given that the hypothesis is that the short form of PIF4 is functioning in a dominant negative fashion, the authors need to show that this short isoform expresses a protein. In addition, they need to show that this form is functioning in a dominant negative fashion with other PIFs, either by showing that this form reduces the DNA binding and/or transcriptional responses of other PIFs.

(2) The two mutant alleles used for this study (pif4-100 and pif4-2) have T-DNA insertion after the AS exon. Do these alleles express any short version of the protein? The previous studies showed no protein production, and thus, they may not function as a dominant negative form. Usually, the T-DNA insertion alleles may express truncated transcripts, but many do not express any protein due to a lack of stop codon and/or degradation of the transcripts. But in this case, the mutants are behaving like WT. The authors need to show that these alleles are expressing a truncated version of the PIF4 protein.

(3) Figure 4 shows phenotypes of independent lines expressing the PIF4 short version. The authors analyzed only the cotyledon and hypocotyl phenotypes, but not Pchlide or bleaching assays. The authors need to do a thorough phenotype analysis, including heat-stress phenotypes of these lines, to test if the data make sense with their hypothesis.

Author response:

We would like to thank the Editor and the three Reviewers for their detailed assessment of our manuscript and their constructive feedback. We found the suggestions valuable for refining our work. Before presenting the fully updated manuscript, we would like to clarify a few points in this initial response. This manuscript identifies a heat-induced, alternativelyspliced short isoform of PIF4 (PIF4-S) that contributes to the physiological responses observed in heat-stressed etiolated seedlings. First, we agree with all Reviewers that including PIF4 protein data will strengthen our findings an more definitely demonstrate the generation of a protein-coding alternative isoform under heat stress. Therefore, this will be one of our main priorities in the revision. Evidence for the functionality of this alternative isoform is clearly demonstrated by the distinct phenotypes exhibited by transgenic lines expressing either the long or the short versions of PIF4. Nevertheless, we agree that a more comprehensive characterization of these lines, as well as of the pif4 mutant lines, will further strengthen the demonstration of the functional relevance of this alternative splicing event. In addition, we will extend the phenotypic analysis of the PIF4-S lines to heat stress conditions. Importantly, the phenotypes observed in these lines suggest that additional molecular mechanisms may act in parallel with this alternative splicing event to regulate development in heat-stressed etiolated seedlings. As proposed by Reviewer #1, other PIFs may be involved in this response, and we will address this possibility. We will also provide new experimental data to show that alternative splicing in this gene is specific to heat stress and does not occur in other PIFs. Finally, we would like to clarify that the main scope of this manuscript is to demonstrate the functional relevance of the alternative isoform generated by splicing in PIF4 under heat stress. A detailed investigation of its molecular mode of action is beyond the scope of the present study. We sincerely appreciate the thoughtful feedback provided by all Reviewers. We will carefully consider their suggestions and use them to guide the inclusion of additional experiments and analyses in our revised manuscript to reinforce and clarify our conclusions.

aims to consider

I suggest we

the

This paper offers a comparative description/analysis of the artistic work by Nora Turato, etc.

virgule

Ideological colonialism.

Reminds me of Braidotti's posthumanism and also some strands of object-oriented ontology.

using this phrase

Extremely long list of references indicating the intense research done on this project and this topic.

This is an important factor to this research however even if those factors did impact learning from the curriculum, a well designed and improved physical education program that could be widely implemented would still being more beneficial than not.

Written assignments in physical education courses may affect students motivation

How are we sure this is true with elementary schoolers.

Results for learner motivation mostly vary but benefits definitely sway towards the experimental condition. Also little data is cited in this section that explains the changes in motivation and reasoning being discussed leaving a little room for confusion on how they made those conclusions.

In class physical activity was similar between the two groups, however students in the experimental group understood more why they were doing the physical activity, a goal of the new curriculum.

This section shows the findings from the experiments using old physical education programs and new concept based ones where the experimental groups experienced notable knowledge gain.

Design of research used to build the concept based curriculum

This makes an adjustment easy for teachers

Last paragraph the many steps this program will go through to ensure its efficacy but also making sure that the information being taught is relevant to students and also being presented in an efficient way.

Alternative, concept based curricula being developed tie closely in to research based knowledge. This way as research in the field of kinesiolgy and physical activity expand, so can K-12 physical education through a concept based program.

Actual example of a concept-based curriculum being developed at UNCG with the author being one of the people developing this program.

Alternative approach

Benefits of this approach are longer lasting

A better approach to the practice of teaching physical education in K-12 schools than the sports based approach currently seen.

Notes a specific demographic most impacted by the lack of an effective physical education program in schools.

This section discusses attempts that have been made to fix the continuing disparities in physical education

Notes a drop in qualified physical education teachers because administrators fail to realize that kinesiology is a science and the importance of it, which could be a factor leading to disalignment.

Highlights that the issues plaguing P.E. curriculum misalignment 35 years ago are still present today.

The curriculum for physical education is losing the scientific aspect as well as failing to evolve as knowledge and research of the topic of kinesiology does.

Discusses the failures to educate students on the scientific aspect of physical activity as well as how to effectively participate in it.

This section discusses what curriculum alignment is so that the reader can understand what misalignment is and how it pertains to P.E.

Highlights the origin of problem

Highlights the importance of physical education specifically in the K-12 curriculum and emphasizes the importance of this disalignment.

Keywords that pop up regularly throughout the article giving the reader and idea for what to look out for.

"Abstract" section, giving a short summary of what the article is about and briefly delves into the misalignment of K-12 physical education, which is the main topic of the article

Only one author

The title describes the broad topics being discussed, kinesiology and physical education, as well as noting that the topics being discussed are a "Perspective"

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As 'QALY valuation methods in detail' clarifies: weights are derived from general population, not patient reports

Only the the QALY framework allow worse-than-dead health states. We should not dismiss QALY from the conversion approach entirely: the QALY weight 0 = 'being dead' is a theoretically sensible anchor for the currently missing 'neutral point' in the 0-10 WELLBY scale

soorten stress: * adaptief: noodzakelijk * druptief: onnodige stress * anticiperend: stress die vooraf optreed * acuut: kort durende stress * chronisch: stress op lange termijn

4:56 PMThis passage argues that India's digitalisation drive is fundamentally a class project serving corporate and imperialist interests. The ruling classes deploy "digital" as a magical catchword to bypass the need for genuine public investment and social transformation, treating digital service delivery as equivalent to actual benefit received. Technology is fetishised to conceal the class interests behind its deployment, while its real costs — lost wages, exclusion from welfare, privatisation burdens — fall entirely on working people. Digitalisation deepens existing inequalities of class, caste, gender and region, atomises collective social institutions into individual consumer relationships, and harvests public data as a subsidy to private capital. The "India Stack" and its sectoral iterations amount to state-funded infrastructure for corporate extraction. Meanwhile, the process accelerates financialisation, enables the withdrawal of the state from healthcare, education, banking and agriculture, and further subordinates the Indian economy to imperialism — all while the very word "imperialism" is scrubbed from mainstream discourse on the subject.

Rapport de Synthèse : Le Contrôle des Armes à Usage Civil en France

Résumé Exécutif

Ce document analyse l'évolution de la politique publique de contrôle des armes à usage civil en France, sur la base du rapport de la Cour des comptes de mars 2026.

Longtemps limitée à un simple cadre réglementaire, cette politique s'est formalisée à partir de 2017 avec la création du Service central des armes et explosifs (SCAE) et le déploiement du Système d’information sur les armes (SIA).

Le constat majeur est celui d'un durcissement significatif de la réglementation, particulièrement concernant la dangerosité des armes et, plus récemment, les armes blanches en lien avec la jeunesse.

Si le contrôle des détenteurs légaux s'est professionnalisé, il subsiste des lacunes graves, notamment l'impossibilité de consulter systématiquement les antécédents psychiatriques.

Par ailleurs, l'impact de ce dispositif sur la criminalité organisée et la circulation illégale reste modeste, alors que les violences avec armes (à feu et blanches) sont en nette progression.

Une attention particulière est désormais portée à la protection des mineurs face à l'émergence de nouvelles typologies d'armes blanches.

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I. Un Cadre Politique et Institutionnel en Mutation

A. Une formalisation récente

Bien que la réglementation des armes soit ancienne, la France ne dispose d'une véritable "politique publique" que depuis le Plan Armes de 2015, impulsé suite aux attentats.

• Acte fondateur : Création du SCAE en 2017 (renforcé en 2021).

• Objectif unique : La sécurité publique et la prévention des atteintes aux personnes et aux biens.

• Coût de la politique : Estimé à un minimum de 161 M€ en 2024, mobilisant plus de 2 000 équivalents temps plein (ETP), principalement dans la police et la gendarmerie.

B. Une complexité réglementaire croissante

Entre 2007 et 2024, 33 textes législatifs et réglementaires ont été adoptés.

Cette instabilité juridique, avec des articles modifiés jusqu'à six fois en douze ans, nuit à la lisibilité pour les usagers et les agents de contrôle, générant parfois des infractions non intentionnelles.

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II. Éducation, Jeunesse et Armes Blanches : Les Nouveaux Enjeux

Le rapport souligne une préoccupation croissante concernant l'accès des mineurs aux armes, particulièrement aux armes blanches, ce qui a conduit à des réformes récentes majeures.

A. La mission parlementaire « Mineurs et armes blanches »

En réponse à la recrudescence des violences impliquant des jeunes, une mission parlementaire a rendu un rapport le 28 mai 2025.

Ses conclusions ont mené à un durcissement immédiat du cadre légal durant l'été 2025.

B. Durcissement des contrôles et interdictions (Décrets de 2025)

Le dispositif cible spécifiquement les objets prisés par un public jeune ou liés à des phénomènes de mode dangereux :

• Interdiction des « couteaux zombies » : Classement en catégorie A (interdiction totale) pour les couteaux à lame fixe présentant des caractéristiques agressives (côté dentelé, pointes acérées, trous dans la lame).

• Réglementation des points de vente : Les commerçants ont désormais l'obligation stricte d'afficher l'interdiction de vente aux mineurs, sous peine de contravention.

• Délais de remise : Les détenteurs d'armes blanches nouvellement surclassées (comme certains poignards ou machettes) avaient jusqu'au 6 décembre 2025 pour les remettre aux forces de l'ordre sans poursuites.

| Type d'arme blanche | Nouveau classement (2025) | Régime juridique | | --- | --- | --- | | Couteaux zombies | Catégorie A | Interdiction totale | | Étoiles de Ninja / Coups de poing américains | Catégorie D | Acquisition et détention réglementées | | Couteaux à cran d'arrêt automatiques | Catégorie D | Port et transport interdits sans motif légitime |

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III. Analyse de la Dangerosité et Contrôle des Détenteurs

A. Le passage au critère de dangerosité

Depuis 2012, la France a abandonné le critère du « calibre militaire » au profit d'une classification (A, B, C, D) basée sur la létalité réelle et la capacité de dissimulation :

• Catégorie A : Armes de guerre et armes interdites (dont les couteaux zombies).

• Catégorie B : Soumise à autorisation préfectorale (tir sportif, protection rapprochée).

• Catégorie C : Soumise à déclaration (chasse, ball-trap).

• Catégorie D : Acquisition et détention libres (sous conditions d'âge).

B. Une défaillance majeure : Le contrôle psychiatrique

Le rapport pointe une "lacune grave" : l'impossibilité pour les préfectures d'accéder de manière exhaustive et fluide aux données d'hospitalisation sans consentement (fichier HOPSYWeb).

Cette faille empêche d'identifier efficacement les détenteurs représentant un risque pour eux-mêmes ou pour autrui.

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IV. Données Statistiques et État de la Menace

Le territoire français compte entre 6 et 8 millions d'armes en circulation.

A. Mortalité et vol d'armes

• Décès par arme à feu : Entre 1 445 et 1 767 par an (incluant suicides et accidents).

• Homicides : Moyenne annuelle de 130 par arme à feu et 123 par arme blanche.

• Vols : Entre 4 000 et 5 000 armes sont déclarées volées chaque année, alimentant les circuits illégaux.

B. Évolution de la délinquance (2014-2024)

Le rapport note une déconnexion entre le contrôle des détenteurs légaux et l'évolution de la criminalité :

• Les faits constatés impliquant une arme ont augmenté de 24 %.

• Les atteintes aux personnes avec arme ont bondi de 45 %.

• Les condamnations liées aux armes de catégorie D (libres d'accès mais souvent utilisées dans la délinquance de voie publique) sont passées de 10 111 en 2007 à 14 445 en 2023.

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V. Outils Numériques et Modernisation : Le SIA

Le Système d’information sur les armes (SIA), déployé en 2022, vise la traçabilité complète de l'arme "du berceau à la tombe".

• Avantages : Dématérialisation des procédures, création d'un "râtelier numérique" pour les chasseurs et tireurs, et simplification pour les armuriers.

• Limites : Un coût de développement ayant dérivé de 76 % (12,9 M€ contre 7,3 M€ prévus) et un problème persistant d'illectronisme (environ 20 % des chasseurs n'auraient pas encore créé leur compte début 2025).

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VI. Recommandations Clés

La Cour des comptes préconise plusieurs mesures urgentes pour renforcer l'efficience de cette politique :

• Fiabiliser HOPSYWeb : Rendre les données psychiatriques consultables via le SIA sous 18 mois.

• Unification des procédures : Fusionner les procédures de "remise" et de "dessaisissement" en une procédure unique de dépossession en cas de danger.

• Contrôle de proximité : Systématiser l'avis des forces de sécurité intérieure (auditions) avant toute délivrance d'autorisation.

• Analyse balistique : Renforcer les moyens d'examen, particulièrement dans la zone Antilles-Guyane où la criminalité est la plus forte.

• Suivi des saisies : Créer un outil statistique national unifié pour suivre les armes saisies par la police, la gendarmerie et les douanes.

balegdah

eLife Assessment

The revised manuscript by Shukla et al. provides important mechanistic insights into kinesin-1 autoinhibition and cargo-mediated activation. Through a convincing integration of protein engineering, computational modeling, biophysical assays, HDX-MS, and electron microscopy, the study delineates how cargo binding induces an allosteric transition that propagates along the coiled-coil stalk to the motor domains, enhancing MAP7 engagement. The revisions substantially improve clarity, figure annotation, and methodological transparency, leaving the remaining limitations, primarily those inherent to conformational heterogeneity and resolution, appropriately acknowledged. Overall, the updated manuscript presents a coherent mechanism for kinesin-1 activation that will be of broad interest to the motor protein, structural biology, and cell biology communities.

Reviewer #1 (Public review):

The authors aim to interrogate the sets of intramolecular interactions that cause kinesin-1 hetero-tetramer autoinhibition and the mechanism by which cargo interactions via the light chain tetratricopeptide repeat domains can initiate motor activation. The molecular mechanisms of kinesin regulation remain a key question with respect to intracellular transport and this study adds important perspectives to our understanding. It has implications for the accuracy and efficiency of motor transport by different motor families, for example the direction of cargos in one or other direction on microtubules.

The authors focus on the response of inactivated kinesin-1 to peptides found in cargos and the cascade of conformational changes that are induced. They also test the effects of the known activator of kinesin-1 - MAP7 - in the context of their model. The study benefits from multiple complementary, albeit relatively low-resolution, methods - structural prediction using AlphaFold3, 2D and 3D analysis of (mainly negative stain) TEM images of several engineered kinesin constructs, biophysical characterisation of the complexes, peptide design, hydrogen/deuterium-exchange mass spectrometry and simple cell-based imaging. Each set of experiments is carefully designed and the intrinsic limitations of each method are offset by other approaches, such that the assembled data convincingly supports the authors' regulatory model of kinesin activation.

This study benefits from prior work by the authors on this system and the tools and constructs they previously accrued, as well as from other recent contributions to the field. This work will be of broad interest to cell and structural biologists, especially those seeking to tackle small and flexible macromolecular complexes, as well as biophysicists and those interested in protein engineering.

Reviewer #2 (Public review):

Summary:

In this paper, Shukla, Cross, Kish, and colleagues investigate how binding of a cargo-adaptor mimic (KinTag) to the TPR domains of the kinesin-1 light chain, or disruption of the TPR docking site (TDS) on the kinesin-1 heavy chain, triggers release of the TPR domains from the holoenzyme. This dislocation provides a plausible mechanism for transition out of the autoinhibited lambda-particle toward the open and active conformation of kinesin-1. Using a combination of negative-stain electron microscopy, AlphaFold modeling, biochemical assays, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and other methods, the authors show how TPR undocking propagates conformational changes through the coiled-coil stalk to the motor domains, increasing their mobility, and enhances interactions with the microtubule-bound cofactor MAP7. Together, they propose a model in which the TDS on CC1 of the heavy chain forms a "shoulder" in the compact, autoinhibited state. Cargo-adaptor binding, mimicked here by KinTag, dislodges this shoulder, liberating the motor domains and promoting MAP7 association, driving kinesin-1 activation.

Strengths:

Throughout the study, the authors use clever construct design - e.g. delta-Elbow, ElbowLock, CC-Di and the high-affinity KinTag - to test specific mechanisms by directly perturbing structural contacts or effecting interactions. The proposed mechanism of releasing autoinhibition via adaptor-induced TPR undocking is also interrogated with a number of complementary techniques that converge on a convincing model for activation that can be further tested in future studies.

