entiendo que aquí se está hablando del gráfico de abajo, pero se linkea el gráfico de arriba.

hay que corregir los labels de los gráficos y cómo se están citando en el texto, pues hay una desconfiguración con las enumeraciones de las figuras, y eso causa confusión

sugiero linkear algún estudio que respalde estos hallazgos, pensando en alguien que no se maneje en estos temas y necesite corroborar esta afirmación

pasar el resumen de la construcción de los índices al gráfico del apartado 1

corregir la sección de los títulos acorde a la estructuración de Andreas en el issue: 1.- medición de seguridad; 2.- seguridad y migración; 3.- seguridad y migración en el tiempo

los colores están invertidos; baja debe ser naranja y alta negra

Poner el caption del plot aquí y eliminar title del labs de los graficos entonces

¡Hola a tod@s!

Con el gusto de saludarles, esperando se encuentren muy bien, les pido que por favor instalen la extensión hypothes.is en su navegador (ver tablón de Classroom).

Posteriormente, en el espacio designado en , identifiquen la hipótesis del autor y la explicación o justificación que ofrece para sustentarla. Coméntenla y emitan su opinión al respecto.

También, encuentren por qué Luis Montaño afirma que la Administración es una disciplina relativamente reciente en México, con poca tradición en investigación y orientada principalmente hacia las áreas docentes.

Finalmente, respondan la siguiente pregunta: ¿Qué opina el autor acerca del liderazgo?

¡Comenzamos!

peer-to-peer writing

If only we could have that in an atuonomous setting that would be the right counterballance

Gives a good target date

Un schwannoma vestibular, o neurinoma del acústico, es un tumor no canceroso que se origina en las células de Schwann que recubren el nervio del equilibrio (nervio vestibular) y del oído interno al cerebro. Suelen ser de crecimiento lento y afectan generalmente a un solo oído, causando síntomas como pérdida de audición, zumbidos (tinnitus) y pérdida de equilibrio.

When it comes to giveaways or events you're a part of, is it possible to have pop ups on your website at all? Or you could add a banner at the top of your page so it's not a part of your regular home page.

Does this then mean that play is a very broad and general term that cannot be restricted to one definition/thing?

I wonder what the differences of play in different cultures is/ Historically each culture has had some form of play or the other. So I wonder what similarities and differences the different forms of play in different cultures had. Every culture is somewhat different, but could play be something common among them all?

This also makes me wonder how many people use play as a way not just to escape their own realities, but to escape all of the laws, rules, and regulations that accompany everyday life.

I am noticing that the author is constantly going back to this theory of competition and how it is in human nature to enjoy competition and thus, enjoy play.

I find it interesting that the author believes that play is never done out of moral duty or as a task, but the author still related play to themes of law and justice? I find it interesting how that the definition of play continues to fluctuate throughout this paper. What exactly is play?

I find it interesting that this author finds a way to related various aspects within society to play. I would not traditionally assume law and order, commerce, profit, and etc to be a part of play so I find it fascinating that play can relate to even the most "non playful" things.

This statement summarizes why people like and want to play games.

One of my favorite factors about game.

I disagree with the statement about wisdom and folly. Even though someone considered smart might not perform as well in certain games, lots of the "good" players are smart people. I agree with the part regrading good and evil.

I think the author pointed out the essential element of the game. There are rules set within the game and players voluntarily follow it, no matter who the players are.

Not sure how this connects with play. I believe this is more closer to origin of literature.

My favorite part about the concept of game, which, I believe, is maximized through modern form of "play," video games.

Essentially, play is something we don't physically need to survive, but spiritually require.

What are these "savage societies"? What are these "tribes" he is referring to?

Perhaps this is why being a part of a team is such a unique experience. You may not otherwise have anything in common with a teammate, and outside of the sport you may never have crossed paths or become friends, but because you were on the same team, there is a unique bond formed that never really disappears.

The idea that the tension element confers a kind of ethical aspect to play is one that I agree with; as the author says, when a game is full of tension, the players character is revealed

I didn't understand what this meant so I looked it up; apparently "warp and woof" refers to the fundamental components of weaving, and as an idiom it refers to the "essential foundation or base" of a structure. So, basically what he's saying is that repetition and alternation are essential components on which play is built.

I am curious to learn more about this link between play and the "sacred sphere." I do understand how the author tied the origins of ritual to play, but how exactly do they connect now? Would praying or going to ones place of worship for service be considered a form of play?

I like that the author is taking time to emphasize that play and non-seriousness cannot be conflated.

I would argue that it can have moral function- think back to the gambler. I would consider gambling to be both a form of play and, when out of control, a kind of vice.

This makes me think about our discussion in our first class; when considering what my favorite game is, I thought about the fact that I play a sport (basketball) but then hesitated to name that as a game. It is a game, of course, but after the years long experience I've had in training for the sport and competing very seriously, calling it a "game" feels in some ways very wrong. To me, it is more than a game; there are instances where some days it feels like work! With this in mind, can it still be considered a form of play?

I don't think this is true. I believe there have been studies done that say that many mammals have the ability to laugh and do so often.

Author response:

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

Reviewer #1 (Public review):

(1) Given the mechanical nature of the device and the propensity for mice to urinate on things, I also wonder how frequently the device breaks/needs to be repaired. Perhaps some details regarding the cost and reliability of the device would be helpful to include, as these are the two things that could make researchers hesitant to adopt immediately.

We thank the reviewer for their astute observations. We also noted the problem of mouse waste and incorporated this concern into the redesign we mention in the text.

“Mouse waste getting on mechanical parts was found to be a major concern for the initial version of the device. As part of the redesign, the linear stages were moved out from under the mice to avoid this problem. Despite this problem, the original version of the device has not had any of its stages break down yet. A common problem though was that stimulus tips would blunt or break if they hit the mesh of the mesh table, requiring replacement. This has been solved in the latest version through a new feature where the mesh is detected via the force sensor, prompting immediate stimulus withdrawal, avoiding damage.”

In regards to cost and adoption, we have added this sentence to the final line of the discussion:

“To promote wide adaptation of this device across as many labs as possible, a company, Tactorum Inc., has been formed.”

(2) The only major technical concern, which is easy to address, is whether the device generates ultrasonic sounds that rodents can hear when idle or operational, across the ultrasonic frequencies that are of biological relevance (20-110 kHz). These sounds are generally alarm vocalizations and can create stress in animals, and/or serve as cues of an impending stimulus (if indeed they are produced by the device).

The reviewer brings up an interesting question. The ARM does not make a lot of noise, but some of the noise it emits does range into the 20-110 kHz range, though besides this does not qualitatively have other similarities to a mouse vocalization. Based on this we tested whether the noise produced by the ARM causes stress in naïve mice.

“A concern was raised that the noise of the ARM may cause stress in the mice tested. To test this, the open field test was performed with naïve mice (n=10) 2 feet from the ARM while the ARM either sat silent or ran through its habituation program, producing noise. The mouse's center point movement was then tracked in relation to the chamber, its edges, and center. No significant differences were found in distance traveled, center entrances, center, time in center, and latency to center entrance based on a student’s two-tailed t-test (Figure S1D-G). Based on this, neither stress nor locomotion differences were detected by this test, indicating the ARM does not induce an increased stress state due to its noise, even in non-habituated mice.”

(3) This sentence in the intro may be inaccurate: "or the recent emergence of a therapeutic targeting voltage-gated sodium channels, that block pain in both rodents and humans such as VX-548 for NaV1.8 (Jones 2023)" Despite extensive searching, I have been unable to find a reference showing that VX-548 is antinociceptive in rodents (rats or mice). As for why this is the case, I do not know. One speculation: this drug may be selective for the human Nav1.8 channel (but again, I have found no references comparing specificity on human vs rodent Nav1.8 channels). To not mislead the field into thinking VX-548 works for rodents and humans, please remove "both rodents and" from the sentence above (unless you find a reference supporting VX-548 as being effective in pain assays with rodents. There is a PK/PD paper with rodents, but that only looks at drug metabolism, not efficacy with pain assays).

We agree with the reviewer and have removed mention of the new Nav1.8 therapeutic also working in rodents.

