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eLife Assessment

This important study provides insights into the role of maternal behavior in the learning and ontogeny of vocalization. It finds evidence that the maternal behavior of sac-winged bats (Saccopteryx bilineata) can influence the learned territorial songs of their pups. The behavioral analyses are convincing, using longitudinal acoustic recordings and behavioral monitoring of individual mother-pup pairs across development and multiple wild bat colonies. The work will be relevant to a broad audience interested in the evolution and development of social behavior as well as sensory-motor learning.

Reviewer #1 (Public review):

Summary:

Fernandez et al. investigate the influence of maternal behavior on bat pup vocal development in Saccopteryx bilineata, a species known to exhibit vocal production learning. The authors performed detailed longitudinal observations of wild mother-pup interactions to ask whether non-vocal maternal displays during juvenile vocal practice, or 'babbling', affect vocal production. Specifically, the study examines the durations of pup babbling events and the developmental babbling phase, in relation to female display rates, as well as pup age and the number of nearby singing adult males. Furthermore, the authors examine pup vocal repertoire size and maturation in relation to maternal display rates encountered during babbling. Statistical models identify female display behavior as a predictor of i) babbling bout duration, ii) the length of the babbling phase, iii) song composition and iv) syllable maturation. Notably, these outcomes were not influenced by the number of nearby adult males (the pups' source of song models) and were largely independent of general maturation (pup age). These findings highlight the impact of non-vocal aspects of social interactions in guiding mammalian vocal development.

Strengths:

Historically, work on developmental vocal learning has focused on how juvenile vocalizations are influenced by the sounds produced by nearby adults (often males). In contrast, this study takes the novel approach of examining juvenile vocal ontogeny in relation to non-vocal maternal behavior, in one of the few mammals known to exhibit vocal production learning. The authors collected an impressive dataset from multiple wild bat colonies in two Central American countries. This includes longitudinal acoustic recordings and behavioral monitoring of individual mother-pup pairs, across development.

The identified relationships between maternal behavior and bat pup vocalizations have intriguing implications for understanding the mechanisms that enable vocal production learning in mammals, including human speech acquisition. As such, these findings are likely be relevant to a broad audience interested in the evolution and development of social behavior as well as sensory-motor learning.

Weaknesses:

The authors qualitatively describe specific patterns of female displays during pup babbling, however, subsequent quantitative analyses are based on aggregate measures of female behavior that pool across display types. Consequently, it remains unclear how certain maternal behaviors might differentially influence pup vocalizations (e.g. through specific feedback contingencies or more general modulation of pup behavioral states).

Comments on revisions:

(1) More detailed analyses of female behavior may be beyond the scope of this study, given the nature of the dataset/recordings. I look forward to the authors' future work on this aspect.

By addressing the important distinction between display number vs. display rate, the authors have provided more direct support for the claim that babbling behavior is related to female displays.

(2) The additional information regarding exposure to adult male song is appreciated.

(3) Added discussion of pup sex differences provides useful context and intriguing speculation about the role of female pup babbling.

(4) The authors' additions have significantly improved the clarity of their acoustic terminology and syllable analyses.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

Fernandez et al. investigate the influence of maternal behavior on bat pup vocal development in Saccopteryx bilineata, a species known to exhibit vocal production learning. The authors performed detailed longitudinal observations of wild mother-pup interactions to ask whether non-vocal maternal displays during juvenile vocal practice or 'babbling', affect vocal production. Specifically, the study examines the durations of pup babbling events and the developmental babbling phase, in relation to the amount of female display behavior, as well as pup age and the number of nearby singing adult males. Furthermore, the authors examine pup vocal repertoire size and maturation in relation to the number of maternal displays encountered during babbling. Statistical models identify female display behavior as a predictor of i) babbling bout duration, ii) the length of the babbling phase, iii) song composition, and iv) syllable maturation. Notably, these outcomes were not influenced by the number of nearby adult males (the pups' source of song models) and were largely independent of general maturation (pup age). These findings highlight the impact of non-vocal aspects of social interactions in guiding mammalian vocal development.

We thank Reviewer 1 for the time and effort dedicated to the revision of our study. The suggestions for the revision of our manuscript were very helpful and have improved our manuscript considerably. 

Strengths:

Historically, work on developmental vocal learning has focused on how juvenile vocalizations are influenced by the sounds produced by nearby adults (often males). In contrast, this study takes the novel approach of examining juvenile vocal ontogeny in relation to non-vocal maternal behavior, in one of the few mammals known to exhibit vocal production learning. The authors collected an impressive dataset from multiple wild bat colonies in two Central American countries. This includes longitudinal acoustic recordings and behavioral monitoring of individual mother-pup pairs, across development.

The identified relationships between maternal behavior and bat pup vocalizations have intriguing implications for understanding the mechanisms that enable vocal production learning in mammals, including human speech acquisition. As such, these findings are likely to be relevant to a broad audience interested in the evolution and development of social behavior as well as sensory-motor learning.

We thank reviewer 1 for this assessment. 

Weaknesses:

The authors qualitatively describe specific patterns of female displays during pup babbling, however, subsequent quantitative analyses are based on two aggregate measures of female behavior that pool across display types. Consequently, it remains unclear how certain maternal behaviors might differentially influence pup vocalizations (e.g. through specific feedback contingencies or more general modulation of pup behavioral states).

In analyzing the effects of maternal behavior on song maturation, the authors focus on the most common syllable type produced across pups. This approach is justified based on the syllable variability within and across individuals, however, additional quantification and visual presentation of categorized syllable data would improve clarity and potentially strengthen resulting claims.

We agree that our analysis of maternal behaviour does not investigate potential contingencies between particular maternal behavioural displays and pup vocalizations (e.g. particular syllable types). Our data collected for this study on maternal behaviour includes direct observations, field notes and/or video recordings. In the future, it will be necessary to work with high-speed cameras for the analysis of potential contingencies between particular maternal behavioural displays and specific pup vocalizations, which allow this kind of fine-detailed analysis. We have planned future studies investigating whether pup vocalizations elicit contingent maternal responses or vice versa. In the revision of our manuscript, we have included a comment pointing out that this special behaviour will be investigated in greater detail in the future. 

As suggested by reviewer 1, in our revised manuscript we have included more information on methods to improve understandability. In particular, we have:

-presented more information on different steps of our acoustic analyses

-provided additional and clearer spectrogram figures representing the different syllable types and categorizations 

-changed the figures accompanying our GLMM analyses following the suggestion of Reviewer 1

Reviewer #2 (Public review):

Summary:

This study explores how maternal behaviors influence vocal learning in the greater sac-winged bat (Saccopteryx bilineata). Over two field seasons, researchers tracked 19 bat pups from six wild colonies, examining vocal development aspects such as vocal practice duration, syllable repertoire size, and song syllable acquisition. The findings show that maternal behaviors significantly impact the length of daily babbling sessions and the overall babbling phase, while the presence of adult male tutors does not.

The researchers conducted detailed acoustic analyses, categorizing syllables and evaluating the variety and presence of learned song syllables. They discovered that maternal interactions enhance both the number and diversity of learned syllables and the production of mature syllables in the pups' vocalizations. A notable correlation was found between the extent of acoustic changes in the most common learned syllable type and maternal activity, highlighting the key role of maternal feedback in shaping pups' vocal development.

In summary, this study emphasizes the crucial role of maternal social feedback in the vocal development of S. bilineata. Maternal behaviors not only increase vocal practice but also aid in acquiring and refining a complex vocal repertoire. These insights enhance our understanding of social interactions in mammalian vocal learning and draw interesting parallels between bat and human vocal development.

We thank reviewer 2 for his/her time and effort dedicated to the revision of our study. The suggestions were very helpful in improving our manuscript. 

Strengths:

This paper makes significant contributions to the field of vocal learning by looking at the role of maternal behaviors in shaping the vocal learning phenotype of Saccopteryx bilineata. The paper uses a longitudinal approach, tracking the vocal ontogeny of bat pups from birth to weaning across six colonies and two field seasons, allowing the authors to assess how maternal interactions influence various aspects of vocal practice and learning, providing strong empirical evidence for the critical role of social feedback in non-human mammalian vocal learners. This kind of evidence highlights the complexity of the vocal learning phenotype and shows that it goes beyond the right auditory experience and having the right circuitry.

The paper offers a nuanced understanding of how specific maternal behaviors impact the acquisition and refinement of the vocal repertoire, while showing the number of male tutors - the source of adult song - did not have much of an effect. The correlation between maternal activity and acoustic changes in learned syllable types is a novel finding that underscores the importance of non-vocal social interactions in vocal learning. In vocal learning research, with some notable exceptions, experience is often understood as auditory experience. This paper highlights how, even though that is one important piece of the puzzle, other kinds of experience directly affect the development of vocal behavior. This is of particular importance in the case of a mammalian species such as Saccopteryx bilineata, as this kind of result is perhaps more often associated with avian species.

Moreover, the study's findings have broader implications for our understanding of vocal learning across species. By drawing parallels between bat and human vocal development (and in some ways to bird vocal development), the paper highlights common mechanisms that may underlie vocal practice and learning in both humans and other mammals. This interdisciplinary perspective enriches the field and encourages further comparative studies, ultimately advancing our knowledge of the evolutionary and developmental processes that shape vocal productive learning in all its dimensions.

We thank reviewer 2 for this assessment. 

Weaknesses:

Some weaknesses can be pointed out, but in fairness, the authors acknowledge them in one way or another. As such, these are not flaws per se, but gaps that can be filled with further research.

Experimental manipulations, such as controlled playback experiments or controlled environments, could strengthen the causal claims by directly testing the effects of specific maternal behaviors on vocal development. Certainly, the strengths of the paper will be consolidated after such work is performed.

The reliance on the number of singing males as a proxy for social acoustic input. This measure does not account for the variability in the quality, frequency, or duration of the male songs to which the pups are exposed. A more detailed analysis of the acoustic environment, including direct measurements of song exposure and its impact on vocal learning, would provide a clearer understanding of the role of male tutors.

Finally, and although it would be unlikely that these results are unique to Saccopteryx bilineata, the study's focus on a single species limits at present the generalizability of some of its findings to other vocal learning mammals. While the parallels drawn between bat and human vocal development are intriguing, the conclusions will be more robust when supported by comparative studies involving multiple species of vocal learners. This will help to identify whether the observed maternal influences on vocal development reported here are unique to Saccopteryx bilineata or represent a broader phenomenon in chiropteran, mammalian, or general vocal learning. Expanding the scope of research to include a wider range of species and incorporating cross-species comparisons will significantly enhance the contribution of this study to the field of vocal learning.

Thank you for your suggestions and comments. 

Regarding your main comment 1: In the future, we plan to implement temporary captivity experiments to investigate how maternal behaviours affect pup vocal development. This study provides the necessary basis for conducting future playback studies investigating specific behaviours in a controlled environment.