Weaknesses:

These reflect limits of what the current data can establish rather than flaws in execution. It remains to be tested if the open state of kinesin-1 initiated by TPR undocking is indeed an active state of kinesin-1 capable of processive movement and/or cargo transport. It also remains to be determined what the mechanism of motor domain undocking from the autoinhibited conformation is. But this important study provides the groundwork for testing these open questions.

Comments on revisions:

My original minor concerns have been addressed in the revision.

Reviewer #3 (Public review):

Summary:

The manuscript by Shukla and colleagues presents a comprehensive study that addresses a central question in kinesin-1 regulation-how cargo binding to the kinesin light chain (KLC) tetratricopeptide repeat (TPR) domains triggers activation of full-length kinesin-1 (KHC). The authors combine AlphaFold3 modeling, biophysical analysis (fluorescence polarization, hydrogen-deuterium exchange), and electron microscopy to derive a mechanistic model in which the KLC-TPR domains dock onto coiled-coil 1 (CC1) of the KHC to form the "TPR shoulder," stabilizing the autoinhibited (λ-particle) conformation. Binding of a W/Y-acidic cargo motif (KinTag) or deletion of the CC1 docking site (TDS) dislocates this shoulder, liberating the motor domains and enhancing accessibility to cofactors such as MAP7. The results link cargo recognition to allosteric structural transitions and present a unified model of kinesin-1 activation. I recommend acceptance of the manuscript subject to the following additions:

Strengths:

(1) The study addresses a fundamental and long-standing question in kinesin-1 regulation using a multidisciplinary approach that combines structural modeling, quantitative biophysics, and electron microscopy.

(2) The mechanistic model linking cargo-induced dislocation of the TPR shoulder to activation of the motor complex is well supported by both structural and biochemical evidence.

(3) The authors employ elegant protein-engineering strategies (e.g., ElbowLock and ΔTDS constructs) that enable direct testing of model predictions, providing clear mechanistic insight rather than purely correlative data.

(4) The data are internally consistent and align well with previous studies on kinesin-1 regulation and MAP7-mediated activation, strengthening the overall conclusion.

Weaknesses:

(1) While the EM and HDX-MS analyses are informative, the conformational heterogeneity of the complex limits structural resolution, making some aspects of the model (e.g., stoichiometry or symmetry of TPR docking) indirect rather than directly visualized.

(2) The dynamics of KLC-TPR docking and undocking remain incompletely defined; it is unclear whether both TPR domains engage CC1 simultaneously or in an alternating fashion.

(3) The interplay between cargo adaptors and MAP7 is discussed but not experimentally explored, leaving open questions about the sequence and exclusivity of their interactions with CC1.

Comments on revisions:

The authors have addressed my comments satisfactorily.

Author response:

The following is the authors’ response to the original reviews

eLife Assessment

The manuscript by Shukla et al. provides important mechanistic insights into kinesin-1 autoinhibition and cargo-mediated activation. Using a convincing combination of protein engineering, computational modeling, biophysical assays, HDX-MS, and electron microscopy, the authors reveal how cargo binding induces an allosteric transition that propagates to the motor domains and enhances MAP7 binding. Despite limitations arising from conformational heterogeneity and structural resolution, the study presents a unified mechanism for kinesin-1 activation that will be of broad interest to the motor protein, structural biology, and cell biology communities.

We are grateful for the time and effort from the reviewers and editors in providing fair and constructive comments that have helped to improve the manuscript. Our point-by-point response is provided below.

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors aim to interrogate the sets of intramolecular interactions that cause kinesin-1 hetero-tetramer autoinhibition and the mechanism by which cargo interactions via the light chain tetratricopeptide repeat domains can initiate motor activation. The molecular mechanisms of kinesin regulation remain an important question with respect to intracellular transport. It has implications for the accuracy and efficiency of motor transport by different motor families, for example, the direction of cargos towards one or other microtubules.

Strengths:

The authors focus on the response of inactivated kinesin-1 to peptides found in cargos and the cascade of conformational changes that occur. They also test the effects of the known activator of kinesin-1 - MAP7 - in the context of their model. The study benefits from multiple complementary methods - structural prediction using AlphaFold3, 2D and 3D analysis of (mainly negative stain) TEM images of several engineered kinesin constructs, biophysical characterisation of the complexes, peptide design, hydrogen/deuterium-exchange mass spectrometry, and simple cell-based imaging. Each set of experiments is thoughtfully designed, and the intrinsic limitations of each method are offset by other approaches such that the assembled data convincingly support the authors' conclusions. This study benefits from prior work by the authors on this system and the tools and constructs they previously accrued, as well as from other recent contributions to the field.

Weaknesses:

It is not always straightforward to follow the design logic of a particular set of experiments, with the result that the internal consistency of the data appears unconvincing in places.

For example, i) the Figure 1 AlphaFold3 models do not include motor domains whereas the nearly all of the rest of the data involve constructs with the motor domains;

We appreciate the reviewer’s comment regarding the absence of the motor domains in the AlphaFold3 models shown in Figure 1. These domains were intentionally excluded to improve visual clarity and to better highlight the interaction between the TPR domains and CC1 in the inhibited kinesin-1 conformation. We felt that this simplified presentation in the main figure helps readers focus on the key mechanistic advance introduced in this work at the outset of the paper. For completeness, we have provided full-length kinesin-1 AlphaFold3 models that include the motor domains in the Supplementary Information (Fig. S1), and they are described in detail in the main text. In addition, we have added a note to the Figure 1 legend to explicitly direct readers to these full-length models.

ii) the kinesin constructs are chemically cross-linked prior to TEM sample preparation - this is clear in the Methods but should be included in the Results text, together with some discussion of how this might influence consistency with other methods where crosslinking was not used.

Thank you. Chemical crosslinking is typically important for obtaining high-quality negative-stain TEM grids of kinesin-1 complexes and has been employed in all prior EM studies by our group and others. While this was described in the Methods, we agree that it should also be stated explicitly in the Results. Accordingly, we have added a sentence to the Results section noting that the proteins were stabilized using the amine-to-amine crosslinker BS3 (“Proteins were also stabilised using the amine-to-amine crosslinker BS3 that was important for achieving reproducibly high-quality samples for imaging.”).

Please see point below for acknowledgement of risks of using crosslinker.

Can those cross-links themselves be used to probe the intramolecular interactions in the molecular populations by mass spec?

We had considered this, however, cross-linking mass spectrometry (XL-MS) has been applied extensively to essentially identical kinesin-1 complexes by Tan et al. (eLife 2023). That work provided important insights into the overall architecture of the complex, including the new head–CC1 interactions. However, as fully acknowledged by the authors, significant ambiguity remained with respect to the positioning of the TPR domains, with many cross-links that could not be straightforwardly rationalized in a single model. These unresolved aspects provided part of the motivation for the present study, as highlighted in the Introduction.

We believe that this ambiguity likely reflects an underlying conformational equilibrium of the kinesin-1 complex (e.g. opening/closing transitions) and/or dynamic docking and undocking of the TPR domains, and lysine-rich features of the TPR domains (most notably the loops that connect the TPR alpha helices) which may make them prone to lock in non-native states, which limits the interpretability of static cross-linking data in this system. In this context therefore, we feel that XL-MS has already been thoroughly explored for kinesin-1 and that its practical limitations in resolving these TPR interactions have been reached.

This consideration was a primary motivation for pursuing cross-linker-free, solution-based approaches, particularly HDX-MS, which we argue provide the most relevant new insights into the assembly and conformational dynamics of the complex. To make this rationale clearer, we have added an explicit note in the HDX-MS section emphasizing that this is a cross-linker-free method. The added text reads:

“To determine how the local structural changes from adaptor binding and shoulder dislocation affected the dynamics of kinesin-1 complexes in solution, as directly and least invasively as possible, and without the risk of cross-linker artefacts.”

In general, the information content of some of the figure panels can also be improved with more annotations (e.g. angular relationship between views in Figure 1B, approximate interpretations of the various blobs in Fig 3F, and more thought given to what the reader should extract from the representative micrographs in several figures - inclusion of the raw data is welcome but extraction and magnification of exemplar particles (as is done more effectively in Fig S5) could convey more useful information elsewhere.

We appreciate these suggestions. We have modified the figures throughout the manuscript in line with the reviewer’s points. Raw data is now provided at higher magnification throughout so the reader can better distinguish individual particles, angular relationships have been added and further annotations provided on 2D class averages. We do not want the reader to draw too many conclusions from images of single closed particles (with the exception of open vs closed in Fig S7) as these require averaging and 2D classification to obtain meaningful insights, and so we have not added zoom panels in these cases. Figure 3F has been annotated as requested.

Reviewer #2 (Public review):

Summary:

In this paper, Shukla, Cross, Kish, and colleagues investigate how binding of a cargo-adaptor mimic (KinTag) to the TPR domains of the kinesin-1 light chain, or disruption of the TPR docking site (TDS) on the kinesin-1 heavy chain, triggers release of the TPR domains from the holoenzyme. This dislocation provides a plausible mechanism for transition out of the autoinhibited lambda-particle toward the open and active conformation of kinesin-1. Using a combination of negative-stain electron microscopy, AlphaFold modeling, biochemical assays, hydrogen-deuterium exchange mass spectrometry (HDX-MS), and other methods, the authors show how TPR undocking propagates conformational changes through the coiled-coil stalk to the motor domains, increasing their mobility and enhancing interactions with the microtubule-bound cofactor MAP7. Together, they propose a model in which the TDS on CC1 of the heavy chain forms a "shoulder" in the compact, autoinhibited state. Cargo-adaptor binding, mimicked here by KinTag, dislodges this shoulder, liberating the motor domains and promoting MAP7 association, driving kinesin-1 activation.

Strengths:

Throughout the study, the authors use a clever construct design - e.g., delta-Elbow, ElbowLock, CC-Di, and the high-affinity KinTag - to test specific mechanisms by directly perturbing structural contacts or affecting interactions. The proposed mechanism of releasing autoinhibition via adaptor-induced TPR undocking is also interrogated with a number of complementary techniques that converge on a convincing model for activation that can be further tested in future studies. The paper is well-written and easy to follow, though some more attention to figure labels and legends would improve the manuscript (detailed in recommendations for the authors).

Weaknesses:

These reflect limits of what the current data can establish rather than flaws in execution. It remains to be tested if the open state of kinesin-1 initiated by TPR undocking is indeed an active state of kinesin-1 capable of processive movement and/or cargo transport. It also remains to be determined what the mechanism of motor domain undocking from the autoinhibited conformation is, and perhaps this could have been explored more here. The authors have shown by HDX-MS that the motor domains become more mobile on KinTag binding, but perhaps molecular dynamics would also be useful for modelling how that might occur.

We are grateful for the reviewer’s comments. We agree that the weaknesses the reviewer has outlined define the limitations of the study and establish important priorities for future work, that includes molecular dynamics simulations. An important prerequisite for the latter is a starting model that one has confidence in. We think that our study and earlier work now provide a good experimentally supported foundation for using AF3 generated assemblies for this purpose, by ourselves and others.

Reviewer #3 (Public review):

Summary:

The manuscript by Shukla and colleagues presents a comprehensive study that addresses a central question in kinesin-1 regulation - how cargo binding to the kinesin light chain (KLC) tetratricopeptide repeat (TPR) domains triggers activation of full-length kinesin-1 (KHC). The authors combine AlphaFold3 modeling, biophysical analysis (fluorescence polarization, hydrogen-deuterium exchange), and electron microscopy to derive a mechanistic model in which the KLC-TPR domains dock onto coiled-coil 1 (CC1) of the KHC to form the "TPR shoulder," stabilizing the autoinhibited (λ-particle) conformation. Binding of a W/Y-acidic cargo motif (KinTag) or deletion of the CC1 docking site (TDS) dislocates this shoulder, liberating the motor domains and enhancing accessibility to cofactors such as MAP7. The results link cargo recognition to allosteric structural transitions and present a unified model of kinesin-1 activation.

Strengths:

(1) The study addresses a fundamental and long-standing question in kinesin-1 regulation using a multidisciplinary approach that combines structural modeling, quantitative biophysics, and electron microscopy.

(2) The mechanistic model linking cargo-induced dislocation of the TPR shoulder to activation of the motor complex is well supported by both structural and biochemical evidence.

(3) The authors employ elegant protein-engineering strategies (e.g., ElbowLock and ΔTDS constructs) that enable direct testing of model predictions, providing clear mechanistic insight rather than purely correlative data.

(4) The data are internally consistent and align well with previous studies on kinesin-1 regulation and MAP7-mediated activation, strengthening the overall conclusion.

Weaknesses:

(1) While the EM and HDX-MS analyses are informative, the conformational heterogeneity of the complex limits structural resolution, making some aspects of the model (e.g., stoichiometry or symmetry of TPR docking) indirect rather than directly visualized.

We agree with the reviewers point. Conformational heterogeneity is a significant challenge, and the model has been developed from multiple complementary approaches. A higher resolution cryoEM study remains a priority, but is challenging because of the size, shape and flexibility of the particle, but we hope that some the approaches used here (e.g. nanobody TPR stabilisation, ElbowLock) will provide a path to achieve this.

(2) The dynamics of KLC-TPR docking and undocking remain incompletely defined; it is unclear whether both TPR domains engage CC1 simultaneously or in an alternating fashion.

We agree that this is a limitation. We strongly suspect that the TPR domains dynamic and are working to overcome experimental challenges to resolve this important outstanding question. We have expanded the discussion section to better highlight this important priority.

(3) The interplay between cargo adaptors and MAP7 is discussed but not experimentally explored, leaving open questions about the sequence and exclusivity of their interactions with CC1.

We agree that this is a limitation but will be an important priority for future studies.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

There are a number of places where the text could be more precise or clear, or the figures could be designed to be more informative:

(1) The word "unitarily" is used in several places, and I don't know what it means in this context.

We have changed the phrasing throughout the manuscript to this term. We were attempting to contrast with presumed cooperative multivalent interactions in the context of the kinesin-1 tetramer but agree that this choice of word doesn’t quite achieve that.

(2) On page 5 the phrase "We focused on the ElbowLock background" is introduced and needs to be explained more clearly.

Thank you. We have amended the text to read “This KIF5C construct contains a short 5 amino acid deletion that restricts flexibility around the elbow and helps maintain particles in their lambda conformation, providing homogenous samples, and facilitating subsequent analysis (34).”

(3) On page 6, the phrase "To improve the resolution of our images, we turned to single-particle cryoEM analysis" is imprecise - what do the authors mean by the resolution of the images? Cryo-EM data does not always guarantee a higher resolution structure, but it offers the possibility of visualising finer structural features. This is probably what is meant here, but needs to be stated more precisely.

We have amended the text to ‘visualise finer structural details’ as suggested.

(4) Page 7 - "suggesting that TPR domains had loosely dissociated from the core" - I don't think the evidence points to dissociation of KLCs from the complex, but the phrase "loosely dissociated" implies this - would benefit from rephrasing.

We have changed this to ‘undocked’ for consistency with other descriptions in the manuscript.

(5) Was the effect of the CC-Di insertion (ΔTDS) detectable by AlphaFold prediction? It would be interesting to include this, partly for completeness and partly because a slightly imperfect and maybe a more dynamic coiled-coil in this region of the molecule may be important in supporting the conformational changes required for activation.

Thank you for this suggestion. Modelling of deltaTDS complex indeed shows displacement of the TPR domains. In the standard 5 output models, the TPR domains now occupy a variety of different positions, all with essentially zero confidence (high position error). Consistent with biochemical data, the CCDi insertion is modelled with with no overall disruption to the architecture or length of CC1 as expected. We think that this is a valuable addition to the study and have included it as a new supplementary figure (Fig S5), with main text reading.

…. “Supporting this, models of ΔTDS complexes using AF3 showed the expected seamless insertion of CCDi into CC1, with displacement of the TPR domains to a variety of different positions, in 5 models, all with high position error with respect to KHC (Fig S5).”

(6) Figure S1 has two sections designated (C) in the legend.

Corrected

(7) Figure S3 - given the resolution and level of interpretation of the 3D reconstructions, it is not relevant to include an FSC curve, but other standard information, such as angular distribution and any evidence of variability from 3D classifications (and how many particles per 3D class) should be included for all structures.

Thank you, a complete workflow for all complexes has now been provided in Figure S8 with the information requested. In each case there were typically two ‘good’ classes. For ElbowLock, this included one without a prominent shoulder, consistent with 2D classification and quantification. We assume this may reflect a docking/undocking equilibrium. For the deltaTDS and KinTag particles, neither class showed the shoulder feature. The main text has been modified to reflect this and reads “For ElbowLock complexes, this resulted in classes with and without a prominent shoulder, in agreement with 2D classification. For ElbowLock-ΔTDS and ElbowLock-KinTag complexes, no prominent shoulder containing classes were observed.”

Reviewer #2 (Recommendations for the authors):

Overall, the figures would benefit from more labels for clarity, some examples and suggestions below:

(1) Figure 1A - Connect motors to the rest of the structure e.g., wiggly lines.

Corrected.

(2) Figure 1B - Add arrows and angles to indicate different views of the model.

Corrected.

(3) Figure 1B - Label TPR1-6 (e.g., inset zoom in).

Corrected.

(4) Figure 2D and 3D - Label the lack of a shoulder in all averages (perhaps with an arrow instead of a circle to not obscure density), include an example average which shows prominent shoulder density.

Corrected. Full sets of classes showing shoulder like features for deltaTDS and KinTag complexes are now shown in Figure S4.