(4) In the intro paragraph where variability in measuring mechanical stimuli is described, there is a new reference from the Stucky lab that further supports the need for an automated way to measure allodynia, as they also found variability between experimenters. This would be a relevant reference to include: Rodriguez Garcia (2024) PMID: 38314814.

Thanks to the reviewer for this relevant citation and we have updated the text to incorporate this:

“Recent studies utilizing the manual highspeed analysis of withdrawal behavior analysis developed by Abdus-Saboor et al. 2019 has reproduced this sizable experimenter effect using the new technique. (Rodríguez García 2024)”

(5) "a simple sin wave motion": should be "sine", correct throughout (multiple instances of "sin")

Corrections made where relevant.

Reviewer #2 (Public review):

(1) ARM seems like a fantastic system that could be widely adopted, but no details are given on how a lab could build ARM, thus its usefulness is limited.

The reviewer raises a good point, unfortunately the authors are constrained by university policies around patent law. That said efforts are being made to make the ARM widely available to interested researchers. As mentioned above to Reviewer 1’s comments, we end the discussion section with this sentence:

“To promote wide adaptation of this device across as many labs as possible, a company, Tactorum Inc., has been formed.”

(2) The ARM system appears to stop short of hitting the desired forces that von Frey filaments are calibrated toward (Figure 2). This may affect the interpretation of results.

The reviewer gives an important observation. We amended the text to include more clarity on the max forces induced, and comments on causes beyond the delivery mechanism. It should be noted that a newly bought fresh set of von Frey’s was used.

“With the same 1.4 and 2 g von Frey filaments Researcher 1 delivered max average forces of 1.5 g and 2.7 g, and Researcher 2 1.35 g and 2.4 g. The ARM delivered average max forces closest to the targeted forces, with 1.36 g and 1.9 g. (Figure 2C) Some of the error observed could be due to the error rate (+/- 0.05 g) in the force gauge and the von Frey set used.”

(3) The authors mention that ARM generates minimal noise; however, if those sounds are paired with stimulus presentation, they could still prompt a withdrawal response. Including some 'catch' trials in an experiment could test for this.

The reviewer makes a very useful suggestion that we incorporated into our carrageenan experiments. This new data can be found in Supplemental Figure 3F.

“For the carrageenan model, three replicates of the force ramp stimulus were delivered to each paw, and catch trials were performed every 3^rd trial to test whether the mice would respond to the noise of the ARM alone. During catch trials, the stimulus was delivered to the open air behind the mouse, and any movement within 5 seconds of stimulus delivery was counted as a response. These trials found a 96% response rate in true trials, with only a 7% rate in catch trials, indicating responses were not being driven by device noise.”

(4) The experimental design in Figure 2 is unclear- did each experimenter have their own cohort of 10 mice, or was a single cohort of mice shared? If shared, there's some concern about repeat testing.

Further clarification was added to avoid confusion on the methods used here.

“Separate cohorts of 10 mice were used for ARM and manual delivery, with a week given between each researcher to avoid sensitization.”

(5) In Figure 5 and S4, the order of the legends does not match the order of the graphs. This can be particularly confusing as the color scheme is not colorblind-friendly. Please consider revising the presentation of these figures.

Corrections made where relevant.

Reviewer #3 (Public review):

(1) Limited details are provided for statistical tests and inappropriate claims are cited for individual tests. For example, in Figure 2, differences between researchers at specific forces are reported to be supported by a 2-way ANOVA; these differences should be derived from a post-hoc test that was completed only if the independent variable effects (or interaction effect) were found to be significant in the 2-way ANOVA. In other instances, statistical test details are not provided at all (e.g., Figures 3B, 3C, Figure 4, Figure 6G).

We would like to thank the reviewer for pointing out the lack of clarity in the text on these statistical methods. We have added further details across the manuscript and shown below here in order to address this concern.

“Both manual delivery and the ARM produced significant paw withdrawal percentage curves, a standard traditional measurement of mechanical sensitivity in the field (von Frey 1896, Dixon 1980, Chaplan 1994)(Figure 2E), with a 2-way ANOVA and a posthoc Tukey test detecting significant increases in comparing the 3 lower force VFH’s (0.02g, 0.07g, 0.16g) to the 2 highest force VFH’s (1g, 1.4g). This demonstrates that the ARM delivers results comparable to highly experienced researchers. However, a 2-way ANOVA and a posthoc Tukey test found that Researcher 2 elicited a significantly higher (p=0.0008) paw withdrawal frequency than Researcher 1 (Figure S2A) which corresponded with Researcher 2’s higher VFH application time as measured by the force sensor (Figure 2B).”

“Adjustments were then made to the PAWS software to automate the measurement of withdrawal latency based on pose tracking data of the withdrawal response and the trajectory of the stimulus delivery encoded into the ARM. Testing of C57/BL6J (n=15) at baseline found significant decreases in withdrawal latency for pinprick compared to cotton swab stimuli delivered in identical ways by the ARM (Figure 3B) based on a 2-tailed student t-test.”

“Mice injected with carrageenan (n=15) showed elevated shaking behavior (p=0.0385, 2-way ANOVA and a posthoc Tukey test) in response to pinprick stimuli in comparison to measurements at baseline (Figure 3C).”

“Remote habituated mice showed a significant decrease (p=0.0217, 2-way ANOVA) in time to rest over the 3 days (Figure 4B), but no significant differences for any single day. The number of turns was measured for each group during the first 10 minutes of day 1 to act as a baseline, and then from 20 to 30 minutes for each day. Turn counts were then compared as a percentage of the baseline count for each group. This period was chosen as it the period when experiments start after the day of habituation on experimental days. It was found that remote-habituated mice showed significantly less turning on day 2 compared to mice habituated with a researcher present (p=0.024, 2-way ANOVA posthoc Tukey test), and that only the remote-habituated mice showed significantly decreased turning behavior on day 3 compared to day 1 (p=0.0234, 2-way ANOVA posthoc Tukey test) (Figure 4C).”

“Sex-dependent differences were found in reflexive and affective behavioral components of the mouse withdrawal response when a researcher was present versus not for both reactions to innocuous and noxious stimuli. A 2-way ANOVA and a posthoc Tukey test found that cotton swab stimuli elicited increased male mouse reflexive paw withdrawal features, including max paw height (p=0.0413) and max paw velocity (Y-axis) (p=0.0424) when Researcher 1 was present compared to when no researcher was present (Figure 4E-F). Pinprick stimuli (Figure 4H-I) on the other hand led to increased max paw height (p=0.0436) and max paw velocity (Y-axis) (p=0.0406) in male mice compared to female mice when Researcher 1 was present.

Analysis of the shaking behavior elicited by cotton swab and pinprick stimuli found no significant differences in shaking behavior duration (Figure 4SA-B) but found sex-dependent differences in paw distance traveled after the initial withdrawal, including during shaking and guarding behaviors. For cotton swab (Figure 4G) male mice showed significantly increased paw distance traveled compared to female mice when Researcher 2 was present (p=0.0468, 2-way ANOVA posthoc Tukey test) but not when Researcher 2 was present or no researcher was present. Pinprick stimuli also elicited sex-based increases in paw distance traveled (Figure 4J) in male mice when Researcher 2 was present compared to both male mice when no researcher was present (p=0.0149, 2-way ANOVA posthoc Tukey test) and female mice when Researcher 1 was present (p=0.0038, 2-way ANOVA posthoc Tukey test).”

(2) In the current manuscript, the effects of the experimenter's presence on both habituation time and aspects of the withdrawal reflex are minimal for Researcher 2 and non-existent for Research 1. This is surprising given that Researcher 2 is female; the effect of experimenter presence was previously documented for male experiments as the authors appropriately point out (Sorge et al. PMID: 24776635). In general, this argument could be strengthened (or perhaps negated) if more than N=2 experiments were included in this assessment.

The reviewer makes an important point regarding this data and the need for further experiments. We designed a new set of experiments to examine the effect of male and female researchers overall. It should be noted that this is rather noisy data given it was collected by three sets of male and female researchers over 3 weeks. That said a significant difference was found between mouse sexes when a male researcher was present. This is consistent with previous data, but as we discuss this does not invalidate previous data as researcher gender appears to be only one of the factors at work in researcher presence effects on mouse behavior, leading to individuals having the potential for greater or lesser effects than their overall gender. Our new results can be found in Figure 4K.