Regarding your main comment 2: We completely agree that the number of singing males only represents a proxy for acoustic input that pups receive during ontogeny. In the future, we plan to investigate in detail how the acoustic landscape influences pup vocal development and learning. This will include quantifying how long pups are exposed to song during ontogeny and assessing the influence of different tutors, including a detailed analysis of song syllables of the adult tutors to compare it to vocal trajectories of song syllables in pups. 

Regarding your main comment 3: We also fully agree that it is unlikely that these results are unique to Saccopteryx bilineata. We are certain that other mammalian vocal learners show parallels to the vocal development and learning processes of S. bilineata. Especially bats are a promising taxon for comparative studies because their vocal production and perception systems are highly sophisticated (due to their ability to echolocate). The high sociability of this taxon also includes a variety of social systems and vocal capacities (e.g. regarding vocal repertoire size, vocal learning capacities, information content, etc.) which support social learning and social feedback – as shown in our study. 

As suggested, in our revised manuscript we have includes information on the validation of the ethogram. Furthermore, we have corrected all the spelling mistakes – thank you very much for pointing them out!

Recommendations for the authors:  

Reviewer #1 (Recommendations for the authors):

The following comments and suggestions are offered to improve clarity and strengthen support for the paper's main claims.

(1) Female displays as feedback:

a) The authors rather broadly describe maternal behavior as feedback based on its occurrence during pup babbling. Feedback typically entails some degree of response contingency, which is not explicitly established here. Although the authors qualitatively describe a variety of female displays that only occur within the babbling context, they also state that "all these behaviors could occur singly or in an interactive way" (Line 102). The authors go on to use aggregate counts of these diverse female displays in their analyses. It would of course be interesting to know whether distinct female displays are evoked differentially by pup behavior and whether specific female behaviors, in turn, predict subsequent pup vocalizations. A display-specific approach might also reveal more about the mechanisms by which the female behavior shapes babbling (e.g. specific reinforcement signals vs. more graded social facilitation or 'audience effect'). However, even without identifying such finegrained contingencies, the main text should at least mention the results shown in Figure 1A. Namely, that pups initiate ~80% of interactive behavioral sequences, suggesting that subsequent maternal displays are likely to be pup-contingent responses (i.e. feedback) and not simply co-occurring behavior.

We fully agree with Reviewer 1 that it would be very informative to investigate whether distinct female displays are evoked differentially by pup behavior, such as specific syllables within babbling. Or conversely, whether specific female behaviors precede particular pup vocalizations. For this study, we documented maternal behavior through direct observations, field notes, and/or video recordings. However, to capture potential contingencies between specific maternal behavioral displays and vocalization occurring in the millisecond range, other data collection methods (e.g. high-speed camera) will be required in the future. 

Related to this, we have included the following statements (see below). Statement 1 also cites a very recent study in zebra finches, demonstrating that female calls can promote song learning success (Bistere et al. 2024, line 57, lines 304-305). 

Lines 297-305: This finding serves as an initial indication that non-vocal interactions with the mother may influence a pup´s individual learning trajectories. Future studies will focus on the relationship between acoustic change, maternal feedback, and learning success, specifically investigating contingencies between particular pup vocalizations and maternal displays in natural settings. Playback experiments are an additional approach to test the impact of contingency on vocal learning. For example, one study in zebra finches demonstrated that contingent non-vocal maternal feedback affects imitation success (Carouso-Peck & Goldstein, 2019), while another recent study found that female calls can promote song learning but the role of contingency remains to be determined (Bistere et al., 2024).  

Lines: 332-334: This might also apply to S. bilineata where pups initiated ~ 80% of social interactions, suggesting that maternal feedback is likely influenced by the pup´s vocal practice.  

b) The authors claim that the number of maternal displays during babbling predicts the duration of babbling bouts (Figure 1D). I find this analysis - and others based on the raw number of behaviors during babbling - difficult to interpret given that the raw number of displays may depend upon the duration of the babbling bout over which they are counted. In other words, might the number of displays reflect the fact that more displays can occur within the interval of longer babbling bouts? It would be relatively straightforward to minimize this potential confound by testing whether female display *rates* predict longer bouts.

We calculated the display rates (maternal displays per bout duration) and conducted a GLMM (the same analysis after log-transformation and scaling) like in our original manuscript (model 1).  

GLMM

summary(vocpracf)

Generalized linear mixed model fit by maximum likelihood (Laplace Approximation) ['glmerMod']  Family: Gamma  ( log )

Formula: bout_dur ~ age.z + behavioural_quotient.log.z + nomales.z + (1 | ID) Data: set1

Author response table 1.

Author response table 2.

Author response table 3.

Author response table 4.

Author response table 5.

Interpretation: Our analysis in the original manuscript shows that the bout duration increases with number of maternal displays. As reviewer 1 points out: more time offers more opportunities for the mother to show displays. The number of displays in longer bouts could just reflect that more displays are possible in a longer period. This could be a potential confounding factor. However, our analysis of display rates as an explaining factor shows that the relationship between bout duration and display rate is negative. This means that in longer bouts the displays increase (as seen in the first scenario), but they happen less frequently per time unit. This could indicate that in longer bouts, the mother takes breaks or longer periods of time between each display, which decreases the frequency of displays. This minimizes the risk of a potential confound, as it shows that the rate of displays tends to decrease rather than increase in longer bouts. In summary: The display rate does not appear to ‘favour’ longer bouts, as longer bouts are associated with a lower display rate. This speaks against the hypothesis that the number of displays only increases due to the longer bout duration. This also means that our analyses, which show that maternal displays influence song syllable production, are not biased or confounded by the bout duration. This suggests that maternal behaviour is targeted and selective, and represents a potentially contingent reaction to the pup´s vocal production, and is not simply determined by the duration of a bout.

We added this analysis in our supplementary material (Table S2) and pointed this out in the revision of our main manuscript (lines 136-138). 

c) The introduction states that "Pup babbling is not tied to a specific function." (Lines 75-78). This may be an important point worth exploring with this unique data set. For example, the termination of a babbling bout is defined in some cases by the onset of nursing. Have the authors (or others) tested whether babbling elicits nursing behavior? If so, this may represent a reinforcement mechanism that affects babbling rates and subsequent song outcomes. Similar functional shifts in developing vocal behavior have been reported in male chipping sparrows, in which juvenile begging calls - which initially elicit parental feeding behavior - can later be incorporated into 'sub-song' (i.e. babbling) during the development of courtship song (Lui, Wada, Nottebohm, PLOS ONE, 2009).

Thank you for pointing out this interesting study on chipping sparrows! 

To address your question: Strauss et al. (2010) conducted a study on pup and maternal behaviors, demonstrating that babbling did not consistently result in nursing.  When denied care, pups often returned to resting or grooming, a pattern we also observed in our study. While nursing might provide an additional reinforcement mechanisms, it is not the cause that evokes babbling – this is what we mean by stating “pup babbling is not tied to a specific function”. Babbling is not a begging behavior as described by Lui et al. 2009. As mentioned in the review of ter Haar et al. 2021, babbling differs structurally from begging in that it is composed of both adult-like and juvenile syllables and lacks context specificity. To solicit care (i.e. begging) pups produce several isolation calls in a fast repetitive manner. We added a more detailed explanation to make this distinction clear (lines 79-83).

Another interesting fact and probably more comparable to the study of the chipping sparrows – in which begging calls are incorporated into subsong practice – might be the isolation call syllables of S. bilineata. Directly after birth, S. bilineata pups produce multisyllabic isolation calls (see Knörnschild & von Helversen 2008, Knörnschild et al. 2012, Fernandez & Knörnschild 2017) that serve to solicit maternal care. For the first 2.5 weeks, pups only produce innate vocalizations, including echolocation and isolation calls (Fernandez et al. 2021). During the babbling phase, the syllables encoding the individual (and group) signature of the isolation call are also incorporated into babbling bouts. The production of isolation calls might also mark an initial step in the vocal learning process. However, in contrast to the subsong of chipping sparrows, babbling bouts in S. bilineata also include syllables acquired through vocal imitation. Thus, although we find similarities in vocal practice and development between chipping sparrows and S. bilineata, there are also distinct differences. 

(2) Are pups exposed to more male songs when the mother is present?

The number of singing males in each colony was used as a reasonable proxy for the amount of social acoustic input. However, I wonder if pups are exposed to more adult male songs when the mother is present and, relatedly, if females tend to remain present for longer if a pup is babbling (potentially increasing its exposure to male songs during the babbling phase).

The mother is always present when males are singing. In S. bilineata, males predominantly engage in territorial song twice daily: at dusk and dawn. After foraging at night, territorial singing males are the first to return to the roost, and females will only return when they hear male song. Pups are either attached to the mother´s belly or – when growing older – will fly into the roost followed by the mother. In the evening, males sing approximately half an hour before leaving for foraging. Females will usually leave first, followed by their pups, and males leave last. Hence, females/mothers are always present when pups are exposed to male acoustic input.  

(3) Pup sex differences:

The authors test for sex differences within a subset of pups and briefly mention that vocal development is considered in both males and females. This presumably means that female pups also exhibit vocal imitation of adult male territorial songs, even though they only produce these vocalizations during the babbling phase, after which they stop singing entirely. If so, this would, to my knowledge, be a unique phenomenon among vocal learners and would be interesting to discuss in greater detail.

We followed your recommendation and discussed this topic in greater detail. We included the following part in our discussion (lines 257-269): An intriguing aspect of this species is that, unlike most song-learning songbird species, female pups show no differences from males in babbling behavior and vocal development (Fernandez et al. 2021). This study corroborated this finding: female pups received the same maternal feedback, and their song syllable imitation did not differ in any way from male pups (as observed as well in Knörnschild et al. 2010). This phenomenon is rare among vocal learners and raises the question of why female pups match male vocal development despite not using the learned vocalizations later in life. One potential explanation might lie in the function of the territorial song for adult females: it serves as an acoustic signal to help females locate new suitable colonies after dispersal. The territorial song exhibits different dialects, with females showing a preference for local over foreign dialects (Knörnschild et al., 2017). The own early practice and production of song might enhance the ability to evaluate male song and support mating decisions.

(4) Characterization of song syllables:

The authors explain their acoustic analyses in detail within the methods, however, descriptions of the syllable classification procedures and acoustic movement analyses need to be presented more clearly in the main text, so readers unfamiliar with bioacoustics or previous work can follow the logic. Also, given the qualitative descriptions of the data and the two spectrogram examples provided (Figures 2 and S1), it is difficult for the reader to fully evaluate the suitability and output of these critical procedures.

Suggestions:  

- Qualitative descriptions of syllable characteristics (i.e. buzz, pulse, trill, ripple, gap, smeared noisy, precursor syllable, mature syllable, adult-like syllable, early vs. late babbling phase, syllable name, etc) should all be clearly-labeled in example spectrograms and used consistently, without using different terms interchangeably (e.g. mature vs. adult-like).