(5) Figure 3D: Label motor domains and elbow as in other figures.

Corrected.

(6) Methods: Include more information on how EM classes were compared to AF projections (e.g., Figure 1D). Was this done visually or computationally? Likewise, more information is needed on how classes were judged to have prominent/weak shoulder density (Figure 2D). In the figure legend, there is a statement that "Full sets of classes are provided in Fig. S4" but this is absent in the supplement.

Thank you. This information has been added to the methods.

“For comparison to the AF3 model, simulated density was generated using the molmap command in ChimeraX (73) filtering to 15 Å, and projections were generated/selected automatically using the Reference Based Auto Selected 2D function in CryoSPARC”.

Full sets of classes are now provided in Figure S4.

(7) Figure 1-3 - Raw micrographs are a very useful inclusion but would benefit from being a more zoomed-in view (e.g., Figure S5 scale). Particularly useful for 3C, where the mixture of open and closed would be good to see.

Higher zoom micrographs have been provided throughout.

(8) Figure 5D: Panels too small to see the result, suggest making full width and moving E below.

Thank you. We have expanded the panel and moved the model to a new Figure 6.

(9) Figure S1: PAE plot convincing, but pLDDT colour models needed.

A representative model coloured for pLDDT has been added to Figure S1. Most of the structure sits within the light blue confident range (90 > pLDDT > 70) with the exception of the disordered regions and neck coil.

(10) Figure 5B: Reason for the variable inputs?

The reviewer raises an interesting point. The slightly reduced expression of deltaElbow and slightly increased expression of ElbowLock is a consistent feature of these experiments. We note that this effect is in the ‘opposite direction’ to the impact on binding to MAP7 and so does not affect our conclusions from the experiment. However, we wonder whether opening and closing of the complex may impact on turnover of kinesin proteins, which could have implications for their normal homeostasis and possible degradation after transport in polarised cells. We are considering how to explore this going forwards. We have added a note to the results section to highlight this interesting observation to the reader.

“We also noted slightly elevated expression of ElbowLock complexes and slightly lower expression of DeltaElbow complexes, suggesting that opening/closing of the complex could impact on kinesin-1 turnover”

(11) Figure legend 5B: Insufficient detail, the end result is stated, but the three separate gels are not described.

Legend has been expanded.

(12) Figure 3F: Currently somewhat problematic. It is unclear if the models are in the same view, and so comparison is difficult. Figure 1C (bottom right) shows class averages with a clear, separate CC density, so the relatively featureless model in this region is puzzling. A statement on how the three model views are related to each other, if aligned with each other, would be useful.

We appreciate the reviewers point. Models were aligned in Chimera, using the fit in map command. Because of the limited features of the models presumably due to flexibility, achieving a good alignment for all three models was challenging, but we think that showing the 180-degree rotations is probably about the best we can achieve here.

(13) The following statement is too strong: "Nonetheless, we obtained reference-free 2D class averages that appeared to show full-length 'side' views of the complex with clear definition of the elbow, hinge 2, and KHC-KLC (coiled-coil) interface features which enabled us to identify CC1 confidently (Fig. 1D)". Given that the negative-stain EM data were collected primarily to validate the AlphaFold model, the assignment of CC1 should be described as consistent with rather than confidently identified from the class averages. The resolution of the EM data does not independently support such an assignment, and the wording needs to be softened.

We appreciate the reviewer’s point, we have softened the wording as suggested. The paragraph now reads.

“To visualise finer structural details, we turned to single-particle cryoEM analysis of frozen-hydrated samples. We were unable to obtain optimal samples suitable for determining the complete structure. Nonetheless, we obtained reference-free 2D class averages that appeared to show full-length ‘side’ views of the complex with clear definition of the elbow, hinge 2, and KHC-KLC (coiled-coil) interface features (Fig. 1D). The motor domains were poorly resolved in these classes, suggesting that the head assembly is somewhat flexible relative to the coiled coil/TPR body. A comparison to low-pass filtered back-projections from the AF3 model (without motor domains) revealed density at a position concurrent with the docked TPR domains (Fig. 1D).”

(14) There is a typo in the figure legend of Figure 3 - (E) and (F) should be (F) and (G).

Corrected

Reviewer #3 (Recommendations for the authors):

I recommend the following additions:

(1) Figure 1 labeling - In panel A, please label the "linker domain" and the "KLC subunits" explicitly to help orient the reader. In panel B, please mark the "TPR shoulder" corresponding to the docked TPR domains on CC1; this will help the reader connect parts B and C.

Thank you, we have modified Figure 1A with this additional information.

(2) The TPR docking site (TDS) is a central structural element, and its sequence boundaries are provided in the Methods. It would help to visualize this directly in Figure 2A or in an inset.

We hope that the reviewer agrees that the zoomed in model in Figure 5A (alongside MAP7) provides a sufficiently detailed view of the structural interface to highlight the orientation of TPR1 with respect to CC1. The side chain contacts in the model are very plausible and confidently predicted (and can be straightforwardly reproduced in AF3 using the sequence information provided in the methods), but as our study has not explored this interaction at the single residue level, we would prefer not to imply this to the reader at this stage.

(3) The authors' model of cargo-induced TPR dislocation is convincing. However, the Discussion could benefit from a clarification on whether both KLC-TPR domains are expected to be bound simultaneously or if a dynamic exchange occurs, as the EM data suggest potential asymmetry.

Thank you, please see point 5 below where we have modified the discussion to reflect the reviewer’s thoughtful comments.

(4) The HDX-MS analysis is comprehensive, but the authors may want to briefly comment on the coverage of low-signal regions (especially within CC2-CC3) to enhance clarity.

We have added an additional supplementary figure (S10) showing sequence coverage. Overall, this is 88% but with some lower coverage around KHC-CC0 (neck) and the acidic linker that connects the KLC coiled-coil to the TPR. We have added a note to the main text to reflect this.

“Sequence coverage was high (overall 88%) with the exception of KHC-CC0 (neck coil) and the acidic-linker region that connects the KLC coiled-coil to the TPR domains where coverage was lower”

(5) In the Discussion, the proposed interplay between MAP7 and cargo adaptors is intriguing, especially considering the results from Anna Akhmanova's lab showing that MAP7 activates kinesin-1 processivity. Do the authors suggest that competition for CC1 is mutually exclusive or sequential? The answer has mechanistic implications.

We have been considering questions for some time, and the short answer is that we don’t fully understand the dynamics yet. However, we appreciate the reviewer’s prompt to clarify our thinking on this. We have attempted to do this in a revised discussion section where we more explicitly outline these outstanding questions.

① Leukemia Lösemi (kan kanseri)

② Granulamatous diseases Granülomatöz hastalıklar (iltihabi granül oluşumlarıyla seyreden hastalıklar)

① Pregnancy Gebelik

② Puberty Ergenlik / Puberte

③ Scurvy Skorbit (C vitamini eksikliği hastalığı)

④ Plasma cell gingivitis Plazma hücreli gingivit

⑤ Nonspecific conditional gingival growths Spesifik olmayan koşullu diş eti büyümeleri

Idiopathic → Nedeni bilinmeyen

Hyperplastic → Hücre veya doku artışı (hiperplazi) ile ilgili

Fibrotic = Fibrotik / bağ dokusu (lifli doku) artışı olan

Yani bir dokuda kollajen ve bağ dokusunun aşırı birikmesi sonucu oluşan sertleşmiş, kalınlaşmış doku durumudur.

ınflammatory = İltihabi / inflamasyonla ilişkili

Now this happens inside Fortnite and Roblox...

And this is much easier on desktop! Also note, for mobile, most games are free, which is a reason why it appears that mobile keeps growing and PC not so much.

eLife assessment

This manuscript provides an important contribution to the field of platelet biogenesis, and the convincing evidence will advance our understanding of signal transduction driving the development of late megakaryopoiesis and platelet reactivity that results in bleeding diathesis. The paper is noteworthy for analyzing two related, either singly or in combination, tyrosine phosphatases in this conditional, stage development gene knockout. Because SHP1 is a negative regulator and SHP2 is an activator, the synergistic effects found in the double knockout were surprising.

Reviewer #1 (Public review):

Barré et al. investigated the role of Shp1 and Shp2 in megakaryocytes (MKs) and platelets by conditional knock-out of Shp1, Shp2, or both under the control of the Gp1ba promoter. Deletion of Shp1 and Shp2 in MKs and platelets was almost complete. The Shp1/Shp2 double knock-out mice displayed macrothrombocytopenia and increased bleeding, whereas the single knock-outs did not show significant defects. Platelet function was aberrant in DKOs, but not in single knock-outs, and so was ligand-induced signaling, particularly Syk phosphorylation.

Megakaryocyte maturation was impaired in Shp1/Shp2 DKO mice. Ligand-induced signaling was impaired in Shp2 knock-out and DKO. Ex vivo formation of platelets and in vivo maturation of MKs were impaired in DKO mice. Pharmacological inhibitors of Shp1 and Shp2 had largely similar effects as observed in the single knock-outs. The authors conclude that Shp1 and Shp2 have synergistic functions in the MK/platelet lineage, and that Shp2 may be a potential therapeutic target in myeloproliferative neoplasms.

Strengths:

The data clearly show effects of the Shp1/Shp2 double knock-out on MKs and platelets.

Weaknesses:

There appears to be a discrepancy between the results with the Shp2 single knock-out and the Shp2 inhibitor: the Shp2 knock-out does not affect MKs and platelets, except Erk1/2 signaling, whereas the Shp2 inhibitors appear to affect MK function.

This work is interesting and may have potential from a therapeutic point of view.

Reviewer #2 (Public review):

Summary:

In this manuscript, Barré et al. investigate the roles of the phosphatases Shp1 and Shp2 in the megakaryocyte and platelet lineage using genetic depletion in mice. By employing Gp1ba-Cre-based models, the study builds on the authors' previous work and addresses some limitations associated with earlier Pf4-Cre approaches. The authors report relatively mild alterations in megakaryocyte and platelet parameters in mice lacking either Shp1 or Shp2 alone, whereas combined deletion of both phosphatases results in macrothrombocytopenia, mild bleeding, and impaired GPVI-dependent platelet aggregation accompanied by reduced Syk phosphorylation. The functional platelet defects are linked to reduced expression of GPVI and integrin α2, while thrombocytopenia is associated with impaired megakaryocyte maturation, reduced ploidy, defective proplatelet formation, and altered TPO-dependent Ras/MAPK signaling. Similar effects on megakaryopoiesis are also observed in vitro following treatment with newly developed Shp2 inhibitors.

Strengths and Weaknesses:

The study addresses an important biological question and presents a substantial dataset that could contribute to a better understanding of Shp1 and Shp2 function in platelet biology. However, several aspects of data presentation and interpretation would benefit from additional clarification. In particular, while the authors conclude that single genetic deletion or pharmacological inhibition of Shp1 has a limited impact and that the major phenotypes are specific to combined Shp1/2 deletion or Shp2 inhibition, some of the data suggest more nuanced effects that may warrant further discussion.

Reviewer #3 (Public review):

Summary:

In this manuscript, Barré et al utilize the Gp1ba-Cre transgenic mouse model to build upon previous findings in a Pf4-Cre system to investigate the effects of individual and combined Shp1 and Shp2 deletion in megakaryocytes and platelets. They report decreased megakaryocyte maturation, macrothrombocytopenia, and increased bleeding primarily in association with the Shp1/Shp2 double-knockout condition. The authors further show that this phenotype appears to be driven primarily by Shp2 and implicate dysregulation of Mpl signaling and downstream Ras/MAPK pathways, including ERK1/2. Given the key role of these pathways in human diseases such as myeloproliferative neoplasms and the challenges associated with modulating such a central pathway, identification of a specific regulator of Mpl signaling poses intriguing questions for future studies on clinical applicability.

Strengths:

Overall, the experiments combine in vitro, in vivo, and ex vivo approaches and appear to have been carefully designed and carried out, with multiple technical and biological replicates where relevant. The authors make a compelling argument for using the Gp1ba-Cre as opposed to the Pf4-Cre system and demonstrate both the dose- and stage-dependent effects of Shp1 and Shp2 on megakaryopoiesis and thrombopoiesis. They find that Shp1 and Shp2 are required in late-stage megakaryocyte maturation and that even low levels of expression compared to baseline are likely sufficient to yield generally normal megakaryocytes. Their findings also lead to specific future directions, such as the mechanism by which Shp1 regulates megakaryopoiesis and thrombopoiesis that is distinct from TPO-mediated signaling.

Weaknesses:

While the experiments have been thoughtfully designed and carried out, there is limited background explanation on relatively complex or niche pathways/mechanisms, such as the relationship between P-selectin, CRP, and PAR4p; the interactions between SFK, Syk, GPVI, and CLEC-2; and TPO, MPL, ERK1/2, AKT, and STAT3, which, while likely intuitive to experts in their respective fields, may be less obvious to a reader approaching this manuscript with a global interest in megakaryopoiesis/thrombopoiesis and thus detract from the impact of the findings.

With regard to the science itself, some of the conclusions feel premature based on the available data.

(1) The section "Aberrant ITAM signaling in Shp1- and Shp2-deficient platelets" is challenging to follow for those not well-versed in ITAM signaling and associated pathways, and may take additional outside reading to follow the conclusion that Syk-dependent signaling is modulated downstream of GPVI and CLEC-2 based on lack of change in Src p-Tyr418, especially considering that Src p-Tyr418 was previously introduced as a measure of SFK rather than Syk. In the introduction, Shp1 is specifically mentioned as a negative regulator of the ITAM/Syk/phospholipase pathway. However, in Figure 4Ai and Bi, Syk phosphorylation/activation in Shp1 knockout cells did not appear to be different from Shp2 knockout cells, and is lower than the control, which is surprising for a negative regulator. It is also not clear why, in the section (Figure 4A-B), there is reduced Syk activation in Shp1 and Shp2 single knockout cells upon CLEC2 stimulation (but apparently not with CRP) when there was no difference in response to CLEC2 (but a difference in response to CRP) in the previous section (Figure 3A, C).

(2) In the section "Reduced Tpo signaling in Shp1/2-deficient MKs," only Western blot data for (p)ERK1/2, AKT, and STAT3 are presented before concluding that decreased ERK1/2 activity is a mechanistic explanation for thrombocytopenia seen in the Shp1/2 double-knockout condition. Such a statement would benefit from additional experiments, such as protein or transcriptional levels of ERK1/2 targets specifically relevant to megakaryopoiesis, such as ETS, FOS, and JUN, to assess the consequences of decreased phosphorylated ERK1/2.

(3) Suggesting that "inhibiting Shp2 will not hav[e] any bleeding consequence in patients" and that Shp2 may be a therapeutic target in myeloproliferative neoplasms when none of these studies have been carried out in a human model is a bold conclusion. There are no data presented on, for example, whether Shp2 inhibition can help reverse the MPL/JAK/STAT pathway in the setting of gain-of-function mutations specifically associated with myeloproliferative neoplasms.

Author response:

eLife Assessment

This manuscript provides an important contribution to the field of platelet biogenesis, and the convincing evidence will advance our understanding of signal transduction driving the development of late megakaryopoiesis and platelet reactivity that results in bleeding diathesis. The paper is noteworthy for analyzing two related, either singly or in combination, tyrosine phosphatases in this conditional, stage development gene knockout. Because SHP1 is a negative regulator and SHP2 is an activator, the synergistic effects found in the double knockout were surprising.

We thank the reviewer for acknowledging the importance and novelty of our findings.

Public Reviews:

Reviewer #1 (Public review):

Barré et al. investigated the role of Shp1 and Shp2 in megakaryocytes (MKs) and platelets by conditional knock-out of Shp1, Shp2, or both under the control of the Gp1ba promoter. Deletion of Shp1 and Shp2 in MKs and platelets was almost complete. The Shp1/Shp2 double knock-out mice displayed macrothrombocytopenia and increased bleeding, whereas the single knock-outs did not show significant defects. Platelet function was aberrant in DKOs, but not in single knock-outs, and so was ligand-induced signaling, particularly Syk phosphorylation.

Megakaryocyte maturation was impaired in Shp1/Shp2 DKO mice. Ligand-induced signaling was impaired in Shp2 knock-out and DKO. Ex vivo formation of platelets and in vivo maturation of MKs were impaired in DKO mice. Pharmacological inhibitors of Shp1 and Shp2 had largely similar effects as observed in the single knock-outs. The authors conclude that Shp1 and Shp2 have synergistic functions in the MK/platelet lineage, and that Shp2 may be a potential therapeutic target in myeloproliferative neoplasms.

Strengths:

The data clearly show effects of the Shp1/Shp2 double knock-out on MKs and platelets.

Weaknesses:

There appears to be a discrepancy between the results with the Shp2 single knock-out and the Shp2 inhibitor: the Shp2 knock-out does not affect MKs and platelets, except Erk1/2 signaling, whereas the Shp2 inhibitors appear to affect MK function.

This work is interesting and may have potential from a therapeutic point of view.

Pharmacological effects do not always correlate with congenital anomalies arising for genetic defects. The Shp2 allosteric inhibitors used in our study only inhibit catalytically inactive Shp2, whereas targeted deletion of Ptpn11 results in a loss of total Shp2 expression, including catalytic and non-catalytic related functions, with developmental consequences. Further, Gp1ba-Cre+;Shp2fl/fl megakaryocytes express approximately 22% of normal Shp2 level, which likely also contributes to differences observed between pharmacological inhibition and genetic ablation of Shp2.