“These results indicate that researcher presence at baseline can lead to significant differences in reflexive and affective pain behavior. In this case, male mice showed increased behavioral responses to both touch and pain behavior depending on whether the researcher was present. This led to sex differences in the affective and reflexive component of the withdrawal response when a researcher is present, which disappears when no researcher is present, or a different researcher is present. For this set of researchers, the female researcher elicited the greater behavioral effect. This appeared at first to contradict previous findings (Sorge 2024, Sorge 2014), but it was hypothesized that the effect of an individual researcher could easily vary compared to their larger gender group. To test this, 6 new researchers, half male and half female, were recruited and a new cohort of mice (n=15 male, n=15 female) was tested in each of their presence over the course of 3 weeks, controlling for circadian rhythms (Figure 4K). The newly added force ramp stimulus type was used for these experiments, with three replicates per trial, to efficiently measure mechanical threshold in a manner comparable to previous work. It was found that female mice showed significantly decreased mechanical threshold compared to male mice (p=0.034, Šídák's multiple comparisons test and student’s t-test) when a male researcher was present. This did not occur when a female researcher or no researcher was present. In the latter case of slight trend towards this effect was observed, but it was not significant (p=0.21), and may be the result of a single male researcher being responsible for handling and setting up the mice for all experiments.”

“These findings indicate that sex-dependent differences in evoked pain behavior can appear and disappear based on which researcher/s are in the room. There is a trend towards male researchers overall having a greater effect, but individuals may have a greater or lesser effect on mouse behavior, independent of the gender or sex. This presents a confound that must be considered in the analysis of sex differences in pain and touch behavior which may explain some of the variation in findings from different researchers. Together, these results suggest that remote stimulus delivery may be the best way to eliminate variation caused by experimenter presence while making it easier to compare with data from researchers in your lab and others.”

(3) The in vivo BLA calcium imaging data feel out of place in this manuscript. Is the point of Figure 6 to illustrate how the ARM can be coupled to Inscopix (or other external inputs) software? If yes, the following should be addressed: why do the up-regulated and down-regulated cell activities start increasing/decreasing before the "event" (i.e., stimulus application) in Figure 6F? Why are the paw withdrawal latencies and paw distanced travelled values in Figures 6I and 6J respectively so much faster/shorter than those illustrated in Figure 5 where the same approach was used?

Thanks to the reviewer for bringing up this concern. We have included further text discussing this behavioral data and how it compares to previous work in this study.

“Paw height and paw velocity were found to be consistent with data from figures 4E-I (male researcher and male mice) and 5C (stimulus intensity 2.5 and 4.5) for similar data, with slightly elevated measures of paw distance traveled and decreased paw withdrawal latency for the pinprick stimulus. This was likely caused by sensitization due to multiple stimulus deliveries over the course of the experiment, as due to logistics, 30 stimulus trials were delivered per session due to logistical constraints vs the max of 3 that were performed during previous experiments.”

“This data indicates that the ARM is an effective tool for efficiently correlating in vivo imaging data with evoked behavioral data, including sub-second behavior. One limitation is that the neural response appears to begin slightly before stimulus impact (Figure 6F, 6SB). This was likely caused by a combination of the imprecise nature of ARM v1 paw contact detection and slight delays in the paw contact signal reaching the Inscopix device due to flaws in the software and hardware used, slowing down the signal. Improvements have been made to eliminate this delay as part of the ARM v2, which have been shown to eliminate this delay in vivo fiber photometry data recorded as part of new projects using the device.”

(4) Another advance of this manuscript is the integration of a 500 fps camera (as opposed to a 2000 fps camera) in the PAWS platform. To convince readers that the use of this more accessible camera yields similar data, a comparison of the results for cotton swabs and pinprick should be completed between the 500 fps and 2000 fps cameras. In other words, repeat Supplementary Figure 3 with the 2000 fps camera and compare those results to the data currently illustrated in this figure.

The reviewer makes a good point about the need for direct comparison between 500 fps and 2000 fps data. To address this we added data from same mice, from 2 weeks prior with a comparable set up. These new results can be found in Supplemental Figure 3.

“Changes were made to PAWS to make it compatible with framerates lower than 2000 fps. This was tested using a 0.4 MP, 522 FPS, Sony IMX287 camera recording at 500 fps, and data recorded at 2000 fps by the previously used photron fastcam (Figure 3SC-F). The camera paired with PAWS was found to be sufficient to separate between cotton swab and pinprick withdrawal responses, suggesting it may be a useful tool for labs that cannot invest in a more expensive device. PAWS features measured from 500 fps video data were not significantly different from the 2000 fps data based on a 2 way ANOVA.”

(5) In Figure 2F, the authors demonstrate that a von Frey experiment can be completed much faster with the ARM vs. manually. I don't disagree with that fact - the data clearly show this. I do, however, wonder if the framing of this feature is perhaps too positive; many labs wait > 30 s between von Frey filament applications to prevent receptive field sensitization. The fact that an entire set of ten filaments can be applied in < 50 s (< 3 s between filaments given that each filament is applied for 2 s), while impressive, may never be a feature that is used in a real experiment.

The reviewer makes an important point about how different researchers perform these tests and the relevant timings. We have moderated the framing of these results to address this concern.

“Further, we found that the ARM decreased the time needed to apply a stimulus 10 times to a mouse paw by 50.9% compared to manual delivery (Figure 2F). This effect size may decrease for researchers who leave longer delays between stimulus delivery, but the device should still speed up experiments by reducing aiming time and allowing researchers to quickly switch to a new mouse while waiting for the first.”

(6) Why are different affective aspects of the hindpaw withdrawal shown in different figures? For example, the number of paw shakes is shown in Figure 3C, whereas paw shaking duration is shown in Figure 5D. It would be helpful - and strengthen the argument for either of these measures as being a reproducible, reliable measure of pain - if the same measure was used throughout.

Thanks to the reviewer for pointing out this discrepancy. We have adjusted the figures and text to only use the Number of Paw Shakes for better consistency (Figure 5D and Figure 5-figure supplement 1C).

(7) Is the distance the paw traveled an effective feature of the paw withdrawal (Figure 5E)? Please provide a reference that supports this statement.

A relevant citation and discussion of this metric based on previous studies has been added.

“Mice injected with carrageenan (n=15) showed elevated shaking behavior (p=0.0385) in response to pinprick stimuli in comparison to measurements at baseline (Figure 3C). This aligned with previous findings where PAWS has detected elevations in shaking and/or guarding behavior, examples of affective pain behavior, and post-peak paw distance traveled, which correlates with these behaviors in carrageenan pain models and has been to found to be a good measure of them in past studies (Bohic et al. 2023).”

(8) Dedek et al. (PMID: 37992707) recently developed a similar robot that can also be used to deliver mechanical stimuli. The authors acknowledge this device's ability to deliver optogenetic and thermal stimuli but fail to mention that this device can deliver mechanical stimuli in a similar manner to the device described in this paper, even without experimenter targeting. Additional discussion of the Dedek et al. device is warranted.

We would like to thank the reviewer for identifying  this omission. Discussion of this as well as further discussion of Dedek et al.’s automation prototyping work has been added.

“Previous attempts at automating mechanical stimulus delivery, including the electronic von Frey (Martinov 2013) and dynamic plantar asthesiometer (Nirogi 2012), have focused on eliminating variability in stimulus delivery. In contrast to the ARM, both of these devices rely upon a researcher being present to aim or deliver the stimulus, can only deliver vFH-like touch stimuli, and only measure withdrawal latency/force threshold. Additionally, progress has been made in automating stimulus assays by creating devices with the goal of delivering precise optogenetic and thermal stimuli to the mouse’s hind paw (Dedek 2023, Schorscher-Petchu 2021). The Prescott team went farther and incorporated a component into their design to allow for mechanical stimulation but this piece appears to be limited to a single filament type that can only deliver a force ramp. As a result these devices and those previously discussed lack of customization for delivering distinct modalities of mechanosensation that the ARM allows for. Moreover, in its current form the automated aiming of some of these devices may not provide the same resolution or reliability of the ARM in targeting defined targets (Figure 1C), such as regions of the mouse paw that might be sensitized during chronic pain states. Due to the nature of machine learning pose estimation, substantial work, beyond the capacity of a single academic lab, in standardizing the mouse environment and building a robust model based on an extensive and diverse training data set will be necessary for automated aiming to match the reliability or flexibility of manual aiming. That said, we believe this work along with that of that of the other groups mentioned has set the groundwork from which a new standard for evoked somatosensory behavior experiments in rodents will be built.”