We understand that we should provide a clearer description of the various terms essential to understanding this study. We added a “Terminology” box (line 158) to the main manuscript, defining the acoustic terms we are using throughout our study. Additionally, we enhanced Figure S1 by providing more detailed information on the spectrogram that displays the five distinct song syllable types. Moreover, we included an additional spectrogram in the supplementary material (Fig. S2) displaying examples of precursor and mature syllables for syllable B2. In the method section, “The acoustic movement during ontogeny”, we added a sentence clarifying the terms “early” and “late babbling phase” (Lines 605-606). 

- Show as you tell. Plot the data, at least from a representative pup, for each major step in the analyses (labeled spectrogram, PCA plots with distinct syllable clusters, high vs. low versatility, precursor vs. mature variants, early vs. late syllables with Euclidean distances between centroids and relation to "generic" adult male syllables, etc.)

To illustrate the acoustic analysis more comprehensively, we have made the following additions:

-we included a Figure (Fig. S3) in the supplementary materials showing an excerpt of a babbling bout with labelled syllables to illustrate how we analyzed a) total song syllable count per bout, b) versatility per bout, and c) the number of precursor versus mature B2 syllables (the most common syllable type).

-Additionally, we included a spectrogram with three exemplary B2 syllables to illustrate the acoustic parameter extraction with Avisoft SASLab Pro software for subsequent analysis of vocal change during development (Fig. S4 A).

Lastly, we included a DFA for one of the colonies with three exemplary pups to illustrate how we calculated each pup's acoustic change during ontogeny (Fig. S4 B). 

(5) Minor Comments and Corrections:

- Modeled data are log-transformed, however, the raw data are plotted on linear scales, and in most cases, data points are densely clustered and overlapping at lower values. Plotting the data on log scales would likely aid visibility.

We appreciate this suggestion and changed the plots accordingly. 

- Figure 1E displays 18 data points, (legend says n=19).

The legend is correct; the figure includes 19 data points. Two mothers have the same activity score, so their points are at the same location and it looks like there are only 18 data points. 

- Line 482: Is "VCL" media player meant to refer to "VLC" player?

Yes, thank you for spotting that. We corrected it.  

Reviewer #2 (Recommendations for the authors):

I have only a couple of comments:

- Perhaps it would be useful to briefly go over the validation used for the ethogram in Table S1.

The behaviors listed in the ethogram were defined based on Strauss et al. (2010) and expanded based on our own observations. For consistency, we developed these definitions and trained the students analyzing behavioral data for this study. During the training phase, we validated their analyses until the inter-observer-reliability reached 100% (lines 507-508).  

- The paper seems to be generally written in American English, yet there are some instances of British English spelling, e.g. "standardised"/"standardisation": table 1, table 2, lines 143, 228, 524, 525, 531, 546, 547, 554, 560, 561.

Thank you for spotting these errors, we corrected them.  

- Line 343: "at libitum" should be "ad libitum".

Thank you for spotting this error. We corrected it.

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second page note

test annotation

This situates Islam within the broader historical context of colonialism and the formation of modern nation-states.

This highlights the tension between traditional Islamic practices and modernist calls for reform or complete overhaul.

This quote depicts a important perspective on Islam's role in traditional societies and how it has been viewed by reformists and modernists.

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apprentissage inconscient

Oui c'est vrai. On aura de plus en plus besoin

De plus en plus présent dans les société aujourd'hui : Maintenant les écrans sont: - au coeur de nos sociétés - au coeur de nos vie - au coeur de l'administration - au coeur de la scolarité

Quand le milieu social-culturelle et économique le permette sinon c'est plutôt l'inverse

Peu d'outil numérique, oui en effet. Les causes sont aussi du au manque de moyen des écoles. Les écoles en milieu rural on moins de moyen : - moins d'éléves donc moins de moyens de financement car le budget est souvent attribué par des élèves - certain établissement ont du mal à financer le matériel

Peu de formations, les enseignants au vu des restrictions budgétaires.

Il existe également des difficulté de recrutement car le milieu rural attire moins de monde que les côtes, les îles ou encore le milieu urbain. Comme il y a peu d'éléve beaucoup moins d'enseignants.

discipline récente, depuis uniquement une quarantaine d'années

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"Migration has proved to be a powerful force for development, improving the lives of hundreds of millions of migrants, their families, and the societies in which they live across the world." Thought: The author is emphasizing that migration is not just about movement—it’s a key driver of economic and human development. This connects directly to today’s inquiry question about the role of migration in shaping global societies. The author is trying to challenge the notion that migration is mainly a burden and instead reframe it as an opportunity for growth, both for individuals and countries. I think this sets up the framework for the rest of the report, which seems focused on showing how migration, if managed well, can benefit all parties involved.

"Migration is necessary for all countries... Yet moving has costs that most poor people cannot afford." Question: If poor people can't afford to migrate, how can migration still be considered a tool for reducing global inequality? If migration is mostly an option for the middle class, are we leaving out the poorest populations who might benefit the most? This complicates the idea that migration is an equalizing force. It also made me wonder what policies could lower these barriers for the very poor and make migration more accessible. This ties to the inquiry question by pushing us to ask: Who gets to migrate, and why? Are the people who migrate the ones who truly need to, or the ones who simply can?

"Migration is also just one of many forces transforming societies in an age of rapid change, alongside modernization, secularization, technological progress, shifts in gender roles and family structures, and the emergence of new norms and values." Epiphany: I’d never thought about migration as part of a broader set of global transformations. We usually hear about migration in isolation—as a “crisis” or a policy issue—but seeing it grouped with tech, gender norms, and modernization helped me realize it’s part of a much bigger social shift. That changes how I think about it—migration is not just reacting to change, it is one of the forces driving it. According to the International Organization for Migration (IOM), “migration has become a defining feature of our globalized world” and is increasingly interwoven with “climate change, economic restructuring, and demographic change.” This supports the epiphany by showing that migration is embedded in other global processes—not separate from them. That changes how we should approach it: not as a standalone challenge, but as part of broader transformation strategies. https://www.iom.int/global-compact-migration

Thoughts: "Migration has proved to be a powerful force for development, improving the lives of hundreds of millions of migrants, their families, and the societies in which they live across the world" Here, it is emphasized that migration is not just a humanitarian issue but a major driver of development. This directly relates to today’s inquiry question by stating that migrants contribute economically and socially to both their origin and destination countries. It suggests that properly managed migration policies could enhance development globally—especially when matched with trade and labor demands.

Question: "Responsibility-sharing can also be part of broader bilateral negotiations, such as on trade access under the Jordan Compact or invesstment under the Ethiopia Job Compact." This raises an important question: How effective are trade agreements like the Jordan Compact in improving conditions for migrants and refugees? Do these deals prioritize development outcomes, or are they primarily political tools? This section connects to the inquiry by highlighting how free trade agreements can influence migration policies and potentially support better development strategies. related article: https://www.cgdev.org/blog/jordan-compact-three-years-on

Epiphanie: "Regardless of politics, wealthy countries will need foreign workers to sustain their economies and honor their social commitments to older citizens." This statement made me rethink migration not just as a social issue but as a demographic and economic necessity. With aging populations in many high-income countries, migration becomes essential for maintaining labor forces. It ties directly to how both migration and free trade (in labor) are vital for sustainable development, especially in countries facing workforce shortages.

What would classify as good and bad trusts in this case? And what was necessary? What was the criteria that Roosevelt followed in this case?

TEST: I chose x as the dimesions for reason Y

Alt text for image below: 2024 Mazda CX-30 in the Carbon Turbo trim.

right, because it is just a result of a system that is designed to oppress individual groups. Revolution is the language of the oppressed.

white people probably beat the black people as a form of agency. Violence in opressive regimes is usually not just one sided, just asymmetric. and people just seem so committed to get revenge.

Nongoloza could see how disingenuous this move from the guards was. He immediately gives some BS answer ("lost his footing on the softer ground of sympathy") and then just terrorized the guards from the inside. In other words, his disingenuity was a direct result of the guards'. The language of the oppressed often is just a mirror of the oppressors'.

Too little too late. Nongoloza is not going to change his perception of the guards just because they switch up his treatment. Must start with persuasion. Cannot coerce first and the persuade because then it is obviously disingenuous.

elitism is the embarrassing secret that democracy hides?

the so called "class war" and the "race war": are tightly intertwined. I am a white guy talking about this, but I do believe that the race war is often wrongly prioritized over the class war. Even though this white guy was his boss, they are still in a very similar economic position. There is a million other factors that influence this point, but largely, the problems that face minorities are often compounded by economic ones. Increasing the well being of the lower classes would ALSO increase the well being of minorities because they are doubly impacted by both racial and economic hardship.

"righteous" violence often goes too far. It is easy to lose your grip on violence tendencies. Is it part of human nature to have this limitless anger that we seemingly cant control? Why is it so easy for us to get carried away?

Violence is instant gratification, cooperation is delayed gratification

why? what makes a soldier truly ready to die for their cause? to become a martyr?

this is what is holding us back in the US. the fact that we don't have an umbrella group to actually represent the lower in the class war. Culture war has made people increasingly divided and a unified coalition cannot emerge because people MUST have their specific interest met or else they will lose interest. There is no semblance of solidarity or patience within US politics. It is an all or nothing mindset.

violence is the language of both oppressors and the oppressed. however this violence is often framed as "revolutionizing" or "senseless" depending on the context. Radicalization following senseless violence leads to a revolutionary spirit within the oppressed. Afghan fighters are radicalized by the senseless violence committed by US soldiers.

This is the crux of my argument

test

I dont think I'd use this word - I think it could be construed as political.

This feels...awkwardly stated? Can we use a word other than "Thwarted"?

I would not use underline - it makes it look like a "link" to other material.

The Connecticut river goes from southern Connecticut up to the Canadian border. The river is the border between Vermont and New Hampshire. Even today, it is a big factor in agriculture.

Religion was such a big deal that the founding fathers discussed it in the constitution. Not only that, but they put it first. Seperation between church and state within the first amendment, long with the freedom of assembly, among others.

Tobacco was one of the most, if not the most, grown crop in the Southern United States and in the colonies. Tobacco was an export of the colonies, and they were a major and expensive crop in the area. It was also closely related to slavery, along with cotton.

Hot take, but I feel like forcing people to join the military is a human rights violation. The military, and being a servicemember, is a dangerous job, and humans have the right to safety. If we do not have enough soldiers for a war, then why are we in the war? Solve it through diplomacy.

Definition: the practice of forcing individuals into military service, particularly naval service, against their will, often using intimidation and coercion

The bacons rebellion was an uprising in Virginia, led by Nathanial Bacon, where Jamestown was burned down. It was fueled by native american related grievences.

It is true that religion and politics have always been closely related. Seperation between church and state. Even today, religion and politics are too close for comfort.

I wonder, how much independence? Were certain colonies given more independence than others? Were some granted less freedom becuase of their beleifs? What about treason?

In the new world, colonists often settled due to religion. Many in Europe saw the new world as a place to spread religion. Columbus said it in his journals that he wanted the natives to be christians. They antagonize the local populations, and forcefully change their religion in the northeast.