We thank the reviewer for recognizing the therapeutic potential of our findings.

Reviewer #2 (Public review):

Summary:

In this manuscript, Barré et al. investigate the roles of the phosphatases Shp1 and Shp2 in the megakaryocyte and platelet lineage using genetic depletion in mice. By employing Gp1ba-Cre-based models, the study builds on the authors' previous work and addresses some limitations associated with earlier Pf4-Cre approaches. The authors report relatively mild alterations in megakaryocyte and platelet parameters in mice lacking either Shp1 or Shp2 alone, whereas combined deletion of both phosphatases results in macrothrombocytopenia, mild bleeding, and impaired GPVI-dependent platelet aggregation accompanied by reduced Syk phosphorylation. The functional platelet defects are linked to reduced expression of GPVI and integrin α2, while thrombocytopenia is associated with impaired megakaryocyte maturation, reduced ploidy, defective proplatelet formation, and altered TPO-dependent Ras/MAPK signaling. Similar effects on megakaryopoiesis are also observed in vitro following treatment with newly developed Shp2 inhibitors.

Strengths and Weaknesses:

The study addresses an important biological question and presents a substantial dataset that could contribute to a better understanding of Shp1 and Shp2 function in platelet biology. However, several aspects of data presentation and interpretation would benefit from additional clarification. In particular, while the authors conclude that single genetic deletion or pharmacological inhibition of Shp1 has a limited impact and that the major phenotypes are specific to combined Shp1/2 deletion or Shp2 inhibition, some of the data suggest more nuanced effects that may warrant further discussion.

We thank the reviewer for raising this point. The manuscript is being revised accordingly, including highlighting the potential role of Shp1 in megakaryopoiesis and thrombopoiesis under steady-state and stressed conditions, requiring more detailed investigation.

Reviewer #3 (Public review):

Summary:

In this manuscript, Barré et al utilize the Gp1ba-Cre transgenic mouse model to build upon previous findings in a Pf4-Cre system to investigate the effects of individual and combined Shp1 and Shp2 deletion in megakaryocytes and platelets. They report decreased megakaryocyte maturation, macrothrombocytopenia, and increased bleeding primarily in association with the Shp1/Shp2 double-knockout condition. The authors further show that this phenotype appears to be driven primarily by Shp2 and implicate dysregulation of Mpl signaling and downstream Ras/MAPK pathways, including ERK1/2. Given the key role of these pathways in human diseases such as myeloproliferative neoplasms and the challenges associated with modulating such a central pathway, identification of a specific regulator of Mpl signaling poses intriguing questions for future studies on clinical applicability.

We thank the reviewer for acknowledging the importance and novelty of our findings.

Strengths:

Overall, the experiments combine in vitro, in vivo, and ex vivo approaches and appear to have been carefully designed and carried out, with multiple technical and biological replicates where relevant. The authors make a compelling argument for using the Gp1baCre as opposed to the Pf4-Cre system and demonstrate both the dose- and stagedependent effects of Shp1 and Shp2 on megakaryopoiesis and thrombopoiesis. They find that Shp1 and Shp2 are required in late-stage megakaryocyte maturation and that even low levels of expression compared to baseline are likely sufficient to yield generally normal megakaryocytes. Their findings also lead to specific future directions, such as the mechanism by which Shp1 regulates megakaryopoiesis and thrombopoiesis that is distinct from TPO-mediated signaling.

Weaknesses:

While the experiments have been thoughtfully designed and carried out, there is limited background explanation on relatively complex or niche pathways/mechanisms, such as the relationship between P-selectin, CRP, and PAR4p; the interactions between SFK, Syk, GPVI, and CLEC-2; and TPO, MPL, ERK1/2, AKT, and STAT3, which, while likely intuitive to experts in their respective fields, may be less obvious to a reader approaching this manuscript with a global interest in megakaryopoiesis/thrombopoiesis and thus detract from the impact of the findings.

We thank the reviewer for raising this point. The manuscript is being revised, to better explain the rationale and molecular mechanisms linking these pathways and functions.

With regard to the science itself, some of the conclusions feel premature based on the available data.

(1) The section "Aberrant ITAM signaling in Shp1- and Shp2-deficient platelets" is challenging to follow for those not well-versed in ITAM signaling and associated pathways, and may take additional outside reading to follow the conclusion that Syk-dependent signaling is modulated downstream of GPVI and CLEC-2 based on lack of change in Src p-Tyr418, especially considering that Src p-Tyr418 was previously introduced as a measure of SFK rather than Syk. In the introduction, Shp1 is specifically mentioned as a negative regulator of the ITAM/Syk/phospholipase pathway. However, in Figure 4Ai and Bi, Syk phosphorylation/activation in Shp1 knockout cells did not appear to be different from Shp2 knockout cells, and is lower than the control, which is surprising for a negative regulator. It is also not clear why, in the section (Figure 4A-B), there is reduced Syk activation in Shp1 and Shp2 single knockout cells upon CLEC2 stimulation (but apparently not with CRP) when there was no difference in response to CLEC2 (but a difference in response to CRP) in the previous section (Figure 3A, C).

We thank the reviewer for raising these important points. The manuscript is being revised accordingly, including clarifying the roles of SFKs, Shp1 and Shp2 in the ITAM-Syk-PLCg2 signaling pathway.

Briefly, SFKs are essential for phosphorylating ITAMs, allowing SH2-dependent docking of Syk. Reduced reactivity of Shp1/2 DKO platelets to CRP and collagen is likely due to downregulation of the ITAM-containing GPVI-FcR g-chain complex and integrin a2 subunit, and concomitant reduction in Syk phosphorylation.

However, the marginal albeit significant reduction in Syk phosphorylation downstream of CLEC-2 in Shp1 and Shp2 KO platelets was not determined and was insufficient to impact CLEC-2-mediated platelet aggregation under the conditions tested.

Differences in the stoichiometry and docking of Syk to phosphorylated GPVI-FcR g-chain and CLEC-2 likely contribute to the differences in platelet reactivity and Syk phosphorylation downstream of the two receptors in the absence of Shp1 and Shp2.

(2) In the section "Reduced Tpo signaling in Shp1/2-deficient MKs," only Western blot data for (p)ERK1/2, AKT, and STAT3 are presented before concluding that decreased ERK1/2 activity is a mechanistic explanation for thrombocytopenia seen in the Shp1/2 doubleknockout condition. Such a statement would benefit from additional experiments, such as protein or transcriptional levels of ERK1/2 targets specifically relevant to megakaryopoiesis, such as ETS, FOS, and JUN, to assess the consequences of decreased phosphorylated ERK1/2.

We thank the reviewers for these constructive comments. Further experiments are being planned to determine the biological and transcriptional consequences of reduced ERK1/2 phosphorylation during megakaryopoiesis and thrombopoiesis.

(3) Suggesting that "inhibiting Shp2 will not have any bleeding consequence in patients" and that Shp2 may be a therapeutic target in myeloproliferative neoplasms when none of these studies have been carried out in a human model is a bold conclusion. There are no data presented on, for example, whether Shp2 inhibition can help reverse the MPL/JAK/STAT pathway in the setting of gain-of-function mutations specifically associated with myeloproliferative neoplasms.

This conclusion is being tempered in the revised manuscript. Genetic- and pharmacological-based approaches will be used to establish the therapeutic potential of inhibiting Shp1 and Shp2 in mouse models of MPN, including Jak2 gain-of-function mice. Bleeding and thrombotic complications of inhibiting Shp1 and Shp2 will be explored as part of these studies.

That's also a hallucination, no paper with Langford. Maybe a human should look at this stuff?

eLife Assessment

This study provides valuable findings in the study of enhancer biology by identifying and dissecting a minimal enhancer regulating dlx2b expression during zebrafish tooth development, supported by promoter dissection, reporter assays, and genome-editing approaches. The work offers a resource and extends previous findings but has limited broader impact, with several conclusions about general cis-regulatory principles and functional consequences remaining only partially supported. Accordingly, the strength of evidence is at present incomplete, as additional functional validation would be needed to fully substantiate some of the claims.

Reviewer #1 (Public review):

Summary:

Jackman et al report the analysis of a cis-regulatory region upstream of the dlx2b gene in zebrafish, that is hypothesised to control gene expression in the developing tooth. To demonstrate this, the authors performed solid promoter bashing analysis to assess the gene expression driven by the regulatory region, and validated the expression against a GFP-reporter knock-in. They narrowed down the tooth-specific enhancer activity to the MTE, which was sufficient to drive gene expression. Interestingly, they have identified a vertebrate conserved region which contained four predicted transcription factor binding sites, which when mutated individually, did not alter the reported gene expression. However, in combination, the expression was disrupted. The authors propose a putative upstream regulator cebpa binding one of the predicted TFBS, using in situ hybridisation to show overlapping gene expression domains.

Strengths:

The experiments presented in this paper were rigorously executed and the authors' effort to systematically dissect the different elements of the enhancer are commendable. The discussion and limitations of the study were very well-balanced.

First, the results represent important findings first for the enhancer biology field to sustain evidence of the role of redundant TFBSs. Too often, only TFBS mutations that are sufficient and necessary to drive gene expression patterns are reported, but work providing evidence that some TFBS are necessary but not sufficient by themselves to drive expression is rarer. TFBS redundancy is a crucial concept in enhancer biology but also a difficult concept to prove that hinders the accurate prediction of enhancer function. In an era where increasingly more powerful machine learning models are developed to predict enhancer function, this work is a reminder of the complexity of enhancer biology and provides ground truths for experimental validation.

Second, the results present valuable findings for the field of tooth development. While the authors have comprehensively described work performed in this space, there are still not many tooth-specific enhancers identified and accurately described. The work also presents further avenues for studying upstream regulators.

Weaknesses:

It seems to me that one of the greatest outcomes of this work is demonstrating the collective action of mutated TFBSs where individual mutations are not affecting gene expression. These findings fall into the realm of enhancer redundancy but this concept was not thoroughly discussed in the introduction of the paper.

The claimed results are generally well-supported by the experiments performed, and hypothesis and speculations have been clearly stated. However, some speculative statements remain that should be addressed, for example in the abstract line 33 "These findings suggest that loss of MTE function permits alternative cis-regulatory elements to gain control of the promoter". There is no data indicating what these cis-regulatory elements could be, hence this sentence might be better suited in the discussion.

The manuscript could be strengthened by further exploration of the wider region upstream of dlx2b to support the recruitment of other TFBSs: Were there any other vertebrate-conserved regulatory regions just outside of the MTE? Were there any other family members of the predicted TFs expressed in the tooth? Transcription factor binding sites identity remains a prediction; it could be expanded to other TFs within the same family.

Reviewer #2 (Public review):

The manuscript by Jackman et al. explores the role of a candidate enhancer of dlx2b in zebrafish tooth formation.

They have mapped the dental epithelium and mesenchyme activity of a 4kb promoter proximal region previously identified as a candidate enhancer region. They identified candidate TFBS and candidate transcription factors regulating this enhancer and proposed that their findings reveal principles of enhancer function during vertebrate organogenesis (tooth development) and the power of dissecting cis regulatory architecture. The study offer valuable genetic tagging resource for studying tooth development while further experiments and analyses would be needed to support the suggestion for novel findings on in cis-regulatory principles of tooth development. In the lack of functional evidence on endogenous target gene pr tooth development, some of the claims of the paper may need rephrasing.

(1) The candidate enhancer region has previously been published, this study narrows the enhancer effect to a well-conserved region within. To what degree the element is unique in the locus for tooth development and to what degree this element is required for tooth morphogenesis, is not addressed.

(2) The knock-in approach is convenient for reporter activity based analyses, however it lacks the precision that would be necessary to conclude on enhancer- autonomous effects or enhancer effects on the endogenous target promoter. The HSP promoter inserted in within a 5kb(?) insert in the UTR region of dlx2b creates an chimeric E-P context. The expression profile of the knock-in reporter is substantially different from the endogenous gene (Figure 1B and C) suggesting E-P interaction dependent expression profile, which may confuse what in the expression comes solely from the enhancer and not as a result of the HSP promoter interaction with the enhancer. An alternative heterologous promoter would help in defining the enhancer specific effects.

(3) Function of the candidate enhancer: The MTE enhancer effect is measured by gain of function towards dlx2b regulation. The deletion assays are limited to plasmids designed to test the enhancer in isolation from the endogenous enhancer architecture, or to a deletion in the knock-in, which may be impacted by the chimeric regulatory interaction with a heterologous HSP promoter. As a result we do not learn whether the enhancer targets or needs for endogenous target gene activity. This design allows a conclusion on tissue activity of the enhancer but not the requirement for tooth development.

(4) Since the locus is scattered by candidate enhancers (see genome annotation resources) it is feasible that additional E-P interactions lead to potential enhancer redundancies with the MTE. For a conclusive functional test/requirement of the MTE enhancer, the authors would need to delete it in the endogenous locus context. The knock-in could theoretically be used for an enhancer function on dlx2b activity, if the authors show that there is interaction with the endgogenous promoter (3C type experiment); and that the MTE enhancer-driven GFP activity was identical to the endogenous tagged dlx2b activity. This does not appear to be the case, as ectopic expression in Fig 1C as compared to B is shown. Of note, RNA detection by WISH would be more precise for comparisons. The figure likely compares protein (legend is unclear, but text suggests protein) to mRNA, which is imprecise.

(5) There is an experimental design question arising with generating the MTE deletion in the knock-in (line 391): the authors describe generating the transgenic lines by screening for reduced reporter activity first. This suggests the authors pre-emptively looked for an effect as result they predicted when generating the transgenic lines, which would create a circular argument. All transgenic lines carrying the deletion (tested by sequencing first) would need to be assayed for activity change and then can conclusion could be made on effect of MTE loss by statistical analyses of reporter activities in the generated lines.

(6) Most transgenic work described are based on single transgenic lines. Enhancer promoter contexts may be affected either by position effects (in case of the reporter constructs) or by the heterologous promoter context of the knock which may be affected by unexpected recombination events. Such unintended confound effects can be excluded by replicates.

(7) GFP protein detection does not allow precise spatio-temporal resolution due to varying protein stability in tissues, which potentially impacts endogenous gene activity comparison, and accurate determination of activity dynamics towards conclusions on lineage determining/maintenance roles of the dlx2b enhancer.

(8) The expression pattern change upon MTE loss (retention of mesenchyme, loss of epithelium) is an interesting observation, which would benefit from more comprehensive analysis of the grammar (TFBS contributions) to the pattern variation by dissection of the combination of TFBS contributions. Without such, enhancer grammar remains mostly unclear, thus, principles of morphogenesis may not have been uncovered.

(9) The diagrammatic models of the conclusions are illustrating simple logic which does not add to the text.

(10) Author contributions need to be explained in more detail to be sufficiently granular for fair credit.

Reviewer #3 (Public review):

In the manuscript entitled "A Minimal tooth Enhancer Regulates dlx2b Expression During Zebrafish Tooth 1 Formation: Insights into Cis-Regulatory Logic in Organogenesis", the authors explore the cis-regulatory logic of a dlx2b minimal enhancer capable of directing dlx2b gene expression to the developing tooth germs. The study combines (1) CRISPR-mediated GFP knock-in to track endogenous gene expression; (2) a promoter-bashing approach to identify a minimal tooth enhancer (MTE); (3) site-directed mutagenesis coupoled with transgenesis to assess the individual role of conserved TF binding sites; and (4) in vivo deletgion of the MTE to examine the consequences for gene expression. Overall, this is a technically solid study that provides some novel insights into tooth development and extends previous observations by the authors (Jackman & Stock, 2006; PNAS). However, the added value of the manuscript is limited by both the narrow experimental scope and the relatively modest impact of the findings for the broader field of developmental biology.

Main concerns:

(1) My main concern is that the study restricts the search for cis-regulatory information to the 5' region 4kb upstream of the TSS of the gene, rather than encompassing the full genomic locus. This is particularly limiting given that a knock-in allele was generated, which in principle allows interrogation of regulatory elements across the entire locus, and that the authors acknowledge the availability of genome-wide regulatory datasets (e.g. DANIO-CODE) in the Discussion. Despite this, no systematic effort is made to test additional regulatory elements beyond the proximal promoter/enhancers.<br /> This has important implications for the interpretation of the current work as: (a) dlx2b, as many developmental genes, resides in a gene desert enriched in open chromatin regions that may function as distal enhancers, and (b) the deletion of the MTE unmasked a cis-regulatory activity which nature cannot be explained with the information provided, and that may seem relevant for the expression of the gene in the dental mesenchyme.

(2) A second concern is the absence of information on the functional consequences of deleting the gene or the MTE on tooth primordium development. From the description of the KI strategy, it is unclear whether the GFP insertion results in a functional fusion protein. The cytoplasmic GFP distribution and the schematic in Figure S1 instead suggest the presence of a terminal stop codon in the GFP sequence, which would result in a dlx2b loss-of-function allele. If this interpretation is correct, the manuscript does not describe the developmental consequences in homozygous embryos. Similar concerns apply to the MTE deletion: it remains unclear whether loss of this enhancer results in any detectable morphological or developmental defects.

Obstetricians and sonographers instead of maternity units.

How about: Fraiya provides obstetricians and sonographers with AI-powered tools to support their ultrasound screening programs, improving detection of congenital abnormalities and cutting scan time by 42%.