(9) Page 2: von Frey's reference year should be 1896, not 1986.

This typo has been fixed, thanks to the reviewer for noting it.

“For more than 50 years, these stimuli have primarily been the von Frey hair (vFH) filaments that are delivered to the mouse paw from an experimenter below the rodent aiming, poking, and subsequently recording a paw lift (von Frey 1896, Dixon 1980, Chaplan 1994).”

(10) Page 2: Zumbusch et al. 2024 also demonstrated that experimenter identification can impact mechanical thresholds, not just thermal thresholds.

Text has been updated in order to note this important point.

“A meta-analysis of thermal and mechanical sensitivity testing (Chesler 2002, Zumbusch 2024) found that the experimenter has a greater effect on results than the mouse genotype, making data from different individual experimenters difficult to merge.”

(11) Page 2: One does not "deliver pain in the periphery". Noxious stimuli or injury can be delivered to the periphery, but by definition, pain is a sensation that requires a central nervous system.

Text has been updated for improved accuracy.

“Combining approaches to deliver painful stimuli with techniques mapping behavior and brain activity could provide important insights into brain-body connectivity that drives the sensory encoding of pain.”

Like, r = sqrt(x^2 + y^2) distance?

Reviewer #2 (Public review):

Summary:

Bushey et al. investigate the feasibility of using RubyACRs, specifically A1ACR1 and HfACR1 (described previously in (Govorunova et al., 2020)) as red-shifted inhibitory opsins in Drosophila melanogaster. The study employs a wide range of techniques to demonstrate successful neuronal inhibition. Electrophysiology experiments established that HfACR1 was most effective at hyperpolarizing cells, compared to A1ACR1 and GtACR1; both RubyACRs also appeared to be more effective than GtACR1 when the latter was actuated by green light. The authors further demonstrate successful neuronal inhibition using calcium imaging. RubyACRs were also shown to be useful in in vivo behavioral setups, specifically in spontaneous locomotion, associative learning, and courtship paradigms. In the courtship assay, in particular, the authors test multiple wavelengths of light at various light intensities, thus providing a rigorous analysis of the RubyACRs' efficacy under different light conditions.

Strengths:

The work provides the Drosophila field with a promising new tool. Red-shifted opsins are particularly advantageous in behavioral assays as red light penetrates the cuticle better than green or blue light, and provides less visual stimulation to the fly. It is also ideal for imaging as it allows for simultaneous optogenetic stimulation and GCamp imaging. A particular strength of the paper is the direct demonstration of RubyACR's capacity to inhibit neurons via electrophysiology and calcium imaging. Furthermore, inhibition effects in the three behavioral assays are strong and convincing. Given the apparent efficacy of RubyACRs and the advantages of a red-sensitive anion channelrhodopsin, this tool has great potential.

Weaknesses:

This work convincingly demonstrates the efficacy and potential utility of RubyACRs in Drosophila for imaging and behavior. However, the lethality/toxicity of RubyACRs is a relevant concern that should be addressed in-depth rather than glossed over, as it may pose a major obstacle to use. Discussing this issue in the present study will also help guide potential users and will set the stage for potential future efforts to ameliorate RubyACRs as optogenetic inhibitors.

Major concerns:

(1) Table 1 demonstrates high lethality in the RubyACRs compared to GtACR1. For example, in the MI04979-VGlut driver, GtACR1 expression resulted in 32.9% lethality, while HfACR1 expression resulted in 98.7% lethality. This lethality presents an obstacle to the potential adoption of this tool, and should be discussed in detail, rather than in passing. The authors might like to present "% lethality" rather than "% survived", as the former is more relevant when discussing the relative yield and health of flies that can be used in experiments.

(2) In Figure 3D, driver>opsin flies have lower locomotion during the baseline (i.e., dark) phase, compared to opsin-only controls or GtACR1 flies. For some comparisons, flies are walking around 10-fold slower. For example, in the case of VGlut-GAL4>HfACR1, test flies are walking at <1 mm/s, while "Empty" test flies are walking at ~10 mm/s. This suggests that, for these drivers, neuronal and/or network function is affected. It opens the possibility that the lethality and locomotor defects could be due to cell-autonomous toxicity. We ask the authors to provide a description of this effect in the Results and to discuss it in the Discussion. Relatedly, VGlut-GAL4>GtACR1 flies in red light exhibit a locomotion increase, but this data is not mentioned in the text. The use of differing scales for the Y-axes in these panels can be confusing when the reader is expected to compare velocity across different panels. It would be best if the y-axes were set to a single range, e.g., 0 to 12 mm/s.

(3) Lethality in broad drivers could result from cell-autonomous toxicity or neuronal dysfunction resulting from RubyACR expression. Ideally, the authors would address or even investigate the possible mechanisms of toxicity of the RubyACRs. Do cells and/or synapses expressing RubyACRs have normal morphology and function? For example, the authors could compare cell survival between flies with RubyACR expression and flies with a fluorescent protein with no opsin. The authors may also want to present lethality data for other, less broad drivers (such as MB320C, which was used for the associative memory assay) in order to demonstrate whether this problem is confined to broad drivers such as VGlut-GAL4, or if this is a problem with narrow drivers as well. If new experiments are not possible, these issues should at least be mentioned in the Discussion.

Minor concerns

(1) The specific method used for quantifying lethality is mentioned briefly in Table 1 but is not detailed in the Methods. The authors derive lethality by comparing to a sibling control group with either the opsin or the driver alone, but the opsin alone or driver alone may cause some lethality by themselves. We suggest the use of a viability assay, e.g. (Rockwell et al., 2019), which would give potential users a clearer picture of which developmental stage is most affected by opsin expression, as well as allow opsin-only, driver-only and experimental groups to be assessed separately (lethality would then be reported as the % of embryos that reach each stage of development, and eventually enclosure).

(2) For the calcium imaging analysis in Figure 2, the U-shaped curve observed for mean ΔF/F0 for A1ACR1 and HfACR1 may not be due to actual desensitization for the channels, as the authors suggest (lines 143-145), but may be due simply to a shifting baseline. The authors use the 5-s period preceding stimulation onset as F0, but in some cases (e.g., HfACR1 at 250 uW/mm2), calcium fluorescence rises above baseline and remains high post-stimulation (ΔF/F0 of +0.5, which we observe is the same magnitude as the ΔF/F0 of -0.5 observed during inhibition), thus affecting the ΔF/F0 for subsequent trials. The authors should discuss this incomplete recovery in the text, or (if available) use a static channel instead to provide a stable F0 for calculating ΔF/F0. Alternatively, if the authors wish to rigorously test the hypothesis that high light intensity indeed results in desensitization of these channels, they may consider using different flies for each light intensity or longer inter-stimulus intervals.

(3) For Figure 3C (Flybowl assay), the authors mention that "simply expressing the opsins decreased baseline locomotor activity compared to empty driver lines". However, the "Empty" controls in 3C appear to refer to opsin-only controls, not driver-only controls. The driver-only controls are not presented in the figure. The use of "empty" differs between the text and the figure, as the text refers to "empty" driver lines, while the figure uses "empty" to apparently refer to opsin-only controls. We recommend changing the terminology across all figures to be unambiguous, e.g., by using "opsin-only" or "driver-only" as opposed to the ambiguous "empty". In addition, the fact that opsin-only controls move less than driver-only controls may suggest some toxicity as a result of the opsin-only construct; this should be discussed further.

(4) Figures 4 and 5 lack the reporting of driver-only controls.

(5) Figures 3 and 4 lack positive controls; that is, the benchmarking of the efficacy of RubyACRs in their respective behavioral paradigms against a known inhibitor, e.g., GtACR1 with green light. To confirm that this GtACR1 transgene is functional, the authors could include GtACR1 with green light as a positive control for these two figures, as they have done for Figure 5-supplement 2 and 3.