Definition: subject to or liable for the payment of tithes, which are typically a portion of income or resources given to a religious organization

That is crazy to me that at that point the colonies were so young. In my life, the states have always just been "very old", but it is insane to me that the colonies were so young! There were people in the colonies that were older than the colonies themselves.

schemata

Test

1 step a day is easy and achievable... there is no excuse not to.

Another quite chilling and dystopian line.

This list made me completely rethink the way I look at those everyday items and skills, and how they can be utilized for good.

This is great point. The war against these communities isn't a new thing at all.

Is meer de callous-unemotional; psychopathie

Informal Education is the type of education zoos supply to visitors.

Seems that there is much overlap between the three education categories.

Añadir rango de valores que puede tomar en el final de esta diapo

sleeping hours

si el de abajo es determine este debería ser compare

by default?

tbl_summary()

distribution

Afegir al bottom

...life is no so easy

Falta la diapo de comiat

The odds of tumour response is 21% higher in the group treated with drug B than in the group treated with drug A.

The incidence of tumour response is 14% higher in the group treated with drug B than in the group treated with drug A.

is

is

Compte amb la direcció dels > <

Cohens d is a standardized effect size for measuring the difference between two group means:

t_test

is

library(HSAUR3) data("WeightLoss")

sleep hours

in sleep hours

Posaria un exemple de càlcul a sota

E_1 = (33*28)/48 = 19.25

Falten subindex i a O i a E.

Explicarà la lectura d'aquests valors? Per fer-ho més fàcil podries posar una frase a sota.

Delete. És molt interessant per nosaltres però diria que irrellevant per ells en un curs com aquest (falta temps per tot arreu).

Delete

Delete

Aquí explicaria de veu que això és una manera de reportar l'efecte observat.

Caldria identificar quina és Welch. Pots posar un sub index a la t $t_{Welch}$

un título aquí que uno es el equal variancies i el otro no?

Falta S1 i S2

en el summary de la presentación de Pau pone between groups

In a dependent sample, the measures are related.

For example, if you take a sample of patients who have had a painkiller and ask them about their pain before and after taking the medicine

Independent samples are samples that are selected randomly so that its observations do not depend on the values other observations.

For example, if the men's group and the women's group are asked about their health status.

before and after

borrar

Feedback is SO important!

I can see this.

Another thing to think about: An assignment that takes one student 30 min might take another 1 hour. This doesnt indicate how smart they are but different people work at different paces.

PERFECT practice makes perfect. We have to ensure students are practicing correctly otherwise misconceptions will form

AGREED!! However- I beleive if upper level students in grade school should be able to get their work done in class with time management, if not, its homework.

I agree depending on what is assigned. Homework that is not essential to a students learning is busy work to me. EX: unecessary packets, word searches, crosswords, or things with little academic thinking involved.

I can totally see how explicit instructions would be helpful regarding homework. This helps cut down the confusion students might have about how to complete their homework which boosts productivity.

I agree that these tools can undermine effective learning if used incorrectly. However, I strongly beleive if we teach students ethical and productive ways to use AI, we set them up for success. After all, our technology world is evolving.

Qui au bureau ?

voyons ce qu'on fait ici.

faut il introduire ici le soutien financier de la chaire, du crihn ?

Ces points me semblent corrects. Il m'a été évoqué l'idée qu'en tant qu'association, il était possible de toucher des droits de la SOFIA lorsqu'un livre est prêté en bibliothèque (ou quelque chose comme ça, je ne suis pas allé creuser). Je ne pense pas qu'il y ait un intérêt à jouer ce jeu là.

je pense que ce sont les types de membre classiques. Je n'ai pas d'avis sur cet aspect. Ces statuts ne définissent pas de rôle ou de pouvoir particulier pour l'un ou l'autre des membres.

À nouveau, est-ce que l'on crée cette association pour avoir une coquille administrative utile ? Ou est-ce que l'on se dote d'une association pour structurer la gouvernance des Ateliers.

Je penche pour le premier.

En lien avec l'annotation précédente sur le nom (article 1).

Si l'on souhaite distinguer l'association du collectif, il peut être pertinent de trouver un autre nom. J'identifie deux aspects :

1. aspect politique : veut-on que l'association colle au plus près des activités éditoriales, en y structurant aussi les décisions éditoriales par exemple, ou préfère-t-on que l'association soit la moins encombrante possible sur les activités du collectif ?
2. aspect pratique : il y a cependant un certain intérêt pratique à ne pas multiplier les noms et les entités. On peut ainsi en jouer comme l'on veut et invoquer ASP en tant qu'asso ou en tant que collectif au gré des besoin. Et cela n'empêche pas d'acter dans les statuts le fait que l'activité éditoriale du collectif Les Ateliers de [sens public] n'est pas définie par l'association Les Ateliers de [sens public], puis d'élaborer au sein du collectif la charte ou la gouvernance que l'on veut.

Qui le demande ?

Est ce que cela vous convient ?

dat dat iets is gestopt

el salt de 0.69 a 31% és una mica heavy per ells, no? Perquè a més en la teoria els dos exemples que poseu són RR més grans que 1. Pots fer-li el càlcul 1-0.69=0.31 -> 31% reduction.

en las diapos los iguales estan al revés

each

for the variables that showed statistically significant differences between waves.

et falta hemoglobin aquí

em sona raro. no seria after?

ídem ggplot d'abans

canvia el codi perquè no et surti a sota -0.2 i 0.2

x=covid_wave, y=age, fill=covid_wave

the. O per no repetir tant, "whether there are differences in age between groups"

diferent de l'a)

ídem, diferent de la a)

et surt identat diferent de l'a) de dalt

the (these em fa l'efecte com si ja n'haguessis parlat abans)

a

variables

Read the

from?

beaucoup moins que les écritures non-latins. Il faudrait rappeler toutefois les avantages d'une langue romanisée dans ces mêmes traitements

les signes diacritiques ne gênent pas la recherche dans la catalogue informatique. à trouver d'autres exemple plus typiques

Il serait intéressant de préciser la situation documentaire avant la numérisation du fonds Indochinois. Les chercheurs internationaux, vietnamiens se confrontaient-il à un maque de ressources ?

bilingues

???

source

source

la note peut être intégrée dans le corps de l'article avec précision de source

citation ?

bien !

L'idée est intéressante mais les arguments ne sont pas assez solides pour la défendre. Le transfert hors de la BnF peut être dû à la répartition des spécialités / de spécialistes aux différentes institutions. Mais on peut signaler que la BnF n'a pas chercher un titulaire, ou un contrat solide pour le traitement du fonds (en citant les cas des répétiteurs et leur travail bénévole ou peu payé etc.) pour défendre votre avis. A préciser comment les collections chinoises et khmers sont mieux intégrées ?

Vos arguments deviennent plus convaincants quand vous citez les sources comme dans ce passage.

pas tout à fait. On peut comprendre dans le sens contraire, ils sont classés dans un fonds distinct parce qu'ils sont prioritaires. D'autres fonds spécifiques sont considéré comme patrimoines nationaux Cf. https://www.bnf.fr/fr/les-fonds-speciaux-du-departement-litterature-et-art

source

source

source ?

source ?

pourquoi ces cotes ? Quelles significations données par la BnF pour ces lettrages ? à citer : https://editionskime.fr/produit/hanoi-paris/

quelle logique ? à expliciter

à expliciter ce qui constitue le dépôt légal d'Indochine. Ceci dit, les ouvrages sont déposés à la fois à la BnF mais aussi à la bibliothèque centrale de Hanoi.

lesquels ? à préciser

à préciser

lequel ? Source ?

pluriel

pourquoi focaliser sur les communautés vietnamiennes ? Il serait intéressant de le clarifier davantage.

Indochinois

source ?

expression maladroite, à reformuler

eLife Assessment

This valuable contribution to the field evaluated the function of the cytoskeletal protein ABBA in mediating key aspects of mitosis of neuronal precursor cells. The authors provide compelling evidence that ABBA interactions with its signaling partners is related to the development of at least some cases of microcephaly — a developmental anomaly associated with intellectual disability and other neurological findings.

Key claim of the article

compare w spillers

??

look into this footnote

quin sentit té que posi un eix x??

it moves (like Jagger)

aquí també es mouuuuu

de l'anterior a aquesta es mou el gràfic cap a baix

as instead of of?

should seguit de usually em sona raro. Are normally described?

maybe : enlloc de .

canviaria el color de la mean perquè es veu molt fluixet

De ancova

to http://github.com/hypothesis/pdf.js-hypothes.is

eLife Assessment

How misfolded proteins are segregated and cleared is a significant question in cell biology, since clearance of these aggregates can protect against pathologies that may otherwise arise. The authors discover a cell cycle stage-dependent clearing mechanism that involves the ER chaperone BiP, the proteosome, and CDK inactivation, but is curiously independent of the anaphase promoting complex (APC). These are valuable and interesting new observations, but the evidence supporting these claims is only partially complete. New experiments and/or toning down the conclusions and highlighting what has not been learned may be appropriate and can then spur more work in the field.

Reviewer #1 (Public review):

Du et al. address the cell cycle-dependent clearance of misfolded protein aggregates mediated by the endoplasmic reticulum (ER) associated Hsp70 chaperone family and ER reorganisation. The observations are interesting and impactful to the field.

Strength:

The manuscript addresses the connection between the clearance of misfolded protein aggregates and the cell cycle using a proteostasis reporter targeted to ER in multiple cell lines. Through imaging and some biochemical assays, they establish the role of BiP, an Hsp70 family chaperone, and Cdk1 inactivation in aggregate clearance upon mitotic exit. Furthermore, the authors present an initial analysis of the role of ER reorganisation in this clearance. These are important correlations and could have implications for ageing-associated pathologies. Overall, the results are convincing and impactful to the field.

Weakness:

The manuscript still lacks a mechanistic understanding of aggregate clearance. Even though the authors have provided the role of different cellular components, such as BiP, Cdk1 and ATL2/3 through specific inhibitors, at least an outline establishing the sequence of events leading to clearance is missing. Moreover, the authors show that the levels of ER-FlucDM-eGFP do not change significantly throughout the cell cycle, indicating that protein degradation is not in play. Therefore, addressing/elaborating on the mechanism of disassembly can add value to the work. Also, the physiological relevance of aggregate clearance upon mitotic exit has not been tested, nor have the cellular targets of this mode of clearance been identified or discussed.

Reviewer #2 (Public review):

This paper describes an interesting observation that ER-targeted misfolded proteins are trapped within vesicles inside nucleus to facilitate quality control during cell division. This work supports the concept that transient sequestration of misfolded proteins is a fundamental mechanism of protein quality control. The authors satisfactorily addressed several points asked in the review of first submission. The manuscript is improved but still unable to fully address the mechanisms.

Strengths:

The observations in this manuscript are very interesting and open up many questions on proteostasis biology.

Weaknesses:

Despite inclusions of several protein-level experiments, the manuscript remained a microscopy-driven work and missed the opportunity to work out the mechanisms behind the observations.

Reviewer #3 (Public review):

This paper describes a new mechanism for the clearance of protein aggregates associated to endoplasmic reticulum re-organization that occurs during mitosis.