Rapport d'enquête : Maltraitance Infantile et Procédures de la Brigade des Mineurs

Résumé Exécutif

Ce document analyse une enquête menée par la Brigade des mineurs de Clermont-Ferrand concernant un nourrisson de trois mois admis aux urgences dans un état critique.

L'affaire met en lumière la complexité des investigations liées au syndrome du bébé secoué (syndrome de Silverman), où l'absence de témoins directs oblige les enquêteurs à s'appuyer sur des preuves médicales, des enquêtes d'environnement et des techniques d'interrogatoire visant à obtenir des aveux.

Les points clés de l'affaire incluent :

• Constatations médicales : Présence d'hématomes intracrâniens d'âges différents et d'hémorragies rétiniennes, signes pathognomoniques de secousses violentes.

• Évolution des versions : Passage d'un déni initial à l'évocation d'une chute accidentelle, pour finir par l'aveu de gestes violents répétés sous l'influence de l'exaspération liée aux pleurs.

• Résultat judiciaire : Mise en examen du père pour violences sur mineur, encourant 15 ans de réclusion criminelle, tandis que l'enfant subit des séquelles neurologiques irréversibles.

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I. Le Signalement et le Diagnostic Médical initial

L'enquête débute par un signalement du CHU de Clermont-Ferrand concernant un nourrisson de 3 mois présentant un tableau clinique alarmant : pâleur extrême, extrémités froides et troubles graves de la conscience.

Expertise Clinique

Les médecins identifient immédiatement des éléments suspects justifiant l'intervention de la police :

• Hématomes multiples : Un hématome volumineux récent et plusieurs autres plus anciens.

• Lésions cérébrales : Le scanner révèle des hématomes localisés au cerveau ayant causé des dommages neurologiques irréversibles.

• Hémorragie rétinienne : Un symptôme qualifié d'« épidémique » et symptomatique du traumatisme par secouement.

Le Syndrome de Silverman (Bébé Secoué)

Le rapport médical privilégie l'hypothèse d'un traumatisme causé par un adulte.

Le mécanisme identifié est un mouvement de bascule violent d'avant en arrière.

La tête du nourrisson, représentant un poids proportionnellement important par rapport à son corps, provoque des chocs répétés du cerveau contre la boîte crânienne, comprimant l'organe et empêchant la circulation sanguine.

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II. La Méthodologie de l'Enquête Policière

La Brigade des mineurs, représentée par les enquêtrices Dominique, Brigitte et la commandante Marie-Lyne, déploie une stratégie d'investigation multidimensionnelle pour briser le silence entourant la sphère familiale.

1. Mesures de Garde à Vue

Dès l'admission de l'enfant, les deux parents sont placés en garde à vue.

L'objectif est de reconstituer l'emploi du temps minute par minute et d'identifier tout changement de comportement chez l'enfant avant l'hospitalisation à 5h du matin.

2. Enquête d'Environnement

Les enquêteurs procèdent à une « enquête d'environnement » exhaustive pour dresser le profil psychologique du couple :

• Audition des proches : Une dizaine de membres de la famille (oncles, tantes, grands-parents) sont convoqués pour évaluer la dynamique familiale.

• Enquête de voisinage : Des entretiens avec les voisins immédiats visent à déceler d'éventuels bruits de disputes ou des pleurs incessants.

• Perquisition du domicile : Recherche d'indices matériels de négligence ou de violence.

3. Investigation en Pharmacie

Une technique récurrente consiste à vérifier les achats de médicaments.

Dans cette affaire, les enquêteurs découvrent que le couple a acheté des traitements homéopathiques « anti-choc » (type Arnica) dès les premiers jours de vie de l'enfant, suggérant des incidents antérieurs dissimulés.

--------------------------------------------------------------------------------

III. Analyse de la Dynamique des Aveux

L'enquête progresse à travers la confrontation des versions contradictoires des suspects.

| Phase de l'interrogatoire | Version du Père | Réaction de la Mère | | --- | --- | --- | | Déni Initial | Vie de famille idyllique, enfant désiré. | Récit d'une nuit de pleurs et de convulsions. | | L'Accident Prétendu | Avoue avoir "échappé" le bébé, causant une chute sur le canapé puis au sol. | Ignore l'existence de cette chute. | | Aveu Final | Admet avoir secoué l'enfant deux fois par exaspération. | Effondrement lors de la confrontation. |

Le rôle de l'exaspération

Le père finit par admettre que les cris du nourrisson le mettaient « à bout de nerfs ».

Ce facteur déclencheur, combiné à la fatigue des premiers mois, est identifié par les policiers comme la cause fréquente du passage à l'acte chez des parents par ailleurs « sans histoires ».

La stratégie de protection

L'enquête souligne la difficulté de la preuve : la mère du suspect a suggéré que son fils était capable de s'accuser pour protéger sa compagne.

Cela justifie l'usage de la confrontation directe, où les deux parents sont réunis dans la même pièce pour faire jaillir la vérité.

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IV. Conclusions et Conséquences Judiciaires

L'issue de cette enquête confirme la gravité et la fréquence de ces actes de maltraitance au sein de la cellule familiale.

• Bilan Médical : Bien que l'enfant ait survécu grâce à la pose d'un drain crânien pour évacuer le sang et le liquide, il souffrira de séquelles neurologiques et motrices à long terme.

• Situation Juridique : Le père a été mis en examen pour violences sur mineurs.

La loi prévoit une peine pouvant aller jusqu'à 15 ans de prison.

• Données Statistiques : En France, environ 200 cas de bébés secoués sont recensés chaque année.

Pour la seule brigade de Clermont-Ferrand, cette affaire représentait déjà le septième cas depuis le début de l'année.

Citation Clé :

« C’est plus facile de dire qu’on a fait tomber son gamin, que de dire qu’on l’a secoué. » – Brigitte, enquêtrice à la Brigade des mineurs.

English often changes, and is far from a static object. This is one of the main points of the article, if not the main point.

Simply put, there are multiple variations of English, despite the common belief that there is one overarching shared version.

Words themselves can often change meaning within a given English.

Many people hold bias towards different forms of English, especially newer ones. Perhaps they have had less time to adjust to said newer forms, causing more backlash.

Again, the meaning (As well as implication) of words can change between Englishes.

One should find a way to make their version of English understandable by others, while keeping the intricacies of said English.

The constant changing of English alongside the sheer variety of English types makes machine translation ineffective.

(Can't annotate between pages.) These listed Englishes are, as mentioned, only some of them. The sheer variety is quite an important factor in this situation, and new Englishes could theoretically be made at any time.

(Quote selected continues until the end of the sentence.) Americans in the United States tend to be unused to other kinds of Englishes, due to their country's position (I say as an American, but also as someone who doesn't talk to people in person often, so I cannot truly input my personal opinion on this here.)

Not only can English change, English WILL change. It will change easily and often. Trying to fight such a tide is pointless in the long run, so it is best to move with it and learn how English changes.

Logically speaking, a global language should be relatively simple, albeit complicated enough to convey any ideas or messages one wants to convey.

These suggestions reflect my above quote, that English will certainly change. The answer being suggested here overall is to adjust to it, and to teach others how to adjust to it.

The English language (And many others) is(/Are) a complex thing, meaning it would be hard to find many particular words or intonations that prove beyond a doubt that one's language has a major effect on their experience of reality. That is what I have taken from this, but I could be missing something.

High-stakes situations would indeed require proper communication, as without that, things could go awry quickly.

Hur mycket är det här egentligen i dagens värde?

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要gps?

7258能否满足？

变声看平台是否支持

有平台资源？

对讲平台客户有方案？还是需要我们找供应商？

微信语音/视频通话，暂没有方案

微信扫码绑定，用移远云？还是客户方案？

bk7258 双mic 不支持

wifi 和 4g网络切换怎么实现？wifi 做主控，外挂4g标准？

摄像头规格是否支持120w

eLife Assessment

This fundamental study presents experimental evidence on how geomagnetic and visual cues are integrated in a nocturnally migrating insect. The evidence supporting the conclusions is compelling. The work will be of broad interest to researchers studying animal migration and navigation.

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

Reviewer #1 (Public review):

Summary:

The manuscript by Ma et al. provides robust and novel evidence that the noctuid moth Spodoptera frugiperda (Fall Armyworm) possesses a complex compass mechanism for seasonal migration that integrates visual horizon cues with Earth's magnetic field (likely its horizontal component). This is an important and timely study: apart from the Bogong moth, no other nocturnal Lepidoptera has yet been shown to rely on such a dual-compass system. The research therefore expands our understanding of magnetic orientation in insects with both theoretical (evolution and sensory biology) and applied (agricultural pest management, a new model of magnetoreception) significance.

The study uses state-of-the-art methods and presents convincing behavioural evidence for a multimodal compass. It also establishes the Fall Armyworm as a tractable new insect model for exploring the sensory mechanisms of magnetoreception, given the experimental challenges of working with migratory birds. Overall, the experiments are well designed, the analyses are appropriate, and the conclusions are generally well supported by the data.

Strengths:

• Novelty and significance: First strong demonstration of a magnetic-visual compass in a globally relevant migratory moth species, extending previous findings from the Bogong moth and opening new research avenues in comparative magnetoreception.<br /> • Methodological robustness: Use of validated and sophisticated behavioural paradigms and magnetic manipulations consistent with best practices in the field. The use of 5 min bins to study a dynamic nature of magnetic compass which is anchored to a visual cue but updated with latency of several minutes is an important finding and a new methodological aspect in insect orientation studies.<br /> • Clarity of experimental logic: The cue-conflict and visual cue manipulations are conceptually sound and capable of addressing clear mechanistic questions.<br /> • Ecological and applied relevance: Results have implications for understanding migration in an invasive agricultural pest with expanding global range.<br /> • Potential model system: Provides a new, experimentally accessible species for dissecting the sensory and neural bases of magnetic orientation.

Weaknesses:

Overall, this is a strong study, and the authors have completed an excellent major revision.

Reviewer #2 (Public review):

Summary:

The work titled "Geomagnetic and visual cues guide seasonal migratory orientation in the nocturnal fall armyworm, the world's most invasive insect" provided experimental evidence on how geomagnetic and visual cues are integrated, and visual cues are indispensable for magnetic orientation in the nocturnal fall armyworm.

Strengths:

It has been demonstrated that the Australian Bogon moth could integrate global stellar cues with the geomagnetic field for long distance navigation. However, data are lacking for other insects. This study suggested that the integration of geomagnetic and visual cues may represent a conserved navigational mechanism broadly employed across migratory insects.

Weaknesses:

The visual cues used in the indoor experimental system designed by the authors may have some limitations in ecological relevance. The author may need more explanations on this experimental system.

In the revised manuscript, the authors have added explanations in the discussion section. I am fine with the revision.

Author response:

The following is the authors’ response to the previous reviews

Public Reviews:

Reviewer # 1 (Public review):

(1) Structure and Presentation of Results

• I recommend reordering the visual-cue experiments to progress from simpler conditions (no cues) to more complex ones (cue-conflict). This would improve narrative logic and accessibility for non-specialist readers. The authors have chosen not to implement this suggestion, which I respect, but my recommendation stands.

Thank you for this suggestion. We understand your point that presenting the experiments from simpler to more complex conditions may seem more intuitive. However, we have kept the original order because it better reflects the logic of the study itself. Our work first asked whether fall armyworms, like the Bogong moth, use a magnetic compass that is integrated with visual cues. Only after establishing this behavioral feature did we go on to test whether visual cues are required to maintain magnetic orientation. To make this reasoning clearer to readers, we have explicitly stated in the Introduction that magnetic orientation in the Bogong moth depends on the integration of visual cues, which provides clearer context for the experimental design.

(2) Ecological Interpretation

• The authors should expand their discussion on how the highly simplified, static cue setup translates to natural migratory conditions, where landmarks are dynamic, transient, or absent. Specifically, further consideration is needed on how the compass might function when landmarks shift position, become obscured, or are replaced by celestial cues. Additionally, the discussion would benefit from a more consolidated section with concrete suggestions for future experiments involving transient, multiple, or more naturalistic visual cues. This point was addressed partially in one paragraph of the Discussion, which reads as follows:

"In nature, they are likely to encounter a range of luminance-gradient visual cues, including relatively stable celestial cues as well as transient or shifting local features encountered en route. Although such natural cues differ from our simplified laboratory stimulus, they may represent intermittently sampled visual inputs that can be optimally integrated with magnetic information, with the congruency between visual and magnetic cues likely playing a key role in maintaining a stable compass response. Whether the cues are static or changing, brief periods without them may still allow the subsequent recovery of a stable long-distance orientation strategy. Determining which types of natural visual cues support the magnetic-visual compass, and how they interact with magnetic information, including how their momentary alignment or angular relationship is integrated and how such visual cue-magnetic field interactions may require time to influence orientation, together with elucidating the genetic and ecological bases of multimodal orientation, will be important objectives for future research." While this paragraph is informative, the wording remains lengthy, somewhat unclear, and vague. Shorter, clearer statements would improve readability and impact. For example:

• How could moths maintain direction during periods when only the magnetic field is present and visual landmarks are absent?

• Could celestial cues (e.g., stars) compensate, and what happens if these are also obscured?

• What role does saliency play when multiple visual landmarks are present simultaneously?

• How might a complex skyline without salient landmarks affect orientation?

Including simple, concise sentences that pose concrete open questions and suggest experimental designs would strengthen the discussion without creating space issues. In my view, a comprehensive discussion of how the simplified, static cue setup relates to natural migratory conditions-where landmarks are dynamic, transient, or absent-would add significant value to the paper.

Thank you for this constructive and insightful comment. You correctly point out that our articulation of the ecological relevance of the simplified, static cue setup was not sufficiently clear. We also agree that the original wording in the Discussion remained overly general. In the revised Discussion, we updated the manuscript to incorporate recently published findings on the use of light–dark gradients for orientation in fall armyworms. However, we explicitly note that it remains unclear whether fall armyworms can exploit naturally occurring luminance gradients, such as those generated by the moon, for orientation under natural conditions. We further emphasize that during natural migration the visual environment is dynamic, with celestial cues available intermittently and local visual features changing continuously during flight. In this context, we outline several key unresolved questions, including whether celestial cues can compensate when local landmarks are absent; how multiple visual cues are weighted and integrated with geomagnetic information; how transient visual cues (like moving clouds or changing illumination) influence orientation; and how luminance gradients that are common in natural nocturnal environments interact with the geomagnetic field to support orientation. For each of these issues, we briefly suggest experimental approaches to guide future research.

(3) Methodological Details and Reproducibility

• The lack of luminance level measurements should be explicitly highlighted.

Thank you for your helpful suggestion. You are right that luminance level is an important experimental parameter. We have stated this information in the Methods section under Behavioral apparatus: “The ambient light level in the experimental environment was measured to be below 1 lux using a Testo 540 lux meter (Testo SE & Co. KGaA, Titisee-Neustadt, Germany). Further work is still required to compare the illuminance used in this study with that under natural conditions, which are inherently variable.” This point is also clarified in the legend of Figure S3 in the supplementary material.

• The authors chose not to adjust figure legends by replacing "magnetic South" with "magnetic North." While I believe this would be more conventional and preferable, this is ultimately a minor stylistic issue.

Thank you very much for your suggestion. We understand your point and agree that using “magnetic North” would be more conventional. However, because our experiments focus on the orientation behavior of the autumn population, magnetic South is aligned with the landmark direction representing the potential migratory direction, which we believe makes the figures more intuitive for readers. We therefore consider this a minor stylistic issue.

(4) Conceptual Framing and Discussion

• Although the authors made a good attempt to explain the limitations of using an artificial visual cue, I believe there is room or a more explicit argument. For example, it could be stated clearly that this species is unlikely to encounter a situation in nature where a single, highly salient landmark coincides with its migratory direction. Therefore, how these findings translate to real migratory contexts remains an open question. A sentence or two making this point directly would strengthen the discussion.

Thank you for your helpful suggestion. We now address this point explicitly in the Discussion, noting that fall armyworms are unlikely to experience a natural visual environment dominated by a single, static, and highly salient landmark coinciding with their migratory direction. Consequently, how these findings translate to real migratory contexts remains an open question.

(5) Technical and Open-Science Points

• Sharing the R code openly (e.g., via GitHub) should be seriously considered. The code does not need to be perfectly formatted, but making it available would be highly beneficial from an open-science perspective.

Thank you for the suggestion. We agree that making code openly available is valuable from an open-science perspective. The MMRT script used in this study is Moore’s Modified Rayleigh Test, available from the original publication by Massy et al. (2021; https://doi.org/10.1098/rspb.2021.1805). In the previous version, we only cited this reference in the Materials and Methods section; we have now added a direct link to the script to improve clarity and accessibility. We have also provided a public link to the data-recording scripts used in the Flash Flight Simulator (https://doi.org/10.17632/6jkvpybswd.1). This repository additionally includes a map-based optical flow script that was not used in the present study but is shared for completeness.

Reviewer #1 (Recommendations for the authors):

• LL. 133-137 (end of paragraph starting with "The fall armyworm is a migratory crop pest native to the Americas"): Suggest splitting into shorter, clearer sentences. The limitations of this method could be better articulated here and elaborated in the Discussion.

Thank you for this suggestion. We have revised this paragraph by splitting it into shorter, clearer sentences and by articulating the limitations of this method more explicitly. These limitations are further elaborated in the Discussion.