(6) Several citations are missing. In their discussion, the authors highlight that shorter wavelengths of light are more attenuated by tissue (lines 278-281); this should be accompanied by the relevant citations (Inagaki et al., 2014). Similarly, the claim that behavioral experiments exhibit greater sensitivity to shorter wavelengths should be substantiated (lines 281-283).

References:

Govorunova EG, Sineshchekov OA, Li H, Wang Y, Brown LS, Spudich JL. 2020. RubyACRs, nonalgal anion channelrhodopsins with highly red-shifted absorption. Proc Natl Acad Sci U S A 117:22833-22840.

Inagaki HK, Jung Y, Hoopfer ED, Wong AM, Mishra N, Lin JY, Tsien RY, Anderson DJ. 2014. Optogenetic control of Drosophila using a red-shifted channelrhodopsin reveals experience-dependent influences on courtship. Nat Methods 11:325-332.

Rockwell AL, Beaver I, Hongay CF. 2019. A direct and simple method to assess Drosophila melanogaster's viability from embryo to adult. J Vis Exp e59996.

Author response:

Reviewer #1 (Public review):

Summary:

Review of the manuscript titled " Mycobacterial Metallophosphatase MmpE acts as a nucleomodulin to regulate host gene expression and promotes intracellular survival".

The study provides an insightful characterization of the mycobacterial secreted effector protein MmpE, which translocates to the host nucleus and exhibits phosphatase activity. The study characterizes the nuclear localization signal sequences and residues critical for the phosphatase activity, both of which are required for intracellular survival.

Strengths:

(1) The study addresses the role of nucleomodulins, an understudied aspect in mycobacterial infections.

(2) The authors employ a combination of biochemical and computational analyses along with in vitro and in vivo validations to characterize the role of MmpE.

Weaknesses:

(1) While the study establishes that the phosphatase activity of MmpE operates independently of its NLS, there is a clear gap in understanding how this phosphatase activity supports mycobacterial infection. The investigation lacks experimental data on specific substrates of MmpE or pathways influenced by this virulence factor.

We thank the reviewer for this insightful comment and agree that identification of the substrate of MmpE is important to fully understand its role in mycobacterial infection.

MmpE is a putative purple acid phosphatase (PAP) and a member of the metallophosphoesterase (MPE) superfamily. Enzymes in this family are known for their catalytic promiscuity and broad substrate specificity, acting on phosphomonoesters, phosphodiesters, and phosphotriesters (Matange et al., Biochem J., 2015). In bacteria, several characterized MPEs have been shown to hydrolyze substrates such as cyclic nucleotides (e.g., cAMP) (Keppetipola et al., J Biol Chem, 2008; Shenoy et al., J Mol Biol, 2007), nucleotide derivatives (e.g., AMP, UDP-glucose) (Innokentev et al., mBio, 2025), and pyrophosphate-containing compounds (e.g., Ap4A, UDP-DAGn) (Matange et al., Biochem J., 2015). Although the binding motif of MmpE has been identified, determining its physiological substrates remains challenging due to the low abundance and instability of potential metabolites, as well as the limited sensitivity and coverage of current metabolomic technologies in mycobacteria.

(2) The study does not explore whether the phosphatase activity of MmpE is dependent on the NLS within macrophages, which would provide critical insights into its biological relevance in host cells. Conducting experiments with double knockout/mutant strains and comparing their intracellular survival with single mutants could elucidate these dependencies and further validate the significance of MmpE's dual functions.

We thank the reviewer for the comment. In our study, we demonstrate that both the nuclear localization and phosphatase activity of MmpE are required for full virulence (Figure 3D–E). Importantly, deletion of the NLS motifs did not impair MmpE’s phosphatase activity in vitro (Figure 2F), indicating that its enzymatic function is structurally independent of its nuclear localization. These findings suggest that MmpE functions as a bifunctional protein, with distinct and non-overlapping roles for its nuclear trafficking and phosphatase activity. We have expanded on this point in the Discussion section “MmpE Functions as a Bifunctional Protein with Nuclear Localization and Phosphatase Activity”.

(3) The study does not provide direct experimental validation of the MmpE deletion on lysosomal trafficking of the bacteria.

We thank the reviewer for the comment. The role of Rv2577/MmpE in phagosome maturation has been demonstrated in M. tuberculosis, where its deletion increases colocalization with lysosomal markers such as LAMP-2 and LAMP-3 (Forrellad et al., Front Microbiol, 2020). In our study, we found that mmpE deletion in M. bovis BCG led to upregulation of lysosomal genes, including TFEB, LAMP1, LAMP2, and v-ATPase subunits, compared to the wild-type strain. These results suggest that MmpE may regulate lysosomal trafficking by interfering with phagosome–lysosome fusion.

To further validate MmpE’s role in phagosome maturation, we will perform fluorescence colocalization assays in THP-1 macrophages infected with BCG/wt, ∆mmpE, complemented, and NLS-mutant strains. Co-staining with LAMP1 and LysoTracker will allow us to assess whether the ∆mmpE mutant is more efficiently trafficked to lysosomes.

(4) The role of MmpE as a mycobacterial effector would be more relevant using virulent mycobacterial strains such as H37Rv.

We thank the reviewer for the comment. Previously, the role of Rv2577/MmpE as a virulence factor has been demonstrated in M. tuberculosis CDC 1551, where its deletion significantly reduced bacterial replication in mouse lungs at 30 days post-infection (Forrellad et al., Front Microbiol, 2020). However, that study did not explore the underlying mechanism of MmpE function. In our work, we found that MmpE enhances M. bovis BCG survival in both macrophages (THP-1 and RAW264.7) and mice (Figure 2A-B, Figure 6A), consistent with its proposed role in virulence. To investigate the molecular mechanism by which MmpE promotes intracellular survival, we used M. bovis BCG as a biosafe surrogate and this model is widely accepted for studying mycobacterial pathogenesis (Wang et al., Nat Immunol, 2025; Wang et al., Nat Commun, 2017; Péan et al., Nat Commun, 2017).

Reviewer #2 (Public review):

Summary:

In this paper, the authors have characterized Rv2577 as a Fe3+/Zn2+ -dependent metallophosphatase and a nucleomodulin protein. The authors have also identified His348 and Asn359 as critical residues for Fe3+ coordination. The authors show that the proteins encode for two nuclease localization signals. Using C-terminal Flag expression constructs, the authors have shown that the MmpE protein is secretory. The authors have prepared genetic deletion strains and show that MmpE is essential for intracellular survival of M. bovis BCG in THP-1 macrophages, RAW264.7 macrophages, and a mouse model of infection. The authors have also performed RNA-seq analysis to compare the transcriptional profiles of macrophages infected with wild-type and MmpE mutant strains. The relative levels of ~ 175 transcripts were altered in MmpE mutant-infected macrophages and the majority of these were associated with various immune and inflammatory signalling pathways. Using these deletion strains, the authors proposed that MmpE inhibits inflammatory gene expression by binding to the promoter region of a vitamin D receptor. The authors also showed that MmpE arrests phagosome maturation by regulating the expression of several lysosome-associated genes such as TFEB, LAMP1, LAMP2, etc. These findings reveal a sophisticated mechanism by which a bacterial effector protein manipulates gene transcription and promotes intracellular survival.

Strength:

The authors have used a combination of cell biology, microbiology, and transcriptomics to elucidate the mechanisms by which Rv2577 contributes to intracellular survival.

Weakness:

The authors should thoroughly check the mice data and show individual replicate values in bar graphs.

We kindly appreciate the reviewer for the advice. We will update the relevant mice data in the revised manuscript.

Reviewer #3 (Public review):

Summary:

In this manuscript titled "Mycobacterial Metallophosphatase MmpE Acts as a Nucleomodulin to Regulate Host Gene Expression and Promote Intracellular Survival", Chen et al describe biochemical characterisation, localisation and potential functions of the gene using a genetic approach in M. bovis BCG and perform macrophage and mice infections to understand the roles of this potentially secreted protein in the host cell nucleus. The findings demonstrate the role of a secreted phosphatase of M. bovis BCG in shaping the transcriptional profile of infected macrophages, potentially through nuclear localisation and direct binding to transcriptional start sites, thereby regulating the inflammatory response to infection.