Experimental data showing clearance of protein aggregates during mitosis is solid, statistically significant, and very interesting. The authors made several new experiments included in the revised version to address the concerns raised by reviewers. A new proteomic analysis, co-localization of the aggregates with the ER membrane Sec61beta protein, expression of the aggregate-prone protein in the nucleus does not result in accumulation of aggregates, detection of protein aggregates in the insoluble faction after cell disruption and mostly importantly knockdown of ATL proteins involved in the organization of ER shape and structure impaired the clearance mechanism. This last observation addresses one of the weakest points of the original version which was the lack of experimental correlation between ER structure capability to re-shape and the clearance mechanism.

In conclusion, this new mechanism of protein aggregate clearance from the ER was not completely understood in this work but the manuscript presented, particularly in the revised version, an ensemble of solid observations and mechanistic information to scaffold future studies that clarify more details of this mechanism. As stated by the authors: "How protein aggregates are targeted and assembled into the intranuclear membranous structure waits for future investigation". This new mechanism of aggregate clearance from the ER is not expected to be fully understood in a single work but this paper may constitute one step to better comprehend the cell capability to resolve protein aggregates in different cell compartments.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:  

Reviewer #1 (Public Review):

Strengths:

The manuscript utilizes a previously reported misfolding-prone reporter to assess its behaviour in ER in different cell line models. They make two interesting observations:

(1) Upon prolonged incubation, the reporter accumulates in nuclear aggregates.

(2) The aggregates are cleared during mitosis. They further provide some insight into the role of chaperones and ER stressors in aggregate clearance. These observations provide a starting point for addressing the role of mitosis in aggregate clearance. Needless to say, going ahead understanding the impact of aggregate clearance on cell division will be equally important.

Weaknesses:

The study almost entirely relies on an imaging approach to address the issue of aggregate clearance. A complementary biochemical approach would be more insightful. The intriguing observations pertaining to aggregates in the nucleus and their clearance during mitosis lack mechanistic understanding. The issue pertaining to the functional relevance of aggregation clearance or its lack thereof has not been addressed. Experiments addressing these issues would be a terrific addition to this manuscript.

We have performed protein blotting and proteomics to characterize ER-FlucDM-eGFP expressing cells. We have also provided evidence to support the role of ER reorganization in regulating aggregate clearance. Our proteomic analysis provided a global view of the cellular state of cells expressing ER-FlucDM-eGFP, which potentially revealed functional relevance of ER-FlucDM-eGFP. Details are explained in the following comments. 

Reviewer #2 (Public Review):

Summary:

The authors provide an interesting observation that ER-targeted excess misfolded proteins localize to the nucleus within membrane-entrapped vesicles for further quality control during cell division. This is useful information indicating transient nuclear compartmentalization as a quality control strategy for misfolded ER proteins in mitotic cells, although endogenous substrates of this pathway are yet to be identified.

Strengths:

This microscopy-based study reports unique membrane-based compartments of ERtargeted misfolded proteins within the nucleus. Quarantining aggregating proteins in membrane-less compartments is a widely accepted protein quality control mechanism. This work highlights the importance of membrane-bound quarantining strategies for aggregating proteins. These observations open up multiple questions on proteostasis biology. How do these membrane-bound bodies enter the nucleus? How are the singlelayer membranes formed? How exactly are these membrane-bound aggregates degraded? Are similar membrane-bound nuclear deposits present in post-mitotic cells that are relevant in age-related proteostasis diseases? Etc. Thus, the observations reported here are potentially interesting.

Weaknesses:

This study, like many other studies, used a set of model misfolding-prone proteins to uncover the interesting nuclear-compartment-based quality control of ER proteins. The endogenous ER-proteins that reach a similar stage of overdose of misfolding during ER stress remain unknown.

We have included a previous study that showed accumulation of BiP aggregates in the nucleus upon overexpression of BiP (Morris et al., 1997; DOI: 10.1074/jbc.272.7.4327) in the discussion (Line 299).

The mechanism of disaggregation of membrane-trapped misfolded proteins is unclear. Do these come out of the membrane traps? The authors report a few vesicles in living cells. This may suggest that membrane-untrapped proteins are disaggregated while trapped proteins remain aggregates within membranes.

We initially made mStayGold-Sec61β to image the ER structures and ER-FlucDM-eGFP aggregates. However, we could not obtain convincing time-lapse images to show the release of ER-FlucDM-eGFP aggregates from the ER membrane as there are abundant ER structures present close to the aggregates during mitosis, preventing the differentiation of the membrane encapsulating aggregates from the ER structures. 

The authors figure out the involvement of proteasome and Hsp70 during the disaggregation process. However, the detailed mechanisms including the ubiquitin ligases are not identified. Also, is the protein ubiquitinated at this stage?

We performed cycloheximide chase experiments in cells released from the G2/M and found that ER-FlucDM-eGFP protein level did not fluctuate significantly when cells progressed through mitosis and cytokinesis. Thus, we did not consider protein ubiquitination and degradation of ER-FlucDM-eGFP as a major mechanism for its clearance. We have included this observation in the results (Figure S7A; Line 266) and in the discussion (Line 324) of the revised manuscript.

This paper suffers from a lack of cellular biochemistry. Western blots confirming the solubility and insolubility of the misfolded proteins are required. This will also help to calculate the specific activity of luciferase more accurately than estimating the fluorescence intensities of soluble and aggregated/compartmentalized proteins. 

We performed solubility test in cells expressing ER-FlucDM-eGFP and detected insoluble ERFlucDM-eGFP after heat stress (Figure S1E; Line 102). We have also performed protein blotting to detect ER-FlucDM-eGFP to normalize the luciferase activity (Line 609). We have updated the method section for luciferase measurement (Line 494).   

Microscopy suggested the dissolution of the membrane-based compartments and probably disaggregation of the protein. This data should be substantiated using Western blots. Degradation can only be confirmed by Western blots. The authors should try time course experiments to correlate with microscopy data. Cycloheximide chase experiments will be useful.

We performed cycloheximide chase experiments in cells released from the G2/M and found that ER-FlucDM-eGFP protein level did not fluctuate significantly when cells progressed through mitosis and cytokinesis (Figure S7A to S7C). Also, live-cell imaging of cells released from the G2/M indicated no significant change of total fluorescence intensity of ER-FlucDMeGFP (Figure S7D). Thus, we do not think that protein degradation of ER-FlucDM-eGFP is the major mechanism for its clearance. 

The cell models express the ER-targeted misfolded proteins constitutively that may already reprogram the proteostasis. The authors may try one experiment with inducible overexpression.

We have re-transduced fresh MCF10A cells with lentiviral particles to induce expression of ER-FlucDM-eGFP. The aggregates started to form after 24 h post-transduction. We made similar observations as described in the manuscript (e.g. aggregate clearance) two days after re-transduction.

It is clear that a saturating dose of ER-targeted misfolded proteins activates the pathway.

The authors performed a few RT-PCR experiments to indicate the proteostasis-sensitivity.

Proteome-based experiments will be better to substantiate proteostasis saturation.

We have performed proteomic analysis in cells expressing ER-FlucDM-eGFP and observed up-regulation of multiple proteins involved in the ER stress response, indicating that cells expressing ER-FlucDM-eGFP experience proteostatic stress (Figure S4A; Line 179).  

The authors should immunostain the nuclear compartments for other ER-membrane resident proteins that span either the bilayer or a single layer. The data may be discussed.

We have co-expressed ER-FlucDM-mCherry and mStayGold-Sec61β and detected mStayGold- Sec61β around ER-FlucDM-mCherry aggregates (Figure 1B).  

All microscopy figures should include control cells with similarly aggregating proteins or without aggregates as appropriate. For example, is the nuclear-targeted FlucDM-EGFP similarly entrapped? A control experiment will be interesting. Expression of control proteins should be estimated by western blots.

We targeted FlucDM-eGFP to the nucleus by expressing NLS-FlucDM-eGFP (Figure S1A). We found that the nuclear FlucDM-eGFP did not co-localize with the ER-FlucDM-mCherry aggregates (Figure S1B; Line 96). We have also determined the expression levels of NLSFlucDM-eGFP and ER-FlucDM-mCherry (Figure S1C and S1D).

There are few more points that may be out of the scope of the manuscript. For example, how do these compartments enter the nucleus? Whether similar entry mechanisms/events are ever reported? What do the authors speculate? Also, the bilayer membrane becomes a single layer. This is potentially interesting and should be discussed with probable mechanisms. Also, do these nuclear compartments interfere with transcription and thereby deregulate cell division? What about post-mitotic cells? Similar deposits may be potentially toxic in the absence of cell division. All these may be discussed.

Thank you for interesting suggestions for our study. We speculated that ER-FlucDM-eGFP aggregates may derive from the invagination of the inner nuclear membrane given that the aggregates are in close proximity to the inner nuclear membrane in interpase cells (Line 299). We have included a previous study that reported a similar aggregate upon BiP overexpression (Morris et al., 1997; DOI: 10.1074/jbc.272.7.4327; Line 300). Our proteomic analysis showed that cells expressing ER-FlucDM-eGFP have several up-regulated proteins related to cell cycle regulation (Figure S4A; Line 346).  

Reviewer #3 (Public Review):

Summary:

This paper describes a new mechanism of clearance of protein aggregates occurring during mitosis.

The authors have observed that animal cells can clear misfolded aggregated proteins at the end of mitosis. The images and data gathered are solid, convincing, and statistically significant. However, there is a lack of insight into the underlying mechanism. They show the involvement of the ER, ATPase-dependent, BiP chaperone, and the requirement of Cdk1 inactivation (a hallmark of mitotic exit) in the process. They also show that the mechanism seems to be independent of the APC/C complex (anaphase-promoting complex). Several points need to be clarified regarding the mechanism that clears the aggregates during mitosis:

• What happens in the cell substructure during mitosis to explain the recruitment of BiP towards the aggregates, which seem to be relocated to the cytoplasm surrounded by the ER membrane.

We have included images to show that BiP co-localizes with ER-FlucDM-eGFP aggregates in interphase cells (Figure S5C). We think that BiP participates in the formation of ER-FlucDMeGFP during interphase instead of getting recruited to the aggregates during mitosis.  

• How the changes in the cell substructure during mitosis explain the relocation of protein aggregates during mitosis.

We provided evidence to show that clearance of ER-FlucDM-eGFP aggregates involves the ER remodeling process. We depleted ER membrane fusion proteins ATL2 and ATL3 to perturb the distribution of ER sheets or tubules and found that cells were defective in clearing the aggregates (Figure 7A and B; Line 278). 

• Why BiP seems to be the main player of this mechanism and not the cyto Hsp70 first described to be involved in protein disaggregation.

In our proteomic analysis, we found that BiP (HSPA5) but not other Hsp70 family members were up-regulated in cells expressing ER-FlucDM-eGFP (Line 352; Figure S4A). This explains why BiP is the main player of the ER-FlucDM-eGFP aggregate clearance.  