• LL. 181-185 (end of paragraph starting with "To examine if fall armyworms integrate geomagnetic and visual cues for seasonal migratory orientation"): It would be helpful to state explicitly that season-specific headings have been confirmed in the lab using a flight simulator, but destination regions remain unknown without further tracking experiments.

Thank you for this helpful suggestion. We have now clarified in the revised manuscript that season-specific orientation headings have been confirmed in the laboratory using a flight simulator, while the actual migratory destination regions remain unclear in the absence of tracking experiments.

• LL. 230-234 (start of paragraph "Our previous research showed that fall armyworms reared under artificially simulated fall conditions…"): Clarify which migratory season is being referenced.

Thank you for this helpful suggestion. We have clarified in the text that the migratory season referenced here is the autumn migratory season. In addition, we have added information in the Methods to specify the actual calendar season during which the insects were reared under the simulated conditions.

• LL. 270-272 (middle of Fig. 2 caption): Suggest explicitly mentioning that for this population, the seasonally appropriate direction is southbound in autumn and northbound in spring, as this may not be clear to non-specialists.

Thank you for this helpful suggestion. We have now explicitly stated the seasonally appropriate migratory directions for this population, indicating southbound migration in autumn and northbound migration in spring, to improve clarity for non-specialist readers.

• LL. 421 (middle of paragraph starting with "We also considered the limitations of the Rayleigh test…"): Add that the groups lacking visual cues exhibited "lower directedness as per lower vector length (r)" in addition to lower flight stability.

Thank you for this helpful suggestion. We further note that the conclusions drawn from the flight stability analysis are consistent with those based on individual r-value analyses.

• LL. 499-501 ("unlike some vertebrates that can rely solely on magnetic information (Mouritsen, 2018)"): This point is slightly downplayed. It should be emphasized that nearly all tested vertebrates and invertebrates (e.g., birds, mole rats, fish, frogs, and other insects) demonstrate a magnetic compass without requiring visual landmarks. Moths are the only tested invertebrates so far that show landmark-magnetic field dependency for their magnetic compass to be manifested in a behavioural orientation response in Flight Simulator.

Thank you for this important comment. We agree that this point represents a key synthesis in the Discussion, as it concerns how our findings relate to, and differ from, magnetic orientation demonstrated in other animal groups. We have therefore expanded the Discussion to note that studies have shown that some animals can exhibit directional preferences in simplified visual environments solely in response to changes in the magnetic field, and we now cite representative examples from birds and mole rats. At the same time, we also acknowledge important methodological and phenotypic differences among taxa. In particular, moths’ magnetic orientation has been assessed using a flight simulator, a setup in which stable directional behavior must be actively maintained during continuous movement. This is an important difference from orientation assays in birds during take-off or in terrestrial mammals such as mole rats. Moreover, whether birds and other animals rely on visual input to detect or calibrate magnetic information under certain conditions remains an open question. We therefore emphasize here both the phenotypic differences observed across experimental systems and the methodological considerations.

• LL. 560-565 (paragraph starting with "Our flight simulator system (Dreyer et al., 2021) …"): Suggest clarifying what the Flash flight simulator system is and how it differs from the Mouritsen-Frost flight simulator.

Thank you for this suggestion. We have added a brief clarification of the Flash flight simulator and how it differs from the Mouritsen–Frost system.

• LL. 605-608 ("Spectral measurements …"): Explicitly mention that total illuminance was not measured and that further work is required to compare the illuminance used with natural conditions which of course vary.

Thank you for this helpful suggestion. We agree that total illuminance is an important factor. We have now added a statement noting that the ambient light level in the experimental environment was measured to be below 1 lux using a Testo 540 lux meter, and we further acknowledge that additional work is required to compare the illuminance used in this study with that under naturally variable conditions.

• LL. 628-641 (end of paragraph starting with "Electromagnetic noise at the experimental site ... "): Explain why this matters for interpreting behavioural responses. Highlight that although conditions were somewhat magnetically noisy which based on the past work may disrupt magnetic compass as it was shown in birds (eg Engels et al. 2014 Nature), the observed magnetic response under certain conditions indicates that the magnetic sense remained functional when landmark and magnetic field were aligned. This way you can pre-empt this criticism of your magnetic conditions being not ideal and noise on the left handside of the spectrum measured (which is not uncommon).

Thank you for this helpful suggestion. We have now cited Engels et al. (2014, Nature) in this section and expanded the text to explain why electromagnetic noise at the experimental site is relevant for interpreting the behavioural responses. We also clarify the rationale for measuring electromagnetic noise and discuss the observed low-frequency (“left-hand side”) noise in the spectrum.

• Fig. 51: Suggest adapting Y-axes and using violin or box plots (e.g., panels A/B starting from 30 up to 50, etc.).

Thank you for this helpful suggestion. We have revised Fig. 5 accordingly by adapting the Y-axis scaling and replacing the original plots with box plots, as suggested.

eLife Assessment

This valuable study advances our understanding of how organisms respond to chronic oxidative stress. Using the nematode C. elegans, the authors identified key neuronal signaling molecules and their receptors that are required for stress signaling and survival. The evidence supporting the conclusions is solid, including rigorous genetics, stress response analysis, and transcriptional profiling. This research will be of broad interest to neuroscientists and researchers working in the field of oxidative stress regulation.

Reviewer #1 (Public review):

Summary:

The researchers aimed to identify which neurotransmitter pathways are required for animals to withstand chronic oxidative stress. This work thus has important implications for disease processes that are caused/linked to oxidative stress. This work identified specific neurotransmitters and receptors that coordinate stress resilience, both prior to and during stress exposure. Further, the authors identified specific transcriptional programs coordinated by neurotransmission that may provide stress resistance.

Strengths:

The manuscript is very clearly written with a well-formulated rationale. Standard C. elegans genetic analysis and rescue experiments were performed to identify key regulators of the chronic oxidative stress response. These findings were enhanced by transcriptional profiling that identified differentially expressed genes that likely affect survival when animals are exposed to stress.

Weaknesses:

Where the gar-3 promoter drives expression was not discussed in the context of the rescue experiments in Fig 7.

Comments on revisions:

This issue has now been appropriately addressed in the revision.

Reviewer #2 (Public review):

In this paper, Biswas et al. describe the role of acetylcholine (ACh) signaling in protection against chronic oxidative stress in C. elegans. They showed that disruption of ACh signaling in either unc-17 mutant or gar-3 mutants led to sensitivity to toxicity caused by chronic paraquat (PQ) treatment. Using RNA seq, they found that approximately 70% of the genes induced by chronic PQ exposure in wild type failed to upregulate in these mutants. The overexpression of gar-3 selectively in cholinergic neurons was sufficient to promote protection against chronic PQ exposure in an ACh-dependent manner. The study points to a previously undescribed role for ACh signaling in providing organism-wide protection from chronic oxidative stress likely through the transcriptional regulation of numerous oxidative stress-response genes. The paper is well-written, and the data are robust, though some conclusions seem preliminary and are not fully support the current data (see below). While the study identifies the muscarinic ACh receptor gar-3 as an important regulator of the response to PQ, the specific neurons in which gar-3 functions were not unambiguously identified, and the sources of ACh that regulate GAR-3 signaling and the identities of the tissues targeted by gar-3 were not addressed.

Comments on revisions:

The authors addressed my comments adequately in their revised submission. Please include representative images to accompany the quantification of the new results presented in Fig S4A.

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public review):

The researchers aimed to identify which neurotransmitter pathways are required for animals to withstand chronic oxidative stress. This work thus has important implications for disease processes that are caused/linked to oxidative stress. This work identified specific neurotransmitters and receptors that coordinate stress resilience, both prior to and during stress exposure. Further, the authors identified specific transcriptional programs coordinated by neurotransmission that may provide stress resistance.

Strengths:

The manuscript is very clearly written with a well-formulated rationale. Standard C. elegans genetic analysis and rescue experiments were performed to identify key regulators of the chronic oxidative stress response. These findings were enhanced by transcriptional profiling that identified differentially expressed genes that likely affect survival when animals are exposed to stress.

We thank the reviewer for their positive assessment.

Weaknesses:

Where the gar-3 promoter drives expression was not discussed in the context of the rescue experiments in Figure 7.

We now provide information about expression using 7.5 kb gar-3 promoter fragment  and compare directly with our analysis of endogenous gar-3 expression using the genome-modified gar-3::SL2::GFP strain (Page 16, new Figures 8 and S3).

Reviewer #1 (Recommendations for the authors):

(1) Figure 3B is not mentioned in the text.

Fixed. Figure 3B is now called out on page 10 of the revised manuscript.

(2) The rationale for using the specific PQ concentration was not provided.

We selected this concentration based on its use for chronic assays by other studies in the field to allow for direct comparison with our results. We now clarify this point in the Methods section (Page 26 of the revised text).

(3) Transgenic animals injected with the unc-17βp::gar-3 transgene (25 ng/μL) displayed strikingly increased survival in the presence of 4 mM PQ compared to either gar-3 mutants or wild type (should have a Figure cited here)

Fixed. Figure 9E is now referenced on Page 19 of the revised text.

(4) The text describing Figure 7C details a comparison with the gar-3 single mutant but the graph shows the unc-17 single mutant

Figure 7C is a comparison of the survival of gar-3 single mutants with either wild type or gar-3;ric-3 double mutants as described in the text.

Reviewer #2 (public comments)

In this paper, Biswas et al. describe the role of acetylcholine (ACh) signaling in protection against chronic oxidative stress in C. elegans. They showed that disruption of ACh signaling in either unc-17 mutants or gar-3 mutants led to sensitivity to toxicity caused by chronic paraquat (PQ) treatment. Using RNA seq, they found that approximately 70% of the genes induced by chronic PQ exposure in wild type failed to upregulate in these mutants. The overexpression of gar-3 selectively in cholinergic neurons was sufficient to promote protection against chronic PQ exposure in an ACh-dependent manner. The study points to a previously undescribed role for ACh signaling in providing organism-wide protection from chronic oxidative stress, likely through the transcriptional regulation of numerous oxidative stressresponse genes. The paper is well-written, and the data are robust, though some conclusions seem preliminary and do not fully support the current data. While the study identifies the muscarinic ACh receptor gar-3 as an important regulator of the response to PQ, the specific neurons in which gar-3 functions were not unambiguously identified, and the sources of ACh that regulate GAR-3 signaling and the identities of the tissues targeted by gar-3 were not addressed, limiting the scope of the study.

We thank the reviewer for their positive assessment. We provide additional data and discussion of the points raised by the reviewer in the revised manuscript. In particular, as suggested by the reviewer, we conducted additional tissue-specific rescue experiments to try to better define GAR-3 site of action. We found that specific rescue of gar-3 expression in either cholinergic motor neurons or muscles each provide partial rescue. In addition, we quantified the expression of the nhr-185 and fbxa-73 genes, identified as upregulated by PQ in our RNA-seq studies, following oxidative stress (new Fig. S4). We observed increased expression of both genes following PQ exposure, providing independent confirmation for transcriptional upregulation of these genes as part of the stress response. See the responses to points #1 and #3 below for additional details.

Major Comments:

(1) The site of action of cholinergic signaling for protection from PQ was not adequately explored. The authors' conclusion that cholinergic motor neurons are protective is based on studies using overexpression of gar-3 and an unc-17 allele that may selectively disrupt ACh in cholinergic motor neurons (Figure 9F), but these approaches are indirect. To more directly address the site of action, the authors should conduct rescue experiments using well-defined heterologous promoters. Figure 7G shows that gar-3 expressed under a 7.5 kb promoter fragment fully rescues the defect of gar-3 mutants, but the authors did not report where this promoter fragment is expressed, nor did they conduct rescue experiments of the specific tissues where gar-3 is known to be expressed (cholinergic neurons, GABAergic neurons, pharynx, or muscles). UNC-17 rescue experiments could also be useful to address the site of action. Does expression of unc-17 selectively in cholinergic motor neurons rescue the stress sensitivity of unc-17 mutants (or restore resistance to gar-3(OE); unc-17 mutants)? These experiments may also address whether ACh acts in an autocrine or paracrine manner to activate gar-3, which would be an important mechanistic insight to this study that is currently lacking.

We performed additional rescue experiments using heterologous promoters to drive gar-3 expression in cholinergic neurons or muscle and found that each provided a small, but significant degree of rescue as assessed from Kaplan-Meier survival curves. These results are presented in Figure 8 of the revised manuscript. We have not conducted similar unc-17 rescue experiments; however, we point out that cellspecific unc-17 knockdown by RNAi using the unc-17b promoter (expression largely restricted to ventral cord ACh motor neurons) increases sensitivity to PQ in our long-term survival assays (Figure 3A). Combined with our analysis of unc-17(e113) mutants, we believe these results support a requirement for unc-17 expression in cholinergic motor neurons.

(2) The genetic pan-neuronal silencing experiments presented in Figure 1 motivated the subsequent experiments, but the authors did not relate these observations to ACh/gar-3 signaling. For example, the authors did not address whether silencing just the cholinergic motor neurons at the different times tested has the same effects on survival as pan-neuronal silencing.

We used the pan-neuronal silencing to motivate further analysis of various neurotransmitter systems. Our genetic studies implicate both glutamatergic and cholinergic systems in protective responses to oxidative stress. The effects of pan-neuronal silencing on survival during long-term PQ exposure may therefore be derived solely from cholinergic neurons, glutamatergic neurons, or a combination of both neuronal populations. Distinguishing between these possibilities may be quite complicated and is not central to the main message of our paper. We therefore suggest this additional analysis lies outside the scope of this revision. Nonetheless, to address the reviewer’s point, in the revised text we expand our discussion relating the pan-neuronal silencing results to our analysis of ACh signaling (pages 21-22).

(3) It is assumed that protection occurs through inter-tissue signaling of ACh to target tissues, where it impacts gene expression. While this is a reasonable assumption, it has not been directly shown here. It is recommended that the authors examine GFP reporter expression of a sampling of the genes identified in this study (including proteasomal genes that the authors highlight) that are regulated by unc-17 and gar-3. This would serve to independently confirm the RNAseq data and to identify target tissues that are subject to gene expression regulation by ACh, which would significantly strengthen the study.

Agreed. To address this question, we investigated expression of the nhr-185 and fbxa-73 genes implicated as upregulated by oxidative stress in our RNA-seq studies. Consistent with our RNA-seq findings, we observed significantly increased expression of a nhr-185pr::GFP transcriptional reporter, primarily in the pharynx and anterior intestine, following 48 hrs of PQ exposure. These results support transcriptional upregulation of expression in these tissues as part of the stress response. fbxa-73 was among the proteasomal genes implicated as oxidative stress-responsive by RNA-seq. Consistent with this finding, by quantitative RT-PCR we observed a significant increase in fbxa-73 expression in wild type animals following 48 hrs of PQ treatment. These new results provide independent confirmation of the gene expression changes we observed by RNA-seq and are now included in new Figure S4 and discussed on Pages 17-18 of the revised manuscript.

Reviewer #2 (Recommendations for the authors):

(1) As an independent way of addressing whether enhanced ACh signaling is sufficient for protection, the authors could examine stress resistance in ace mutants, as was reported in PMID: 39097618, or in mutants with increased ACh secretion.

We thank the reviewer for this suggestion. We are pursuing the impacts of increased cholinergic activation in a separate study. We are pursuing experiments along the lines the reviewer suggests as one facet of this independent study. Our findings here provide evidence that increasing GAR-3 signaling in ACh motor neurons by cell-specific overexpression enhances protection. 

(2) To address the specificity of ACh signaling by gar-3 for this response, the authors could report survival data for mutants lacking each of the other two mACh receptors, gar-1 and gar-2.

We thank the reviewer for this suggestion. We now include new data showing that gar-3;gar-2 double mutants have similar survival to gar-3 single mutants in the presence of PQ new Figure 7F). We agree that further studies of additional GPCRs (e.g. gar-1 and metabotropic glutamate receptors) will be required to definitively establish specificity for GAR-3 and we now acknowledge this point on page 15 of the revised text.

(3) Do carbonylation levels correlate with toxicity? For example, do gar-3 mutants have more carbonylation and gar-3 OE have less?

This is an interesting question. To try to address this, we performed additional protein carbonylation experiments for unc-17 and gar-3 mutants. We found a similar increase in protein carbonylation following PQ exposure for gar-3 mutants as observed for wild type; however, we also noted a higher level a batch-to-batch variability for gar-3 compared with wild type and are therefore hesitant to draw firm conclusions. We have not included these data in the revised manuscript but provide them for the reviewer’s information here (Author response image 1 shows our prior N2 data for comparison). We were not able to conduct similar experiments for unc-17 mutants because we noted local starvation when the animals were grown at the high density required to obtain the protein quantities needed for these experiments.

Author response image 1.

(4) Citations in text for Figures 4A and 8A are missing.

Fixed. Figures 4A and 8A (now 9A) are cited on pages 10 and 17 of the revised text, respectively.

(5) Figures 4-6 and 8 have limited information content. Condense or move to supplementary.

While we acknowledge the reviewer’s viewpoint here, we believe that the analyses of the transcriptional responses described in Figures 4-6 and 8 are central to the study. To address reviewers’ comments, we have included a new Figure 8 and merged previous Figures 8 and 9 (new Figure 9) in the revised manuscript.