Strengths:

The authors demonstrate using a transient transfection method that MmpE when expressed as a GFP-tagged protein in HEK293T cells, exhibits nuclear localisation. The authors identify two NLS motifs that together are required for nuclear localisation of the protein. A deletion of the gene in M. bovis BCG results in poorer survival compared to the wild-type parent strain, which is also killed by macrophages. Relative to the WT strain-infected macrophages, macrophages infected with the ∆mmpE strain exhibited differential gene expression. Overexpression of the gene in HEK293T led to occupancy of the transcription start site of several genes, including the Vitamin D Receptor. Expression of VDR in THP1 macrophages was lower in the case of ∆mmpE infection compared to WT infection. This data supports the utility of the overexpression system in identifying potential target loci of MmpE using the HEK293T transfection model. The authors also demonstrate that the protein is a phosphatase, and the phosphatase activity of the protein is partially required for bacterial survival but not for the regulation of the VDR gene expression.

Weaknesses:

(1)   While the motifs can most certainly behave as NLSs, the overexpression of a mycobacterial protein in HEK293T cells can also result in artefacts of nuclear localisation. This is not unprecedented. Therefore, to prove that the protein is indeed secreted from BCG, and is able to elicit transcriptional changes during infection, I recommend that the authors (i) establish that the protein is indeed secreted into the host cell nucleus, and (ii) the NLS mutation prevents its localisation to the nucleus without disrupting its secretion.

We kindly appreciate the reviewer for the advice and will include the relevant experiments in the revised manuscript. The localization of WT MmpE and the NLS mutated MmpE will be tested in the BCG infected macrophages.

Demonstration that the protein is secreted: Supplementary Figure 3 - Immunoblotting should be performed for a cytosolic protein, also to rule out detection of proteins from lysis of dead cells. Also, for detecting proteins in the secreted fraction, it would be better to use Sauton's media without detergent, and grow the cultures without agitation or with gentle agitation. The method used by the authors is not a recommended protocol for obtaining the secreted fraction of mycobacteria.

We agree with the reviewer and we will further validate the secretion of MmpE using the tested protocol.

Demonstration that the protein localises to the host cell nucleus upon infection: Perform an infection followed by immunofluorescence to demonstrate that the endogenous protein of BCG can translocate to the host cell nucleus. This should be done for an NLS1-2 mutant expressing cell also.

We will add this experiment in the revised manuscript.

(2) In the RNA-seq analysis, the directionality of change of each of the reported pathways is not apparent in the way the data have been presented. For example, are genes in the cytokine-cytokine receptor interaction or TNF signalling pathway expressed more, or less in the ∆mmpE strain?

We thank the reviewer for pointing this out and fully agree that conventional KEGG pathway enrichment diagrams do not convey the directionality of individual gene expression changes within each pathway. While KEGG enrichment analysis identifies pathways that are statistically overrepresented among differentially expressed genes, it does not indicate whether individual genes within those pathways are upregulated or downregulated.

To address this, we re-analyzed the expression trends of DEGs within each significantly enriched KEGG pathway. The results show that key immune-related pathways, including cytokine–cytokine receptor interaction, TNF signaling, NF-κB signaling, and chemokine signaling, are collectively upregulated in THP-1 macrophages infected with ∆mmpE strain compared to those infected with the wild-type BCG strain. The full list of DEGs will be provided in the supplementary materials. The complete RNA-seq dataset has been deposited in the GEO database, and the accession number will be included in the revised manuscript.

(3) Several of these pathways are affected as a result of infection, while others are not induced by BCG infection. For example, BCG infection does not, on its own, produce changes in IL1β levels. As the author s did not compare the uninfected macrophages as a control, it is difficult to interpret whether ∆mmpE induced higher expression than the WT strain, or simply did not induce a gene while the WT strain suppressed expression of a gene. This is particularly important because the strain is attenuated. Does the attenuation have anything to do with the ability of the protein to induce lysosomal pathway genes? Does induction of this pathway lead to attenuation of the strain? Similarly, for pathways that seem to be downregulated in the ∆mmpE strain compared to the WT strain, these might have been induced upon infection with the WT strain but not sufficiently by the ∆mmpE strain due to its attenuation/ lower bacterial burden.

We thank the reviewer for the comment. We will update qRT-PCR data with the uninfected macrophages as a control in the revised manuscript.

Wild-type Mycobacterium bovis BCG strain still has the function of inhibiting phagosome maturation (Branzk et al., Nat Immunol, 2014; Weng et al., Nat Commun, 2022). Forrellad et al. previously identified Rv2577/MmpE as a virulence factor in M. tuberculosis and disruption of the MmpE gene impairs the ability of M. tuberculosis to arrest phagosome maturation (Forrellad et al., Front Microbiol, 2020). In our study, transcriptomic and qRTPCR data (Figures 4C and G, S4C) show that deletion of mmpE in M. bovis BCG leads to upregulation of lysosomal biogenesis and acidification genes, including TFEB, LAMP1, and vATPase. To further validate MmpE’s role in phagosome maturation, we will perform fluorescence colocalization assays in THP-1 macrophages infected with BCG/wt, ∆mmpE, complemented, and NLS-mutant strains. Co-staining with LAMP1 and LysoTracker will assess whether the ∆mmpE mutant is more efficiently trafficked to lysosomes.

Furthermore, CFU assays demonstrated that the ∆mmpE strain exhibits markedly reduced bacterial survival in both human THP-1 and murine RAW264.7 macrophages, as well as in mice, compared to the wild-type strain (Figures 4A and C, 6A). These findings suggest that the loss of MmpE compromises bacterial survival, likely due to enhanced lysosomal trafficking and acidification. This supports previous studies showing that increased lysosomal activity promotes mycobacterial clearance (Gutierrez et al., Cell, 2004; Pilli et al., Immunity, 2012).

(4) CHIP-seq should be performed in THP1 macrophages, and not in HEK293T. Overexpression of a nuclear-localised protein in a non-relevant line is likely to lead to several transcriptional changes that do not inform us of the role of the gene as a transcriptional regulator during infection.

We thank the reviewer for the comment. We performed ChIP-seq in HEK293T cells is based on the fact that this cell line is widely used in ChIP-based assays due to its high transfection efficiency, robust nuclear protein expression, and well-annotated genome (Lampe et al., Nat Biotechnol, 2024; Marasco et al., Cell, 2022). These features make HEK293T an ideal system for the initial identification of genome wide chromatin binding profiles of novel nuclear effectors such as MmpE.

Furthermore, we validated the major observations in THP-1 macrophages, including (i) RNAseq of THP-1 cells infected with either WT BCG or ∆mmpE strains revealed significant transcriptional changes in immune and lysosomal pathways (Figure 4A); (ii) Integrated analysis of CUT&Tag and RNA-seq data identified 298 genes in infected THP-1 cells that exhibited both MmpE binding and corresponding expression changes. Among these, VDR was validated as a direct transcriptional target of MmpE using EMSA and ChIP-PCR (Figures 5E-J, S5D-F). Notably, the signaling pathways associated with MmpE-bound genes, including PI3K-Akt-mTOR signaling and lysosomal function, substantially overlap with those transcriptionally modulated in infected THP-1 macrophages (Figures 4B-G, S4B-C, S5C-D), further supporting the biological relevance of the ChIP-seq data obtained from HEK293T cells.

(5) I would not expect to see such large inflammatory reactions persisting 56 days postinfection with M. bovis BCG. Is this something peculiar for an intratracheal infection with 1x107 bacilli? For images of animal tissue, the authors should provide images of the entire lung lobe with the zoomed-in image indicated as an inset.

We thank the reviewer for the comment. The lung inflammation peaked at days 21–28 and had clearly subsided by day 56 across all groups (Figure 6B), consistent with the expected resolution of immune responses to an attenuated strain like M. bovis BCG. This temporal pattern is in line with previous studies using intravenous or intratracheal BCG vaccination in mice and macaques, which also demonstrated robust early immune activation followed by resolution over time (Smith et al., Nat Microbiol, 2025; Darrah et al., Nature, 2020).

In this study, the infectious dose (1×10⁷ CFU intratracheally) was selected based on previous studies in which intratracheal delivery of 1×10⁷CFU produced consistent and measurable lung immune responses and pathology without causing overt illness or mortality (Xu et al., Sci Rep, 2017; Niroula et al., Sci Rep, 2025). We will provide whole-lung lobe images with zoomed-in insets in the revised manuscript.