Strengths:

Experimental data showing clearance of protein aggregates during mitosis is solid, statistically significant, and very interesting.

Weaknesses:

Weak mechanistic insight to explain the process of protein disaggregation, particularly the interconnection between what happens in the cell substructure during mitosis to trigger and drive clearance of protein aggregates.

In our revised manuscript, we now provided evidence to show that ER-FlucDM-eGFP aggregate clearance involved remodeling of the ER structures during mitotic exit. This is added as a new Figure 7 in the revised manuscript and is described in the result section (Line 278) and in the discussion section (Line 323). We believe that this addition has provided mechanistic insights into ER-FlucDM-eGFP aggregate clearance.

Recommendations for the authors:

Reviewing Editor comments:

I have read these reviews in detail and would like to recommend that the authors perform the experiments according to the reviewers' suggestions, as well as provide the appropriate controls raised by the reviewers.

I think there are not that many requests and they all seem very reasonable and easily doable. I would recommend that the authors carry out the suggested experiments to develop a stronger story where the evidence transitions from being incomplete presently to a "more complete" standard.

We have addressed questions raised by three reviewers and updated our manuscript (labeled in red in the main text).

Reviewer #1 (Recommendations For The Authors):

The manuscript makes exciting observations about the accumulation of reporter protein aggregates in the nucleus and its clearance during mitosis. It also provides some insight into the role of chaperons in aggregate clearance. These observations provide a good platform to perform in-depth analysis of the underlying mechanism and its functional relevance which perhaps the authors will plan over the long term. However, the below suggestions will help improve the current version of the manuscript:

(1) Although it is assumed that the aggregates are cleared by the protein degradation mechanism, clear evidence supporting this assumption in the author's experiments is lacking and needs to be provided. Is it possible that mitosis induces disassembly of these aggregates instead of degradation?

We performed two experiments to verify whether ER-FlucDM-eGFP aggregates are cleared by the protein degradation mechanism. In the first experiment, we treated cells expressing ER-FlucDM-eGFP released from the G2/M boundary with cycloheximide (CHX) and found that ER-FlucDM-eGFP did not decrease in protein abundance in cells progressing through mitosis (Figure S7A to S7C). In the second experiment, we measured the intensity of ERFlucDM-eGFP in early dividing cells and late dividing cells after release from the G2/M boundary and found that there was no significant difference between early and late dividing cells (Figure S7D). Thus, we concluded that protein degradation of ER-FlucDM-eGFP is not the primary mechanism of its clearance during cell division (Line 324). Furthermore, we included new data to show that the ER-FlucDM-eGFP aggregate clearance depends on ER reorganization during cell division, so mitotic exit induces disassembly of the aggregates instead of protein degradation.

(2) It is intriguing that the aggregates are nuclear. Is the nuclear localization mediated by localization to ER? A time course analysis would reveal this and would provide credence to the idea that the reporter was originally expressed in the ER. It is currently unclear if the reporter ever gets expressed in ER.

We showed that in interphase cells, ER-FlucDM-eGFP co-localizes with mStayGold-Sec61β, which labels the ER structures (Figure 1B). So, ER-FlucDM-eGFP is expressed and present in the ER network and invaginates into the inner nuclear membrane as aggregates. We attempted to image ER-FlucDM-eGFP for its formation; however it was technically challenging as the aggregates appeared very small and not too visible after clearance under our microscopy system.  

(3) It would be expected that the persistence of these aggregates would impact cell division and cellular health. An experiment addressing this hypothesis would be very useful in establishing the functional relevance of this observation in the context of the current study.

We have performed proteomic analysis on cell expressing ER-FlucDM-eGFP and found that multiple proteins involved in the ER stress response were up-regulated (Figure S4A). Additionally, proteins related to cell cycle regulation were up-regulated upon expression of ER-FlucDM-eGFP (Figure S4A). The increase of these proteins may indicate a perturbed cellular health (Line 344). 

(4) A recent report (PMID: 34467852) identified the role of ER tubules in controlling the size of certain misfolded condensates. Would specific ER substructures affect the nuclear localization and/or clearance of the FlucDM aggregates? This is tied to point#2 and would provide insights into the connection between ER and the nuclear aggregates.

Thank you for your suggestions. We perturbed the ER remodeling process by knocking down ATL2 and ATL3, which are ER membrane fusion proteins, and found that clearance of ER-FlucDM-eGFP aggregates was affected (Figure 7A and B). Hence, perturbation of the distribution of ER tubules and ER sheets affects ER-FlucDM-eGFP aggregate clearance. We have also added the recent paper about ER tubule size in regulating the sizes of misfolded condensates in the discussion (Line 321)

Reviewer #2 (Recommendations For The Authors):

I expect that the images indicate z-sections. Should be indicated in legends as applicable.

We have indicated whether the images are Z-stack or single Z-slices in the figure legends.  

Small point: the control region (outside inclusion) that was bleached in 2c may be clearly indicated. 

We have added the explanation in the figure legend of Figure 2C.

The US involvement started because of Japan and Pearl Harbor. This pretty much lead to other tragic events to happen in the future.

eLife Assessment

This important Research Advance presents compelling evidence on the neuroprotective effects of reserpine in a well-established model of retinitis pigmentosa (P23H-1). This study builds on previous work establishing reserpine as a neuroprotectant in models of Leber congenital amaurosis. Here authors show reserpine's disease gene-independent influence on photoreceptor survival and emphasizes the importance of considering biological sex in understanding inherited retinal degeneration and the impact of drug treatments on mutant retinas. The work will be of interest to vision researchers as well as a broad audience in translational research.

Reviewer #1 (Public review):

Summary:

The authors investigate the neuroprotective effect of reserpine in a retinitis pigmentosa (P23H-1) model, characterized by a mutation in the rhodopsin gene. Their results reveal that female rats show better preservation of both rod and cone photoreceptors following reserpine treatment compared to males.

Strengths:

This study effectively highlights the neuroprotective potential of reserpine and underscores the value of drug repositioning as a strategy for accelerating the development of effective treatments. The findings are significant for their clinical implications, particularly in demonstrating sex-specific differences in therapeutic response.

Weaknesses:

The main limitation is the lack of precise identification of the specific pathway through which reserpine prevents photoreceptor death.

Comments on revisions:

Thank you for your thorough revisions. I appreciate the effort you have put into addressing all the concerns I previously raised. Upon reviewing your responses and the updated manuscript, I find that you have adequately clarified the issues and incorporated the necessary modifications. Your revisions have strengthened the paper, and I have no further concerns at this stage.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Public review):

Summary:

The authors investigate the neuroprotective effect of reserpine in a retinitis pigmentosa (P23H-1) model, characterized by a mutation in the rhodopsin gene. Their results reveal that female rats show better preservation of both rod and cone photoreceptors following reserpine treatment compared to males.

Strengths:

This study effectively highlights the neuroprotective potential of reserpine and underscores the value of drug repositioning as a strategy for accelerating the development of effective treatments. The findings are significant for their clinical implications, particularly in demonstrating sex-specific differences in therapeutic response.

We sincerely appreciate the reviewer’s comments.

Weaknesses:

The main limitation is the lack of precise identification of the specific pathway through which reserpine prevents photoreceptor death.

We acknowledge that the exact pathway through which reserpine exerts its protective effects on photoreceptors remains undetermined, yet our findings provide critical insights into potential mechanisms. Together with our previous report [PMID: 36975211], the studies being presented here validate proteostasis (including autophagy) and p53 signaling as the key pathways underlying reserpine-mediated survival of photoreceptors in retinal disease models. We also go a step further by showing an influence of the biological sex.

We emphasize that the primary aim of this study was to demonstrate the effectiveness of reserpine in a different retinal degeneration model—specifically, the autosomal dominant RP model—which shares a retinal disease phenotype with the model used for initial screening but involves different genetic and molecular mechanisms of degeneration.

Reviewer #2 (Public review):

Summary:

In the manuscript entitled "Sex-specific attenuation of photoreceptor degeneration by reserpine in a rhodopsin P23H rat model of autosomal dominant retinitis pigmentosa" by Beom Song et al., the authors explore the transcriptomic differences between male and female wild-type (WT) and P23H retinas, highlighting significant gene expression variations and sex-specific trends. The study emphasizes the importance of considering biological sex in understanding inherited retinal degeneration and the impact of drug treatments on mutant retinas.

Strengths:

(1) Relevance to Clinical Challenges: The study addresses a critical limitation in inherited retinal degeneration (IRD) therapies by exploring a gene-agnostic approach. It emphasizes sex-specific responses, which aligns with recent NIH mandates on sex as a biological variable.

(2) Multi-dimensional Methodology: Combining electroretinography (ERG), optical coherence tomography (OCT), histology, and transcriptomics strengthens the study's findings.

(3) Novel Insights: The transcriptomic analysis uncovers sex-specific pathways impacted by reserpine, laying the foundation for personalized approaches to retinal disease therapy.

We are grateful for highlighting the strengths of our work.

Weaknesses:

Dose Optimization

The study uses a fixed dose (40 µM), but no dose-response analysis is provided. Sex-specific differences in efficacy might be influenced by suboptimal dosing, particularly considering potential differences in metabolism or drug distribution.

We acknowledge the limitation of using a fixed dose (40 µM) of reserpine in this study without conducting a comprehensive dose-response analysis. In the primary screens, the EC₅₀ of reserpine was approximately 20 µM. We doubled the concentration for injection to account for the potential loss of reserpine during the in vivo procedures. As we observed the rescue effect of reserpine in mice, we used the same concentration for rats. The fixed-dose approach was chosen to maintain consistency with previous studies evaluating reserpine in retinal degeneration models and to facilitate comparison across studies. Efforts to identify optimal dosing were deprioritized, as the primary goal was different and this information cannot be directly translated to clinical applications.

We also agree that sex-specific differences in efficacy might be influenced by suboptimal dosing, particularly given potential variations in metabolism, drug distribution, and pharmacokinetics between male and female rats. However, recent pharmacokinetic studies on systemically administered reserpine in rats reported no statistically significant covariates, including body weight, age, breed, or sex, affecting pharmacokinetic (PK) or pharmacodynamic (PD) parameters (Alfosea-Cuadrado, G. M., Zarzoso-Foj, J., Adell, A., Valverde-Navarro, A. A., González-Soler, E. M., Mangas-Sanjuán, V., & Blasco-Serra, A. (2024). Population Pharmacokinetic–Pharmacodynamic Analysis of a Reserpine-Induced Myalgia Model in Rats. Pharmaceutics, 16(8), 1101. https://doi.org/10.3390/pharmaceutics16081101). Furthermore, no evidence of sex-specific differences in reserpine pharmacokinetics has been previously identified in available databases (National Center for Biotechnology Information (2025). PubChem Compound Summary for CID 5770, Reserpine. Retrieved January 13, 2025 from https://pubchem.ncbi.nlm.nih.gov/compound/Reserpine). Importantly, the drug in this study was administered intravitreally, where the ocular compartments are relatively isolated from systemic metabolism or excretion. Under these conditions, where absorption, distribution, metabolism, and excretion have minimal impact, we observed sex differences in efficacy using the same dose of drug.