(6) "expression of" is repeated in "Finally, transgenic expression of expression of a wild-type GAR-3::YFP"

Fixed.

emphasizes that handwriting and digital skills are complementary, and handwriting remains essential for cognitive development and early academic success -- provides argument for tech's place in education

handwriting proficiency is correlated with higher achievement in multiple subjects, not just writing, which displays its broad impact on academic performance across subjects

fluency in handwriting supports writing quality and composition development, meaning handwriting practice can accelerate academic skills in writing

first of all this is an important fact to include in my research, but it talks about the benefits handwriting yields for younger ages which reminds me of my original research question of how handwriting impacts learning across ages, but the fact of the matter is that handwriting isn't any more beneficial at one age as it is the latter or prior, because it's a skill that develops with time according to the cognitive developments of students/people

handwriting strengthens memory and attention, supporting the idea that students retain information better when they write by hand compared to typing

handwriting activates motor and visual areas of the brain to reinforce letter recognition, which typing does not, highlighting a neurological basis for its academic benefits

even though handwriting isn’t always formally tested, it still affects other academic skills, showing that its benefits go beyond just testing and are genuinely crucial for overall student learning

eLife Assessment

This important study shows that orientation tuning of V1 neurons is suppressed during a continuous flash suppression paradigm, especially in neurons with binocular receptive fields. These findings, made using cutting-edge imaging techniques, convincingly implicate early visual processing in continuous flash suppression, in agreement with previous studies suggesting reduced effective contrast of such stimuli in V1.

Reviewer #1 (Public review):

This study makes a fundamental contribution to our understanding of interocular suppression, particularly continuous flash suppression (CFS). Using neuroimaging data from two macaque monkeys, the study provides compelling evidence that CFS suppresses orientation responses in neurons within V1. These findings enrich the CFS literature by demonstrating that neural activity under CFS may prevent high-level visual and cognitive processing.

Comments on revisions:

The authors have addressed all my previous comments.

Reviewer #2 (Public review):

Summary:

The goal of this study was to investigate the degree to which low-level stimulus features (i.e., grating orientation) are processed in V1 when stimuli are not consciously perceived under conditions of continuous flash suppression (CFS). The authors measured the activity of a population of V1 neurons at single neuron resolution in awake fixating monkeys while they viewed dichoptic stimuli that consisted of an oriented grating presented to one eye and a noise stimulus to the other eye. Under such conditions, the mask stimulus can prevent conscious perception of the grating stimulus. By measuring the activity of neurons (with Ca2+ imaging) that preferred one or the other eye, the authors tested the degree of orientation processing that occurs during CFS.

Strengths:

The greatest strength of this study is the spatial resolution of the measurement and the ability to quantify stimulus representations during CSF in populations of neurons preferring the eye stimulated by either the grating or the mask. There have been a number of prominent fMRI studies of CFS, but all of them have had the limitation of pooling responses across neurons preferring either eye, effectively measuring the summed response across ocular dominance columns. The ability to isolate separate populations offers an exciting opportunity to study the precise neural mechanisms that give rise to CFS, and potentially provide insights into nonconscious stimulus processing.

Weaknesses:

However, while this is an impressive experimental setup, the major weakness of this study is that the experiments don't advance any theoretical account of why CFS occurs or what CFS implies for conscious visual perception. There are two broad camps of thinking with regard to CFS. On the one hand, Watanabe et al., 2011 reported that V1 activity remained intact during CFS, implying that CFS interrupts stimulus processing downstream of V1. On the other hand, Yuval-Greenberg and Heeger (2013) showed that V1 activity is in fact reduced during CFS. By using a parametric experimental design, they measured the impact of the mask on the stimulus response as a function of contrast, and concluded that the mask reduces the gain of neural responses to the grating stimulus. They presented a theoretical model in which the mask effectively reduced the SNR of the grating, making it invisible in the same way that reducing contrast makes a stimulus invisible.

In the first submission of the manuscript, the authors incorrectly described the Yuval-Greenberg & Heeger (2013) paper and Watanabe et al. (2011) papers, suggesting that they had observed the same or similar effects of CFS on V1 activity, when in fact they had described opposite results. Reviewer 1 also observed that the authors appeared to be confused in their reading of these highly relevant papers. In the revision, the authors have reworked this paragraph, now correctly describing these sets of opposing results. However, I still do not understand what the authors are trying to argue: "...these studies were not designed to quantify the pure effect of CFS on stimulus-evoked V1 responses." I do not understand what is meant by "pure" in this case. Regardless, it is clear that the measurements in the present study strongly support the interpretation of Yuval-Greenberg & Heeger (i.e., that V1 activity is degraded by CFS, 'akin' to a loss in the contrast-to-noise ratio of neural activity). It would be appropriate for the authors to communicate this clearly.

I continue to be of the opinion that this study is lacking an adequate model of interocular interactions that might explain the Ca2+ imaging. The machine learning results are not terribly surprising - multivariate methods, such as SVMs, are more sensitive than univariate approaches. So it is plausible that an SVM can support decoding of the coarse orientation information, even when no tuning is evident in the univariate analyses. However, the link between this result and the underlying neurophysiology is opaque. The failure to model the neural data with an explicit model is a missed opportunity.

Reviewer #3 (Public review):

Summary:

In this study, Tang, Yu & colleagues investigate the impact of continuous flash suppression (CFS) on the responses of V1 neurons using 2-photon calcium imaging. The report that CFS substantially suppressed V1 orientation responses. This suppression happens in a graded fashion depending on the binocular preference of the neuron: neurons preferring the eye that was presented with the marker stimuli were most suppressed, while the neurons preferring the eye to which the grating stimuli were presented were least suppressed. Binocular neuron exhibited an intermediate level of suppression.

Strengths:

The imaging techniques are cutting-edge.

Weaknesses:

The strength of CFS suppression varies across animals, but the authors attribute this to comparable heterogeneity in the human psychophysics literature.

Comments on revisions:

The authors have addressed my comments from the previous round of review, and I have no further comments

Author response:

The following is the authors’ response to the original reviews

eLife Assessment

This important study shows that orientation tuning of V1 neurons is suppressed during a continuous flash suppression paradigm, especially when the neurons have a binocular receptive field. However, the evidence presented is incomplete and, in particular, does not distinguish whether this suppression is due to reduced contrast or due to masking.

This assessment is primarily based on the critique of Reviewer 2 that our results do not distinguish whether the impact of CFS is due to reduced contrast or due to masking. Reviewer 2 referred to Yuval-Greenberg and Heeger (2013), noting that: “V1 activity is, in fact, reduced during CFS … the mask reduces the gain of neural responses to the grating stimulus … making it invisible in the same way that reducing contrast makes a stimulus invisible.” To be precise, Yuval-Greenberg and Heeger (2013) used “akin to”, instead of “the same way”, in their abstract.

We agree that CFS masking and contrast reduction can both lower the signal-to-noise ratio and thereby reducing visibility. However, these two factors operate in fundamentally different ways. According to gain control models by Heeger and others, reducing the physical contrast of a stimulus decreases the excitatory drive, while dichoptic masking increases the normalization pool. Our findings therefore reflect genuine masking-induced suppression and are not attributable to stimulus contrast reduction.

Public Reviews:

Reviewer #1 (Public review):

Disclaimer: While I am familiar with the CFS method and the CFS literature, I am not familiar with primate research or two-photon calcium imaging. Additionally, I may be biased regarding unconscious processing under CFS, as I have extensively investigated this area but have found no compelling evidence in favor of unconscious processing under CFS.

This manuscript reports the results of a nonhuman-primate study (N=2 behaving macaque monkeys) investigating V1 responses under continuous flash suppression (CFS). The results show that CFS substantially suppressed V1 orientation responses, albeit slightly differently in the two monkeys. The authors conclude that CFS-suppressed orientation information "may not suffice for high-level visual and cognitive processing" (abstract).

The manuscript is clearly written and well-organized. The conclusions are supported by the data and analyses presented (but see disclaimer). However, I believe that the manuscript would benefit from a more detailed discussion of the different results observed for monkeys A and B (i.e., inter-individual differences), and how exactly the observed results are related to findings of higher-order cognitive processing under CFS, on the one hand, and the "dorsal-ventral CFS hypothesis", on the other hand.

Thanks for reviewer’s helpful comments and suggestions. We added new contents discussing the inter-individual differences and the "dorsal-ventral CFS hypothesis" in the revision, and made other changes, which are detailed below.

Major Comments:

(1) Some references are imprecise. For example, l.53: "Nevertheless, two fMRI studies reported that V1 activity is either unaffected or only weakly affected (Watanabe et al., 2011; Yuval-Greenberg & Heeger, 2013)". "To the best of my understanding, the second study reaches a conclusion that is entirely opposite to that of the first, specifically that for low-contrast, invisible stimuli, stimulus-evoked fMRI BOLD activity in the early visual cortex (V1-V3) is statistically indistinguishable from activity observed during stimulus-absent (mask-only) trials. Therefore, high-level unconscious processing under CFS should not be possible if Yuval-Greenberg & Heeger are correct. The two studies contradict each other; they do not imply the same thing.

Sorry we did not make our point clear. Our original concern was that the effects of CFS on V1 activity were underestimated, even in Yuval-Greenberg & Heeger (2013), as both studies compared monocular and dichoptic masking to estimate the influence of visibility. In contrast, in original psychophysical studies, the CFS effect was compared with or with dichoptic masking, which is expected to be stronger. We rewrote the paragraph to clarify.

“Two prominent fMRI studies have examined the impact of CFS on V1 activity (Watanabe et al., 2011; Yuval-Greenberg & Heeger, 2013). Watanabe et al. (2011) compared monocular CFS masking (stimulus visible) and dichoptic CFS masking (stimulus invisible), and reported that V1 BOLD responses were largely insensitive to stimulus visibility when attention was carefully controlled. However, using similar experimental design, Yuval-Greenberg and Heeger (2013) observed reduced BOLD responses in V1 under dichoptic masking, suggesting that V1 activity changed with stimulus visibility. They attributed the difference of results between two studies mainly to differences in statistical power (~250 trials per condition vs. ~90 trials per condition). Nevertheless, these studies were not designed to quantify the pure effect of CFS on stimulus-evoked V1 responses, as they contrasted monocular and dichoptic masking conditions to equate stimulus input while manipulating perceptual visibility. In contrast, original psychophysical studies (Tsuchiya & Koch, 2005; Tsuchiya, Koch, Gilroy, & Blake, 2006) demonstrated CFS masking by contrasting the visibility of the target stimulus with and without the presence of dichoptic mask. It is apparent that the pure CFS impact in above fMRI studies would be the difference of BOLD signals between binocular masking and stimulus alone conditions. In other words, the impact of CFS on V1 activity should be larger than what has been reported by Yuval-Greenberg and Heeger (2013).” (lines 55-71)

(2) Line 354: "The flashing masker was a circular white noise pattern with a diameter of 1.89°, a contrast of 0.5, and a flickering rate of 10 Hz. The white noise consisted of randomly generated black and white blocks (0.07 × 0.07 each)." Why did the authors choose a white noise stimulus as the CFS mask? It has previously been shown that the depth of suppression engendered by CFS depends jointly on the spatiotemporal composition of the CFS and the stimulus it is competing with (Yang & Blake, 2012). For example, Hesselmann et al. (2016) compared Mondrian versus random dot masks using the probe detection technique (see Supplementary Figure S4 in the reference below) and found only a poor masking performance of the random dot masks.

Yang, E., & Blake, R. (2012). Deconstructing continuous flash suppression. Journal of Vision, 12(3), 8. https://doi.org/10.1167/12.3.8

Hesselmann, G., Darcy, N., Ludwig, K., & Sterzer, P. (2016). Priming in a shape task but not in a category task under continuous flash suppression. Journal of Vision, 16, 1-17.

In a previous human psychophysical study, we also used the same noise pattern and the CFS effect appeared to be robust (Xiong et al., 2016, https://doi.org/10.7554/eLife.14614). However, we believe that the reviewer made a good point, and weaker suppression due to the use of our stimulus pattern may have contributed to the weaker suppression in Monkey B. This issue is now discussed in the revision regarding the individual variability in our results.

“In addition, the random-noise masker we used might not be as effective as Mondrian patterns (G. Hesselmann, Darcy, Ludwig, & Sterzer, 2016). If reduced stimulus contrast and a Mondrian masker were used, we predict that CFS suppression in Monkey B would strengthen, potentially approaching the level observed in Monkey A. Nevertheless, it is worth emphasizing that our main conclusions are primarily based on data from Monkey A, who exhibited much stronger CFS suppression.” (lines 321-327)

(3) Related to my previous point: I guess we do not know whether the monkeys saw the CF-suppressed grating stimuli or not? Therefore, could it be that the differences between monkey A and B are due to a different individual visibility of the suppressed stimuli? Interocular suppression has been shown to be extremely variable between participants (see reference below). This inter-individual variability may, in fact, be one of the reasons why the CFS literature is so heterogeneous in terms of unconscious cognitive processing: due to the variability in interocular suppression, a significant amount of data is often excluded prior to analysis, leading to statistical inconsistencies.

Yamashiro, H., Yamamoto, H., Mano, H., Umeda, M., Higuchi, T., & Saiki, J. (2014). Activity in early visual areas predicts interindividual differences in binocular rivalry dynamics. Journal of Neurophysiology, 111(6), 1190-1202. https://doi.org/10.1152/jn.00509.2013

The individual difference issue is now explicitly addressed in the Discussion:

“Interocular suppression under CFS is known to vary substantially across individuals (Blake, Goodman, Tomarken, & Kim, 2019; Gayet & Stein, 2017; Yamashiro et al., 2013). This inter-individual variability may contribute to the heterogeneity observed in the CFS literature. We also found that the strength of V1 response suppression during CFS differed between two monkeys, as reflected by population orientation tuning functions (Fig. 2C), Fisher information (Fig. 2F), and reconstruction performance by the transformer (Fig. 3E). Several experimental factors may have contributed to the relatively weaker suppression observed in Monkey B. Because monkeys viewed the stimuli passively, we could not determine the dominant eye for each monkey (instead we switched the eyes and averaged the results), and the target was presented at relatively high contrast. Both factors are known to reduce the effectiveness of CFS suppression (Yang, Blake, & McDonald, 2010; Yuval-Greenberg & Heeger, 2013). In addition, the random-noise masker we used might not be as effective as Mondrian patterns (G. Hesselmann, Darcy, Ludwig, & Sterzer, 2016). If reduced stimulus contrast and a Mondrian masker were used, we predict that CFS suppression in Monkey B would strengthen, potentially approaching the level observed in Monkey A. Nevertheless, it is worth emphasizing that our main conclusions are primarily based on data from Monkey A, who exhibited much stronger CFS suppression.” (lines 311-327)

Moreover, the authors' main conclusion (lines 305-307) builds on the assumption that the stimuli were rendered invisible, but isn't this speculation without a measure of awareness?

We agree. To correct, we have removed the original lines 305-307 discussing the consciousness perception and reframed the manuscript throughout to focus on the impact of CFS on neural coding rather than on perceptual awareness. For example, the title has been changed to:

“Continuous flashing suppression of neural responses and population orientation coding in macaque V1”,

and the ending line of Introduction was changed to:

“This approach enabled us to investigate the potentially differential impacts of CFS on the responses of V1 neurons with varying ocular preferences, as well as apply machine learning tools to understand the impacts of CFS on V1 stimulus coding at the population level.” (lines 81-83)

(4) The authors refer to the "tool priming" CFS studies by Almeida et al. (l.33, l.280, and elsewhere) and Sakuraba et al. (l.284). A thorough critique of this line of research can be found here:

Hesselmann, G., Darcy, N., Rothkirch, M., & Sterzer, P. (2018). Investigating Masked Priming Along the "Vision-for-Perception" and "Vision-for-Action" Dimensions of Unconscious Processing. Journal of Experimental Psychology. General. https://doi.org/10.1037/xge0000420

This line of research ("dorsal-ventral CFS hypothesis") has inspired a significant body of behavioral and fMRI/EEG studies (see reference for a review below). The manuscript would benefit from a brief paragraph in the discussion section that addresses how the observed results contribute to this area of research.

Ludwig, K., & Hesselmann, G. (2015). Weighing the evidence for a dorsal processing bias under continuous flash suppression. Consciousness and Cognition, 35, 251-259. https://doi.org/10.1016/j.concog.2014.12.010

In the revision, we added a new paragraph to discussion issues related to the dorsal-ventral CFS hypothesis.

“A related issue is the dorsal-ventral CFS hypothesis, which proposes that CFS suppression may disproportionately affect ventral visual processing while relatively preserving dorsal pathways involved in visuomotor functions, potentially allowing category- or action-related information to remain accessible under suppression (Fang & He, 2005). However, subsequent fMRI studies have failed to provide consistent support for this dissociation, reporting either stream-invariant awareness effects (Guido Hesselmann & Malach, 2011; Ludwig et al., 2015; Tettamanti et al., 2017), residual signal in ventral rather than dorsal regions (Fogelson et al., 2014; Guido Hesselmann et al., 2011), or residual low-level feature information/partial visibility rather than preserved dorsal processing (Ludwig et al., 2015). Although our study does not directly test dorsal-ventral dissociations, our V1 results provide a constraint on what information downstream visual pathways could access under suppression. When CFS- induced interocular suppression was strong enough and stimuli reconstruction was markedly reduced, as in the case of Monkey A, the information required for category-level or action-related processing may not be sufficient for high-level cortical representation.” (lines 297-310)

Reviewer #2 (Public review):

Summary:

The goal of this study was to investigate the degree to which low-level stimulus features (i.e., grating orientation) are processed in V1 when stimuli are not consciously perceived under conditions of continuous flash suppression (CFS). The authors measured the activity of a population of V1 neurons at single neuron resolution in awake fixating monkeys while they viewed dichoptic stimuli that consisted of an oriented grating presented to one eye and a noise stimulus to the other eye. Under such conditions, the mask stimulus can prevent conscious perception of the grating stimulus. By measuring the activity of neurons (with Ca2+ imaging) that preferred one or the other eye, the authors tested the degree of orientation processing that occurs during CFS.