(6) For the qRT-PCR based validation, infections should be performed with the MmpEcomplemented strain in the same experiments as those for the WT and ∆mmpE strain so that they can be on the same graph, in the main manuscript file. Supplementary Figure 4 has three complementary strains. Again, the absence of the uninfected, WT, and∆mmpE infected condition makes interpretation of these data very difficult.

We thank the reviewer for the comment. As suggested, we will conduct the qRT-PCR experiment including the uninfected, WT, ∆mmpE, Comp-MmpE, and the three complementary strains infecting THP-1 cells. The updated data will be provided in the revised manuscript.

(7) The abstract mentions that MmpE represses the PI3K-Akt-mTOR pathway, which arrests phagosome maturation. There is not enough data in this manuscript in support of this claim. Supplementary Figure 5 does provide qRT-PCR validation of genes of this pathway, but the data do not indicate that higher expression of these pathways, whether by VDR repression or otherwise, is driving the growth restriction of the ∆mmpE strain.

We thank the reviewer for the comment. The role of MmpE in phagosome maturation was previously characterized. Disruption of mmpE impairs the ability of M. tuberculosis to arrest lysosomal trafficking (Forrellad et al., Front Microbiol, 2020). In this study, we further found that MmpE suppresses the expression of key lysosomal genes, including TFEB, LAMP1, LAMP2, and ATPase subunits (Figure 4G), suggesting MmpE is involved in arresting phagosome maturation. As noted, the genes in the PI3K–Akt–mTOR pathway are upregulated in ∆mmpE-infected macrophages (Figure S5C).

To functionally validate this, we will conduct two complementary experimental approaches:

(i) Immunofluorescence assays: We will assess phagosome maturation and lysosomal fusion in THP-1 cells infected with BCG/wt, ∆mmpE, Comp-MmpE, and NLS mutant strains. Colocalization of intracellular bacteria with LAMP1 and LysoTracker will be quantified to determine whether the ∆mmpE strain is more efficiently trafficked to lysosomes.

(ii) CFU assays: We will perform CFU assays in THP-1 cells infected with BCG/wt or ∆mmpE in the presence or absence of PI3K-Akt-mTOR pathway inhibitors (e.g., Dactolisib), to assess whether activation of this pathway contributes to the intracellular growth restriction observed in the ∆mmpE strain.

(8) The relevance of the NLS and the phosphatase activity is not completely clear in the CFU assays and in the gene expression data. Firstly, there needs to be immunoblot data provided for the expression and secretion of the NLS-deficient and phosphatase mutants. Secondly, CFU data in Figure 3A, C, and E must consistently include both the WT and ∆mmpE strain.

We thank the reviewer for the comment. We will provide immunoblot data for the expression and secretion of the NLS-deficient and phosphatase mutants. Additionally, we will revise Figure 3A, 3C, and 3E to consistently include both the WT and ΔmmpE strains in the CFU assays.

Reference

Branzk N, Lubojemska A, Hardison SE, Wang Q, Gutierrez MG, Brown GD, Papayannopoulos V (2014) Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens Nat Immunol 15:1017-25.

Darrah PA, Zeppa JJ, Maiello P, Hackney JA, Wadsworth MH 2nd, Hughes TK, Pokkali S, Swanson PA 2nd, Grant NL, Rodgers MA, Kamath M, Causgrove CM, Laddy DJ, Bonavia A, Casimiro D, Lin PL, Klein E, White AG, Scanga CA, Shalek AK, Roederer M, Flynn JL, Seder RA (2020) Prevention of tuberculosis in macaques after intravenous BCG immunization Nature 577:95-102.

Forrellad MA, Blanco FC, Marrero Diaz de Villegas R, Vázquez CL, Yaneff A, García EA, Gutierrez MG, Durán R, Villarino A, Bigi F (2020) Rv2577 of Mycobacterium tuberculosis Is a virulence factor with dual phosphatase and phosphodiesterase functions Front Microbiol 11:570794.

Gutierrez MG, Master SS, Singh SB, Taylor GA, Colombo MI, Deretic V (2004) Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages Cell 119:753-66.

Innokentev A, Sanchez AM, Monetti M, Schwer B, Shuman S (2025) Efn1 and Efn2 are extracellular 5'-nucleotidases induced during the fission yeast response to phosphate starvation mBio 16: e0299224.

Keppetipola N, Shuman S (2008) A phosphate-binding histidine of binuclear metallophosphodiesterase enzymes is a determinant of 2',3'-cyclic nucleotide phosphodiesterase activity J Biol Chem 283:30942-9.

Lampe GD, King RT, Halpin-Healy TS, Klompe SE, Hogan MI, Vo PLH, Tang S, Chavez A, Sternberg SH (2024) Targeted DNA integration in human cells without double-strand breaks using CRISPR-associated transposases Nat Biotechnol 42:87-98.

Marasco LE, Dujardin G, Sousa-Luís R, Liu YH, Stigliano JN, Nomakuchi T, Proudfoot NJ, Krainer AR, Kornblihtt AR (2022) Counteracting chromatin effects of a splicing-correcting antisense oligonucleotide improves its therapeutic efficacy in spinal muscular atrophy Cell 185:2057-2070.e15.

Matange N, Podobnik M, Visweswariah SS (2015) Metallophosphoesterases: structural fidelity with functional promiscuity Biochem J 467:201-16.

Niroula N, Ghodasara P, Marreros N, Fuller B, Sanderson H, Zriba S, Walker S, Shury TK, Chen JM (2025) Orally administered live BCG and heat-inactivated Mycobacterium bovis protect bison against experimental bovine tuberculosis Sci Rep 15:3764.

Péan CB, Schiebler M, Tan SW, Sharrock JA, Kierdorf K, Brown KP, Maserumule MC,

Menezes S, Pilátová M, Bronda K, Guermonprez P, Stramer BM, Andres Floto R, Dionne MS (2017) Regulation of phagocyte triglyceride by a STAT-ATG2 pathway controls mycobacterial infection Nat Commun 8:14642.

Pilli M, Arko-Mensah J, Ponpuak M, Roberts E, Master S, Mandell MA, Dupont N, Ornatowski W, Jiang S, Bradfute SB, Bruun JA, Hansen TE, Johansen T, Deretic V (2012) TBK-1 promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation Immunity 37:223-34.

Shenoy AR, Capuder M, Draskovic P, Lamba D, Visweswariah SS, Podobnik M (2007) Structural and biochemical analysis of the Rv0805 cyclic nucleotide phosphodiesterase from Mycobacterium tuberculosis J Mol Biol 365:211-25.

Smith AA, Su H, Wallach J, Liu Y, Maiello P, Borish HJ, Winchell C, Simonson AW, Lin PL, Rodgers M, Fillmore D, Sakal J, Lin K, Vinette V, Schnappinger D, Ehrt S, Flynn JL (2025) A BCG kill switch strain protects against Mycobacterium tuberculosis in mice and non-human primates with improved safety and immunogenicity Nat Microbiol 10:468-481.

Wang J, Ge P, Qiang L, Tian F, Zhao D, Chai Q, Zhu M, Zhou R, Meng G, Iwakura Y, Gao GF, Liu CH (2017) The mycobacterial phosphatase PtpA regulates the expression of host genes and promotes cell proliferation Nat Commun 8:244.

Wang J, Li BX, Ge PP, Li J, Wang Q, Gao GF, Qiu XB, Liu CH (2015) Mycobacterium tuberculosis suppresses innate immunity by coopting the host ubiquitin system Nat Immunol 16:237–245

Weng Y, Shepherd D, Liu Y, Krishnan N, Robertson BD, Platt N, Larrouy-Maumus G, Platt FM (2022) Inhibition of the Niemann-Pick C1 protein is a conserved feature of multiple strains of pathogenic mycobacteria Nat Commun 13:5320.

Xu X, Lu X, Dong X, Luo Y, Wang Q, Liu X, Fu J, Zhang Y, Zhu B, Ma X (2017) Effects of hMASP2 on the formation of BCG infection-induced granuloma in the lungs of BALB/c mice Sci Rep 7:2300.