Nonetheless, we agree with the reviewer and plan to pursue dose-response and other studies in future investigations.

Statistical Analysis

In my opinion, there is room for improvement. How were the animals injected? Was the contralateral eye used as control? (no information in the manuscript about it!, line 390 just mentions the volume and concentration of injections). If so, why not use parametric paired analysis? Why use a non-parametric test, as it is the Mann-Whitney U? The Mann-Whitney U test is usually employed for discontinuous count data; is that the case here?<br /> Therefore, please specify whether contralateral eyes or independent groups served as controls. If contralateral controls were used, paired parametric tests (e.g., paired t-tests) would be statistically appropriate. Alternatively, if independent cohorts were used, non-parametric Mann-Whitney U tests may suffice but require clear justification.

We apologize for the lack of clarity. In line 124, we described the injection as “bilateral intravitreal injections of 5 µL of either vehicle or 40 µM reserpine,” and in Figure 1A, we annotated the bilateral injection as DMSO for both eyes and RSP for both eyes. To address this uncertainty, we added the clarification, “with each group receiving bilateral injections of either vehicle or reserpine” (lines 404–405). Since the results are not paired and involve continuous data for which the normality assumption cannot be confidently met or verified, we used the Mann-Whitney U test for statistical analysis.

Sex-Specific Pathways

The authors do identify pathways enriched in female vs. male retinas but fail to explicitly connect these to the changes in phenotype analysed by ERG and OCT. The lack of mechanistic validation weakens the argument.

The study does not explore why female rats respond better to reserpine. Potential factors such as hormonal differences, retinal size, or differential drug uptake are not discussed.

It remains open, whether observed transcriptomic trends (e.g., proteostasis network genes) correlate with sex-specific functional outcomes.

We acknowledge that, while we identified pathways enriched in female versus male retinas, we did not explicitly connect these findings to the functional phenotypes measured by ERG and OCT. Although our transcriptomic data suggest that reserpine differentially influences pathways such as proteostasis and p53 signaling, we did not conduct mechanistic experiments to validate a causal relationship between these pathways and the observed outcomes.

In practice, designing a study to validate the mechanisms of a small molecule modulating multiple pathways presents significant challenges. If the pathways cannot be specifically modulated or if modulation could result in irreversible outcomes, the mechanistic validation becomes difficult to achieve. Drugs demonstrating mutation-agnostic efficacy are often investigated primarily through outcome measures and the analysis of affected pathways rather than through direct mechanistic validation (Leinonen, H., Zhang, J., Occelli, L. M., Seemab, U., Choi, E. H., L P Marinho, L. F., Querubin, J., Kolesnikov, A. V., Galinska, A., Kordecka, K., Hoang, T., Lewandowski, D., Lee, T. T., Einstein, E. E., Einstein, D. E., Dong, Z., Kiser, P. D., Blackshaw, S., Kefalov, V. J., Tabaka, M., … Palczewski, K. (2024). A combination treatment based on drug repurposing demonstrates mutation-agnostic efficacy in pre-clinical retinopathy models. Nature communications, 15(1), 5943. https://doi.org/10.1038/s41467-024-50033-5).

As recommended, we added potential factors that might influence the differential response to reserpine, based on other studies (lines 353–362) highlighting differences in dopamine storage capacity and estrogen independence. We also added a discussion on the possibility of sex-related differences in basal ERG response levels (lines 363–366).

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

The study presents compelling findings on the neuroprotective effects of reserpine in a well-established model of retinitis pigmentosa (P23H-1). The use of ERG, optomotor assays, OCT, immunohistochemistry, and transcriptomic techniques provides a good exploration of the treatment's effects, particularly highlighting the differential response in females. The study underscores the potential of drug repurposing to expedite the availability of therapeutic interventions for patients.

Thanks for your generous comments.

While the manuscript presents an important contribution, I would like to highlight a few points that need clarification or further elaboration to strengthen the work:

(1) Please include the photopic a-wave data in your analysis or provide a justification for its omission. Specifically, it would be valuable to know whether there is an improvement in this parameter under reserpine treatment.

We appreciate the reviewer’s suggestion to include photopic a-wave data in our analysis and acknowledge the importance of this parameter in evaluating cone photoreceptor function. However, we did not analyze the photopic a-wave amplitude in our study because we found the photopic a-wave has low amplitude and high variability, consistent with findings in other studies with P23H-1 rats (Orhan E, Dalkara D, Neuillé M, Lechauve C, Michiels C, et al. (2015) Genotypic and Phenotypic Characterization of P23H Line 1 Rat Model. PLOS ONE 10(5): e0127319. https://doi.org/10.1371/journal.pone.0127319) or even with wild type rats (V.L. Fonteille, J. Racine, S. Joly, A.L. Dorfman, S. Rosolen, P. Lachapelle; Do Rats Generate a Photopic a–Wave? . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2246). We added the description (lines 435-437) explaining why the photopic a-wave was not analyzed. Studies with P23H-1 did not analyze the photopic a-wave, probably for similar reasons.

(2) In Figure 1, it would be helpful to include data from normal control animals to provide a benchmark for retinal degeneration in P23H-1 animals and to better contextualize the effects of reserpine treatment.

Thanks. As suggested, we have included data from normal control animals to Figure 1.

(3) The manuscript states that "Treated female retinas have significantly higher expression of the gene for P62 (SQSTM1), indicating a potential key route for reserpine's activity" (Line 331). Please explain how this difference in expression might translate into a better photoreceptor response in females compared to males.

The difference in P62 (SQSTM1) expression between treated female and male retinas could have important implications for the photoreceptor response. We have identified in our previous study that reserpine increased P62 that mediates proteome balance between ubiquitin-proteasome system (UPS) and autophagy. Together with the role of P62 in the regulation of oxidative stress, P62 might be important for photoreceptor survival and function. Higher expression of P62 in treated females could suggest more efficient cellular maintenance and a better ability to cope with stress, leading to improved photoreceptor survival and function.

(4) Numerous studies have shown that animal models of Parkinson's disease (e.g., those treated with MPTP or rotenone) or retinal tissue from Parkinson's patients exhibit dopaminergic cell death and associated vision loss. Please discuss how these findings relate to your results. Can you hypothesize how dopamine depletion by reserpine may lead to improved photoreceptor responses in your model?

We appreciate the reviewer’s insightful comments. Both MPTP and rotenone act via inhibition of complex I of the respiratory chain, causing cell death and leading to dopamine depletion. In contrast, reserpine acts by inhibiting the vesicular monoamine transporter, depleting catecholamines by preventing their storage and facilitating their metabolism by monoamine oxidase. Although reserpine and other agents can induce animal models of Parkinson's disease, reserpine differs from the others in several aspects: (i) reserpine do not induce neurodegeneration and protein aggregation; (ii) motor performance, monoamine content, and TH staining are partially restored after treatment interruption; and (iii) reserpine lacks specificity regarding dopaminergic neurotransmission (Leão, A. H., Sarmento-Silva, A. J., Santos, J. R., Ribeiro, A. M., & Silva, R. H. (2015). Molecular, Neurochemical, and Behavioral Hallmarks of Reserpine as a Model for Parkinson's Disease: New Perspectives to a Long-Standing Model. Brain pathology (Zurich, Switzerland), 25(4), 377–390. https://doi.org/10.1111/bpa.12253). We have discussed the various effects of catecholamine depletion on retinal diseases (lines 331–337). Both dopamine receptor antagonists and agonists, as well as catecholamine depletion, can exert protective effects on the retina. The reduction in scotopic b-wave amplitude observed at P54, followed by a lack of further progression in degeneration, may support the hypothesis that reduced neuronal activity due to catecholamine depletion could have mitigated damage to retinal neurons.

(5) For readers who may not be familiar with the P23H-1 mutation, it would be beneficial to include a brief description of the timeline and progression of retinal degeneration in this model.

As the progression varies among studies, we have provided our description on observations from the same facility where the animals were housed. The timeline and progression of retinal degeneration are briefly described in the results section (lines 112–115) and Supplementary Figure 1.

(6) Do you have any data on the effects of reserpine treatment in older animals? If available, this could provide additional insight into the potential applicability of reserpine in later stages of disease progression.

Unfortunately, we do not have data from older animals. As described in the results section (lines 116–124), we set the timepoint for interventions before functional impairment peaked, aiming to harness the remaining potential for rescue and promote functional improvement. Our approach focused on developing a gene-agnostic therapy that can delay disease progression and be delivered at an earlier stage than AAV-based therapies, using FDA-approved drugs.

(7) Molecular Basis of Sex Differences: The molecular mechanisms underlying the differential responses in males and females should be elaborated upon. If possible, include a discussion or hypothesis that addresses these sex-specific differences at the molecular level.

We thank the reviewer for highlighting the importance of addressing the molecular basis of sex-specific differences. In our study, we observed distinct transcriptomic responses to reserpine between male and female rats, particularly in molecular pathways related to proteostasis and p53 signaling. While the sex-specific differences in these molecular pathways remain to be fully evaluated, we have added a discussion on sex differences in reserpine responses, incorporating findings from other studies (lines 353–366).

Reviewer #2 (Recommendations for the authors):

(1) There is no mention in the manuscript about the fact that the transgene rats have several copies of rhodopsin and how this can affect these sex differences. Would it be the same in the P23H KO mouse? Or in other models with a single copy of the mutation?

We have described in the Materials and Methods section how they were bred, but we did not specifically mention the allele status in the manuscript. Hemizygous P23H-1 rats used in this study carry a single P23H transgene allele with a transgene copy number of 9, in addition to the normal two wild-type opsin alleles. We added this description to clear the uncertainty (lines 384-387.

(2) This sentence: in abstract lines 26 to 29: "Recently, we identified reserpine as a lead molecule for maintaining rod survival in mouse and human retinal organoids as well as in the rd16 mouse, which phenocopy Leber congenital amaurosis caused by mutations in the cilia-centrosomal gene CEP290 (Chen et al. eLife 2023;12:e83205. DOI: https://doi.org/10.7554/eLife.83205)", to my vew, does not belong to the abstract, maybe in the introduction as stage of art.

Thank you for asking. According to the guidelines for the research advance articles (that follow previously published studies), a reference to the original eLife article should be included in the abstract. As specified in the guidelines, we have updated the citation format to (author, year) for referencing eLife articles (line 29).

(3) Lines 167-170: "Histologic evaluation of the retinas also demonstrated more prominent ONL thinning in the dorsal retina and increased ONL thickness in the dorsal retina measured at 1,000, 1,250, and 1,500 µm distant from the optic nerve head in reserpine-treated group compared with control group (Figure 3C)". I do not understand this sentence. Is it a more prominent thinning or an increased thickness?

We apologize for the confusion caused by this sentence. The histological evaluation showed that ONL thinning was more pronounced in the dorsal retina of control group, which was consistent with OCT findings in Figure 3A. Reserpine treatment increased the ONL thickness in the dorsal retina at specific distances from the optic nerve head (1,000, 1,250, and 1,500 µm). We have revised the sentence for clarity (lines 165-168).