Strengths:

The greatest strength of this study is the spatial resolution of the measurement and the ability to quantify stimulus representations during CSF in populations of neurons, preferring the eye stimulated by either the grating or the mask. There have been a number of prominent fMRI studies of CFS, but all of them have had the limitation of pooling responses across neurons preferring either eye, effectively measuring the summed response across ocular dominance columns. The ability to isolate separate populations offers an exciting opportunity to study the precise neural mechanisms that give rise to CFS, and potentially provide insights into nonconscious stimulus processing.

Weaknesses:

While this is an impressive experimental setup, the major weakness of this study is that the experiments don't advance any theoretical account of why CFS occurs or what CFS implies for conscious visual perception. There are two broad camps of thinking with regard to CFS. On the one hand, Watanabe et al. (2011) reported that V1 activity remained intact during CFS, implying that CFS interrupts stimulus processing downstream of V1. On the other hand, Yuval-Greenberg and Heeger (2013) showed that V1 activity is, in fact, reduced during CFS. By using a parametric experimental design, they measured the impact of the mask on the stimulus response as a function of contrast and concluded that the mask reduces the gain of neural responses to the grating stimulus. They presented a theoretical model in which the mask effectively reduced the SNR of the grating, making it invisible in the same way that reducing contrast makes a stimulus invisible.

We used multi-class SVM (as suggested by reviewer 3) and a transformer-based model to examine the impact of CFS on the classification of 12 orientations spaced in 15o gaps, which resembles coarse orientation discrimination, as well as on stimulus reconstruction, which resembles stimulus perception necessary for high-level cognitive tasks, respectively. The results suggest that under CFS, an observer may still be able to perform coarse orientation discrimination but not high-level cognitive tasks. These findings provide new insights into the implications of CFS for conscious visual perception from a population decoding perspective.

In the revision, we also added a new paragraph discussing the implications of our findings for the dorsal-ventral CFS hypothesis, as suggested by reviewer 1. We previously presented a gain control model for our neuronal data in a VSS talk. However, we later decided that, since there are already nice models by Heeger and others, it would be better present something more unique and novel (i.e., machine learning results), which has now become a major component of the manuscript. We welcome the reviewer’s comments on this part.

An important discussion point of Yuval-Greenberg and Heeger is that null results (such as those presented by Watanabe et al.) are difficult to interpret, as the lack of an effect may be simply due to insufficient data. I am afraid that this critique also applies to the present study.

We are very much puzzled by the reviewer’s critique. First, our main result is not a null effect. A null effect would mean that CFS masking had no impact on population orientation responses. Instead, we observed a significant suppression or abolished tuning, which clearly indicates a strong effect of dichoptic masking. Second, our findings are based on large neural populations recorded using two-photon imaging, providing extensive sampling and statistical power. Thus, we believe that the reviewer’s critique about “insufficient data” are not applicable to our study.

Here, the authors report that CFS effectively 'abolishes' tuning for stimuli in neurons preferring the eye with the grating stimulus. The authors would have been in a much stronger position to make this claim if they had varied the contrast of the stimulus to show that the loss of tuning was not simply due to masking.

We are sorry that we cannot follow the logic here either. Even if “the mask effectively reduced the SNR of the grating, making it invisible in the same way that (“akin to”, to be more precise according to the abstract of Yuval-Greenberg and Heeger (2013)) reducing contrast makes a stimulus invisible”, it does not necessarily mean that dichoptic masking and contrast reduction are the same process or are based on the same neuronal mechanisms. According to gain control models by Heeger and others, reducing the stimulus contrast decreases the excitatory drive, while dichoptic masking increases the normalization pool via interocular suppression, both of which lower SNR, but are two fundamentally distinct processes.

Therefore, varying the stimulus contrast might reveal a main effect of contrast, and possibly an interaction between contrast and dichoptic masking, but it would neither prove nor disprove the main effect of dichoptic masking.

So, while this is an incredibly impressive set of measurements that in many ways raises the bar for in vivo Ca2+ imaging in behaving macaques, there isn't anything in the results that constitutes a real theoretical advance.

We sincerely hope that the reviewer would have a better judgment after reading our responses.

Reviewer #3 (Public review):

Summary:

In this study, Tang, Yu & colleagues investigate the impact of continuous flash suppression (CFS) on the responses of V1 neurons using 2-photon calcium imaging. The report that CFS substantially suppressed V1 orientation responses. This suppression happens in a graded fashion depending on the binocular preference of the neuron: neurons preferring the eye that was presented with the marker stimuli were most suppressed, while the neurons preferring the eye to which the grating stimuli were presented were least suppressed. The binocular neuron exhibited an intermediate level of suppression.

Strengths:

The imaging techniques are cutting-edge, and the imaging results are convincing and consistent across animals.

Weaknesses:

I am not totally convinced by the conclusions that the authors draw based on their machine learning models.

Thanks for pointing this issue. We have used a new multi-class SVM suggested by the reviewer to reanalyze the data and found similar results, which is detailed later.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) Lines 56-63: "As a result, the dichoptic CFS masking, which is cortical, could be substantially stronger than monocular masking when accounting for the pre-cortical effects of monocular masking." I don't quite understand this argument. Could you please elaborate?

We have revised our writing to address the reviewer’s first major comment, which the current issue is related. The elaboration is highlighted in the paragraph below.

“Two prominent fMRI studies have examined the impact of CFS on V1 activity (Watanabe et al., 2011; Yuval-Greenberg & Heeger, 2013). Watanabe et al. (2011) compared monocular CFS masking (stimulus visible) and dichoptic CFS masking (stimulus invisible), and reported that V1 BOLD responses were largely insensitive to stimulus visibility when attention was carefully controlled. However, using similar experimental design, Yuval-Greenberg and Heeger (2013) observed reduced BOLD responses in V1 under dichoptic masking, suggesting that V1 activity changed with stimulus visibility. They attributed the difference of results between two studies mainly to differences in statistical power (~250 trials per condition vs. ~90 trials per condition). Nevertheless, these studies were not designed to quantify the pure effect of CFS on stimulus-evoked V1 responses, as they contrasted monocular and dichoptic masking conditions to equate stimulus input while manipulating perceptual visibility. In contrast, original psychophysical studies (Tsuchiya & Koch, 2005; Tsuchiya, Koch, Gilroy, & Blake, 2006) demonstrated CFS masking by contrasting the visibility of the target stimulus with and without the presence of dichoptic mask. It is apparent that the pure CFS impact in above fMRI studies would be the difference of BOLD signals between binocular masking and stimulus alone conditions. In other words, the impact of CFS on V1 activity should be larger than what has been reported by Yuval-Greenberg and Heeger (2013).” (lines 55-71)

(2) Line 13 low-level stimulus (properties).

Fixed, thanks.

Reviewer #3 (Recommendations for the authors):

Major comments:

(1) My main comment is regarding the SVM classifiers. The pair-wise (adjacent orientation pairs) decoding approach is unrealistic in my opinion and likely explains the very high accuracies that are reported. I believe that a multi-way classification approach - Linear Discriminant Analysis, Decision Trees, etc. - is needed to draw reasonable conclusions. Even SVMs can be adapted for multi-way classification (e.g., Allwein et al., 2000, J. Machine Learning Research).

Following the reviewer’s advice, we reanalyzed the data using a multi-class SVM with a one-vs-one (OvO) scheme to classify 12 orientations (Allwein et al., 2000), which yielded similar results.

“For orientation classification, we trained an all-pair multiclass support vector machine (SVM) classifier to discriminate 12 orientations based on trial-by-trial population neural responses from all trials (Allwein, Schapire, & Singer, 2000). Decoders for different FOVs, ipsilateral/contralateral target presentations, and baseline vs. CFS conditions were trained separately. Under the baseline condition, the decoders achieved mean classification accuracies of 89.5 ± 2.0% and 91.5 ± 2.1% across ipsilateral and contralateral eye conditions in Monkeys A and B, respectively, in contrast to a chance level of 8.3% (1 out of 12). Under CFS, decoding accuracy slightly decreased in Monkey A (81.7 ± 1.9%) but remained stable in Monkey B (90.4 ± 2.1%, Fig. 3A). These results suggest that under CFS, there is still sufficient information for coarse orientation discrimination, even for Monkey A whose V1 neuronal responses were substantially suppressed.” (lines 171-181)

(2) The inconsistent modeling results (Figure 3E,F) are puzzling and need to be adequately addressed.

SSIM and orientation error in original Fig. 3E, F measured the same reconstruction quality, but these two indices go in opposite directions for the same modeling results. To avoid confusion, we have removed the orientation error metric and now only report SSIM.

“We used a structural similarity index (SSIM) (Brunet, Vrscay, & Wang, 2012) to quantify the reconstruction performances. Across the grating-presenting ipsilateral and contralateral eyes, the baseline models reconstructed the grating with median SSIMs of 0.52 and 0.61 for the two FOVs of Monkey A, and 0.57 and 0.63 for the two FOVs of Monkey B, respectively, while the corresponding SSIMs for the CFS models were 0.16 and 0.19 for Monkey A, and 0.55 and 0.53 for Monkey B (Fig. 3E).” (lines 200-206)

Minor points:

(1) The phrase "perceptual consequences" in the title is somewhat strong and possibly misleading, since there are no behavioral measures in this study.

To address this concern from this reviewer and reviewer 1, we now focus on the impact of CSF on population orientation coding rather than perceptual consequences, which is more appropriate describing our modeling results. For example, we changed the title to: “Continuous flashing suppression of neural responses and population orientation coding in macaque V1“. Other changes are also made throughout the manuscript accordingly.

(2) Figure 4: Panel "F" is not marked in the figure.

Fixed, thanks.

Before students paraphrase, I often have them annotate the reading using the collaborative annotation tool Hypothesis. This allows students to highlight key claims and discuss them with classmates, helping them focus on understanding the author’s ideas before attempting to rewrite them

authors explain that handwriting is being replaced by digital tools in classrooms and how it raises concerns about how the shift to typing might affect learning and brain development

emphasizes that handwriting and typing engage the brain differently and students benefit most when they know when to use handwriting versus typing when approaching a learning task

typing did not produce the same beneficial brain activity patterns as handwriting which supports the argument that handwriting is more beneficial for learning and academic performance

explanation of figure that displays how handwriting creates strong brain network connections in important cognitive frequency ranges and how these patterns suggest that handwriting engages the brain more deeply than typing

Researchers used high-density EEG technology to measure detailed brain activity during handwriting and typing which is a scientific method that provides reliable evidence about how writing methods affect neural processes

study directly compares handwriting and typing by having participants complete the same task using both methods, this controlled comparison allows researchers to measure how each writing method affects brain activity

Handwriting requires precise and detailed movements that engage the brain more deeply than pressing keys which is why deeper engagement can help students learn and retain information more effectively

explains that the precise hand movements required for handwriting play an important role in learning & how these complex motor actions activate brain processes that help students understand and retain information

encourages schools to continue teaching handwriting because it helps develop brain connections that support effective learning

researchers found that handwriting produces more complex brain connectivity than typing suggesting handwriting engages the brain more deeply during learning tasks

study that compares handwriting and typing by measuring brain activity in students using EEG technology, a scientific approach that provided evidence about how each writing method affects the brain

authors introduce the growing shift from handwriting to digital typing and explain why it is important to study how this change affects brain function also establishes the relevance of researching handwriting’s role in learning

handwriting creates stronger connections between different brain regions than typing and this stronger brain connectivity helps students process information more effectively and supports learning

This suggests that dhimmis voluntarily chose Muslim courts and were not forced to rely solely on their own communal courts.

The author aims to challenge the idea that dhimmis were always legally separated and discriminated against by showing how they actually experienced justice in Muslim courts.

detta förklarar förra påståendet.

현 최고매수제안가보다 더 높은 금액으로만 수정 가능. 내용 추가

Discover how AI-enabled sales coaching tools help remote teams analyze conversations, improve call quality, and deliver measurable performance gains.

Massive Divergence: One looks flat while the other rockets up.

Perfect Correlation: They appear to move in sync.

Dominance: One appears to stay "above" the other.

Total Stagnation: Both appear as flat, parallel lines.

Society has "umbrella-ed" a lot of categories to make it more digestible to the readers, but often it makes other fall through the cracks. When doing qualitative data would it be best for us to gain as much demographic information as possible to ensure that there is no erasure?

Should this be co-constructed? Should inquiry and all future researcher ask the subjects definitions before a study begins? Can this be done with just observer data? or all data?

Something accepted as authorized, standard, or orthodox, often representing the definitive version of a rule, text, or form

Two types of knowledge and discourse

Endogamy contributes to language diveristy by acting as a chamber for preserving linguistic identities, whereas exogamy significantly promotes to diversity in languages, multinguilism and bilingualism due to its polyethnic beliefs of marriage in different ethnicities.

Men did not have as much restrictions as women. The girls who got married outside of her village was considered insincere.

Women struggled when they got married outside of their clans or villages because they lacked family support and would face violence and abuse.

Easier accessibility of transport promoted to the almost extintion of local use of traditional languages as the Russian language demonstrated ease of communication between people. barter trade was no longer useful, as the Russians brought in new ways of handling the economy.

Learning multiple languages was a powerful tool for trading, socializing and for convenience. It was not necessary for the villagers to have a global language because they were committed to one ethnic identity

though its a small region, daghestan has fascinating facts of how of preserves linguistic identity through cultural beliefs beliefs and inter-marriages.

the newly Weds learn the language from their in laws and that's what's passed to their children; thereby continuing language diveristy

marriage between the same group of ethnic identities

I do kind of wish NPR could be a little more heavy-handed than this Editorially Flavored Juxtaposition

Society values standardized English because of how much it has been reinforced.

p. 9: The author also contributes to this problem

Paragraph 5: Learning standardized English can take many years. One's ability at a language shouldn't measure their intellect.

By having a research paper it can show your audience what you have learned and what it took to learn the information about your topic.

When writing a thesis it is important to show that you are confident in what you are, because you are also trying to convince your reader in what you are writing about.

A thesis is a small description explaining your argument to your readers.

One influential philosophical approach to understanding happiness is utilitarianism, which argues that moral decisions and public policies should aim to maximize overall happiness. This approach treats happiness as a central measure of well-being and uses it to evaluate the consequences of actions and laws. Utilitarian philosophers claim that ethical decisions should focus on outcomes rather than intentions, emphasizing the idea that what matters most is how actions affect people’s overall well-being. For example, one explanation of utilitarian thinking states that policies should be directed at “the greatest happiness for the greatest number” (“Act and Rule Utilitarianism”). This principle suggests that moral choices should consider the well-being of everyone affected, not just individuals or specific groups. In other words, decisions should be evaluated by how much happiness or well-being they create for society as a whole. Another source further explains that utilitarianism evaluates actions based on their consequences, stating that “Utilitarianism is a form of consequentialism because it rests on the idea that it is the consequences or results of actions, laws, policies, etc. that determine whether they are good or bad, right or wrong” (“Act and Rule Utilitarianism”). This evidence highlights how utilitarianism connects morality directly to the outcomes produced by decisions. When actions increase overall well-being, they are considered morally justified, but if they decrease happiness, they may be viewed as ethically problematic.

Research on happiness also supports the idea that happiness can be studied and used to evaluate human well-being. One article explains that “‘Happiness’ is conceptualized as ‘subjective appreciation of life as a whole’ in this article” (“The Death Penalty and Happiness: Evidence from US States”). This definition shows that happiness is not just a temporary emotion but a broader evaluation of a person’s life satisfaction and overall experience. By measuring how people perceive their lives, researchers can better understand whether social policies and conditions contribute to human well-being. Together, these ideas help explain why utilitarian philosophers emphasize happiness when evaluating ethical decisions. If happiness reflects people’s overall well-being, then maximizing happiness becomes a logical goal for moral reasoning and public policy. At the same time, philosophical discussions about happiness suggest that the concept is complex and can be interpreted in different ways. Some scholars argue that happiness is closely tied to human flourishing and emotional fulfillment, meaning it reflects deeper aspects of a person’s life rather than simple pleasure. These perspectives show that happiness is both a philosophical and practical concept, linking ethical theory with real-world research. By combining utilitarian philosophy with studies of happiness, scholars can better understand how ethical decisions and policies affect human well-being. This synthesis of ideas helps illuminate the central research question of how happiness should be defined and whether it should serve as a guiding principle for moral and political decision-making.

This is why I sided more with White than with Jenkins in week 7 when we were going over both of the authors texts that we had looked at in week 6. White (2010) defends his argument by stating how It is peoples "ATTITUDES" (White, 2010) what makes people "vulnerable to prejudice". White actually starts this article by calling out and opposing Fish's New York Times "What Should Colleges Teach?". Where Fish asks the question "Who could object learning a second language?" to the reader. But White calls fish hypocritical stating " And Fish himself acquiesce to this linguistic prejudice when he come saying that people make theyselves targets for racism if and when they dont write and speak like he do" (White 2010).