(4) Lines 182-185 and Figure 4B: FL is not the best approach to quantify rhodopsin levels. Since the DAPI staining is overexposed, it is hard to evaluate the staining of RHO in the ONL. From the visible staining in the OS, it is only possible to affirm that the OS are longer in RSP-treated retinas... more is not to be affirmed based on these figures. I suggest using WB.

We acknowledge the reviewer’s concern regarding the use of fluorescence imaging to quantify rhodopsin levels. While our current data highlight structural preservation, such as the length of the outer segments, we agree that drawing conclusions about rhodopsin levels from fluorescence staining is limited. As we do not have samples for WB and fluorescence imaging cannot quantify rhodopsin, we have revised the description (lines 180-184).

(5) Lines 188-190 and Figure 4C: The images in 4C showed an extreme divergence between treated and untreated retina concerning the amount of stained cones, which is not observed at the quantification at 1000µm statistic. Are the images not representative?

We agree with the reviewer that the images in Figure 4C may not adequately represent the quantified data. To address this, we have changed the figure to reflect the quantification results accurately.

(6) Figures 6C-6D and 6G. Why do the authors not use any statistical analysis? Or are the differences not statistically significant? Why do authors use only WT and DMSO controls? What about untreated P23H controls (no DMSO)?

Thanks for checking, and we apologize for the oversight. We have updated figures 5, 6 and S5 to include adjusted p-value in relevant plots. In addition, details of significance threshold are available in supplementary tables. Regarding controls, untreated P23H retinas (without DMSO) were not included in the current analysis, as our experience shows that DMSO injection itself does not cause functional or structural changes. The key data demonstrating the effect of reserpine involve a comparison between the group treated with reserpine and the control group treated with DMSO, as the only difference between these groups is the involvement of the drug.

(7) Validation of findings by testing key genes (e.g., p62/SQSTM1, Nrf2) using qPCR or immunohistochemistry will strengthen the findings.

We appreciate the reviewer’s suggestion to validate key findings using qPCR or immunohistochemistry, as such experiments are crucial for further strengthening our conclusions. While this was not feasible in the current study due to various constraints, we fully recognize their importance and plan to incorporate these in our follow-up studies.

eLife Assessment

In this valuable study, the authors integrate several datasets to describe how the genome interacts with nuclear bodies across distinct cell types and in Lamin A and LBR knockout cells. They provide convincing evidence to support their claims and particularly find that specific genomic regions segregate relative to the equatorial plane of the cell when considering their interaction with various nuclear bodies.

Reviewer #3 (Public review):

Summary:

Golamalamdari, van Schaik, Wang, Kumar Zhang, Zhang and colleagues study interactions between the speckle, nucleolus and lamina in multiple cell types (K562, H1, HCT116 and HFF). Their datasets define how interactions between the genome and the different nuclear landmarks relate to each other and change across cell types. They also identify how these relationships change in K562 cells in which LBR and LMNA are knocked out.

Strengths:

Overall, there are a number of datasets that are provided, and several "integrative" analyses performed. This is a major strength of the paper, and I imagine the datasets will be of use to the community to further probed and the relationships elucidated here further studied. An especially interesting result was that specific genomic regions (relative to their association with the speckle, lamina, and other molecular characteristics) segregate relative to the equatorial plane of the cell.

Weaknesses:

The experiments are primarily descriptive, and the cause-and-effect relationships are limited (though the authors do study the role of LMNA/LBR knockdown with their technologies).

Comments on revisions:

I have no additional comments. I appreciate the authors responding to my previous comments. I anticipate the datasets and concepts raised will be helpful to many investigators in the field.

Author response:

The following is the authors’ response to the previous reviews

Response to Public Reviews:

We would like to thank the reviewers and editors once more for their time and effort in reviewing our manuscript. Below we discuss specifically our response to the recommendations of Reviewer 2, which were the only substantial changes we made to the manuscript.

Reviewer 2 recommendation:

"My only remaining suggestion is that the authors acknowledge and cite the work of other groups which have similarly found different subsets of LADs based on various molecular/epigenetic features:

(1) doi.org/10.1101/2024.12.20.629719

(2) PMID: 25995381

(3) PMID: 36691074

(4) PMID: 23124521 (fLADs versus cLADs, as described by the authors themselves) The exact subtypes of LADs might be different based on the features examined, but others have found/implicated the existence of different types of LADs. Hence, the pwv-LAD should be contextualized within these findings (which they do relative to v-fiLADs)."

We thank the reviewer for this suggestion and for these references. We think that the best place to go into depth about how our work relates to these references would be in an appropriate review article.

However, we did read these references carefully and responded, as described below, by adding additional clarifying text in the manuscript as well as mention of articles specifically relevant to our description of our results.

(1) Reviewer 2 wrote specifically, "Hence, the pwv-LAD should be contextualized within these findings (which they do relative to v-fiLADs)"

We are not sure exactly what Reviewer 2 means here. In this manuscript we defined p-w-v iLADs, not LADs. So, it would be inappropriate to compare a subset of iLAD regions with different types of LADs.

If this was the meaning of Reviewer 2, then other readers might have similar confusion. Therefore, we added the following clarifying text in red:

"Several previous studies have used varying approaches to subdivide LADs further into distinct subsets of LADs with different biochemical and/or functional properties (Martin et al., 2024; Meuleman et al., 2013; Shah et al., 2023; Zheng et al., 2015). However, in this Section we focused instead on asking whether regions specifically within iLADs might show differential localization relative to the lamina and/or nucleoli and, if so, whether these regions would show different levels of gene expression. More specifically, analogously to how gene expression hot-zones appeared as local maxima in speckle TSA-seq with early DNA replication timing, we asked whether iLAD regions that appeared as local maxima in lamina proximity mapping signals would correspond to iLAD regions with locally reduced gene expression levels and later DNA replication timing relative to their flanking iLAD sequences. Our rationale was that these iLAD regions might represent chromatin domains that together with their flanking iLAD regions would typically localize well within the nuclear interior but in a fraction of the cell population would loop back and attach at the nuclear periphery."

(2) We also added the following text near the end of the section about p-w-v iLADs to place them in the context of one class of "LADs" identified by ChIP-seq rather than DamID. We use quotation marks since the approach used produced a segmentation that included a nearly 50/50 mix of iLAD and LAD regions, as identified by DamID, for this class of domains.

"We note that in a previous study a three-state Hidden Markov Model (HMM) segmented lamin B ChIP-seq data into two chromatin domain states with extensive overlap with LADs defined by lamina DamID (Shah et al., 2023). Whereas the late replicating, low gene density/expression "T1 LAD" state showed very high overlap (98%) with LADs defined by DamID, the intermediate replicating, intermediate gene expression "T2 LAD" state showed only 47% overlap with LADs defined by DamID. This was partly a result of the HMM segmentation algorithm but also due to substantial differences between the lamina ChIPseq versus DamID signals for reasons that remain unclear. The subset of p-w-v iLADs included in T2 comprise only a small percentage of the total T2 LAD coverage, which includes both other iLAD and LAD regions. Thus, the p-w-v iLADs we identified here represent a novel and distinct class of iLAD chromatin domains, not previously described."

(3) Alternatively, what Reviewer 2 might be suggesting implicitly is that we should start with the regions identified as p-w-v iLADs in one cell type and then identify all of those p-w-v iLADs which instead exist as LADs in a second cell type. Once we have identified their LAD equivalents in a second cell type we could then ask whether they possess special characteristics such that they correspond to a specific type of LAD subset. Finally, we could then ask how that specific type of LAD subset compared to the different subtypes of LADs identified by other groups and, in particular, the references Reviewer 2 provided.

We agree that would be an interesting future direction, but we consider that as outside the scope of this current manuscript. We note that we did no such analysis of the characteristics of LADs which existed as p-w-v iLADs in a different cell line. We save that for a possible future analysis, ideally in the same cell types as used in the cited references to allow a more direct comparison.

(4) Finally, we added text in the Discussion that relates our analysis of the differential SON and LMNB1 TSA-seq signals for different LAD regions, and how these correlate with different histone modifications, with results from the recent preprint cited by Reviewer 2. Note that we could not directly correlate our results from human cells with the three classes of LADs described in MEFs by this preprint.

"Fourth, we show how LAD regions showing different histone marks- either enriched in H3K9me3, H3K9me2 plus H2A.Z, H3K27me3, or none of these marks- can differentially segregate within nuclei. These results support the previous suggestion of different "flavors" of LAD regions, based on the sensitivity of the autonomous targeting of BAC transgenes to the lamina to different histone methyltransferases (Bian et al., 2013). Differential nuclear localization also was recently inferred by the appearance of different Hi-C Bsubcompartments, which similarly were differentially enriched in either H3K9m3, H3K27me3, or the combination of H3K9me2 and H2A.Z (Spracklin et al., 2023). More recently, and while this paper was in revision, a new study described segmenting mouse embryonic fibroblast LADs into three clusters using histone modification profiling (Martin et al., 2024). Interestingly, these three LAD clusters also most notably differed by their dominant enrichment of either H3K9me3, H3K9me2, or H3K27me3. Thus, three orthogonal approaches have converged on identifying different LAD regions showing differential enrichment either of H3K9me3, H3K9me2, or H3K27me3. Here, our use of TSA-seq directly measures and assigns the intranuclear localization of these different LAD regions to different nuclear locales."

bijv iemand die depressief is en niet denkt aan de toekomst, maar alleen over simpele dingen zoals eten en drinken

eLife Assessment

Using multiple techniques previously validated by the authors, this study identified INTS12, a component of the Integrator complex involved in 3' processing of small nuclear RNAs U1 and U2, as a factor promoting HIV-1 latency. The work is valuable, based on a sound strategy for screening targets to activate HIV latency and the solid mechanistic insights it provides on INTS12 repression of transcriptional elongation. Future studies are needed to explore INTS12 as a drug target against HIV/AIDS.

Reviewer #1 (Public review):

Gray and colleagues describe the identification of Integrator complex subunit 12 (INTS12) as a contributor to HIV latency in two different cell lines and in cells isolated from the blood of people living with HIV. The authors employed a high-throughput CRISPR screening strategy to knock down genes and assess their relevance in maintaining HIV latency. They had used a similar approach in two previous studies, finding genes required for latency reactivation or genes preventing it and whose knockdown could enhance the latency-reactivating effect of the NFκB activator AZD5582. This work builds on the latter approach by testing the ability of gene knockdowns to complement the latency-reactivating effects of AZD5582 in combination with the BET inhibitor I-BET151. This drug combination was selected because it has been previously shown to display synergistic effects on latency reactivation.

The finding that INTS12 may play a role in HIV latency is novel, and the effect of its knock down in inducing HIV transcription in primary cells, albeit in only a subset of donors, is intriguing.

In this revised version, the authors have included new data and clarifications which help strengthen their conclusions.