DOI: 10.1101/2025.07.29.667315

Resource: RRID:BDSC_24650

Curator: @maulamb

SciCrunch record: RRID:BDSC_24650

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_30002

Curator: @maulamb

SciCrunch record: RRID:BDSC_30002

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_30034

Curator: @maulamb

SciCrunch record: RRID:BDSC_30034

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_51958

Curator: @maulamb

SciCrunch record: RRID:BDSC_51958

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_79033

Curator: @maulamb

SciCrunch record: RRID:BDSC_79033

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_35600

Curator: @maulamb

SciCrunch record: RRID:BDSC_35600

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_28816

Curator: @maulamb

SciCrunch record: RRID:BDSC_28816

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_39038

Curator: @maulamb

SciCrunch record: RRID:BDSC_39038

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_67229

Curator: @maulamb

SciCrunch record: RRID:BDSC_67229

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_61955

Curator: @maulamb

SciCrunch record: RRID:BDSC_61955

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_44621

Curator: @maulamb

SciCrunch record: RRID:BDSC_44621

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_5905

Curator: @maulamb

SciCrunch record: RRID:BDSC_5905

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_6409

Curator: @maulamb

SciCrunch record: RRID:BDSC_6409

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_58430

Curator: @maulamb

SciCrunch record: RRID:BDSC_58430

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_24472

Curator: @maulamb

SciCrunch record: RRID:BDSC_24472

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_28817

Curator: @maulamb

SciCrunch record: RRID:BDSC_28817

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_59012

Curator: @maulamb

SciCrunch record: RRID:BDSC_59012

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_11724

Curator: @maulamb

SciCrunch record: RRID:BDSC_11724

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_38962

Curator: @maulamb

SciCrunch record: RRID:BDSC_38962

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_64752

Curator: @maulamb

SciCrunch record: RRID:BDSC_64752

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_30043

Curator: @maulamb

SciCrunch record: RRID:BDSC_30043

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_3953

Curator: @maulamb

SciCrunch record: RRID:BDSC_3953

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_10462

Curator: @maulamb

SciCrunch record: RRID:BDSC_10462

What is this?

DOI: 10.1038/s44318-025-00489-y

Resource: RRID:BDSC_64968

Curator: @maulamb

SciCrunch record: RRID:BDSC_64968

What is this?

DOI: 10.1038/s44318-025-00497-y

Resource: RRID:BDSC_63204

Curator: @scibot

SciCrunch record: RRID:BDSC_63204

What is this?

DOI: 10.1038/s44318-025-00497-y

Resource: RRID:BDSC_44106

Curator: @scibot

SciCrunch record: RRID:BDSC_44106

What is this?

DOI: 10.1038/s44318-025-00497-y

Resource: RRID:BDSC_7108

Curator: @scibot

SciCrunch record: RRID:BDSC_7108

What is this?

DOI: 10.1038/s44318-025-00497-y

Resource: RRID:BDSC_38403

Curator: @scibot

SciCrunch record: RRID:BDSC_38403

What is this?

DOI: 10.1038/s44318-025-00497-y

Resource: RRID:BDSC_29866

Curator: @scibot

SciCrunch record: RRID:BDSC_29866

What is this?

DOI: 10.1038/s44318-025-00497-y

Resource: RRID:BDSC_29863

Curator: @scibot

SciCrunch record: RRID:BDSC_29863

What is this?

DOI: 10.1038/s44318-025-00497-y

Resource: RRID:BDSC_43632

Curator: @scibot

SciCrunch record: RRID:BDSC_43632

What is this?

DOI: 10.1038/s44318-025-00497-y

Resource: RRID:BDSC_33775

Curator: @scibot

SciCrunch record: RRID:BDSC_33775

What is this?

DOI: 10.1038/s44318-025-00497-y

Resource: RRID:BDSC_66958

Curator: @scibot

SciCrunch record: RRID:BDSC_66958

What is this?

DOI: 10.1038/s44318-025-00497-y

Resource: RRID:BDSC_33769

Curator: @scibot

SciCrunch record: RRID:BDSC_33769

What is this?

DOI: 10.1038/s44318-025-00497-y

Resource: RRID:BDSC_33779

Curator: @scibot

SciCrunch record: RRID:BDSC_33779

What is this?

DOI: 10.1038/s44318-025-00497-y

Resource: RRID:BDSC_27498

Curator: @scibot

SciCrunch record: RRID:BDSC_27498

What is this?

DOI: 10.1038/s44318-025-00497-y

Resource: RRID:BDSC_28043

Curator: @scibot

SciCrunch record: RRID:BDSC_28043

What is this?

DOI: 10.1038/s44318-025-00497-y

Resource: RRID:BDSC_58256

Curator: @scibot

SciCrunch record: RRID:BDSC_58256

What is this?

DOI: 10.1038/s44318-025-00497-y

Resource: RRID:BDSC_26302

Curator: @scibot

SciCrunch record: RRID:BDSC_26302

What is this?

DOI: 10.1038/s44318-025-00497-y

Resource: RRID:BDSC_32039

Curator: @scibot

SciCrunch record: RRID:BDSC_32039

What is this?

DOI: 10.1038/s44318-025-00497-y

Resource: RRID:BDSC_52905

Curator: @scibot

SciCrunch record: RRID:BDSC_52905

What is this?

Why not sample a vector of size n, instead of sampling 4000 vectors of size 1 in a for loop?

DOCUMENT DE SYNTHÈSE : Les Politiques d'Accompagnement à la Parentalité en France

Source : Rapport d’information N° 1638, Assemblée Nationale, Délégation aux droits des femmes et à l’égalité des chances entre les hommes et les femmes, sur les politiques d’accompagnement à la parentalité, présenté par Mme Sarah Legrain et Mme Delphine Lingemann, enregistré le 24 juin 2025.

Synthèse Exécutive

Ce rapport de la Délégation aux droits des femmes et à l’égalité des chances entre les hommes et les femmes met en lumière les inégalités persistantes dans la répartition des charges domestiques et parentales en France, majoritairement assumées par les femmes.

Il révèle que la parentalité, loin d'être neutre en matière de genre, est une cause majeure des inégalités économiques, professionnelles et sociales entre les hommes et les femmes. La "pénalité parentale" affecte de manière significative la carrière et les revenus des femmes, tandis que les hommes en sont largement épargnés.

Les rapporteures identifient plusieurs axes clés pour favoriser une répartition plus égalitaire des tâches parentales et promouvoir une vision positive et égalitaire de la parentalité, formulant 44 recommandations pour y parvenir.

Ces recommandations couvrent l'éducation et l'information, la prise en compte de la parentalité au travail, l'accompagnement des parents dès le désir d'enfant, la refonte des systèmes de congés parentaux et des modes d'accueil, le soutien aux parents d'adolescents et l'accompagnement des familles monoparentales.

Thèmes Principaux et Idées Clés

1. La Charge Domestique et Parentale Inégalitaire : Un Frein à l'Égalité des Femmes

• Division Sexuée Persistante : Malgré une impression d'égalité, les femmes continuent d'assumer la majeure partie des responsabilités domestiques et parentales. En moyenne, elles réalisent 71% des tâches domestiques et 65% des tâches parentales du foyer. Cette division est profondément enracinée dans un héritage historique et des stéréotypes de genre tenaces.
• Stéréotypes de Genre : L'idée que "les mères savent mieux répondre aux besoins et attentes des enfants que les pères" est très présente chez les adultes (60% y adhèrent) et se perpétue chez les jeunes (54% des 18-24 ans). Ces stéréotypes contribuent à une dévalorisation sociale des tâches considérées comme féminines.
• "Double Journée" des Mères : L'arrivée des enfants aggrave cette inégalité. Pour les femmes, cela représente environ "cinq heures de travail supplémentaire", tandis que pour les hommes, cela "réduit leur temps domestique et parental de deux heures". Les femmes salariées cumulent travail professionnel, domestique et parental, totalisant "onze heures par jour contre moins de dix heures pour les hommes".
• Impact du Système de Congés : La différence de durée entre le congé maternité (16 semaines) et le congé paternité (28 jours) renforce la dynamique d'une "mère 'parent principal' et d'un père 'auxiliaire'". Le congé parental est également majoritairement pris par les mères (94% des cas), ce qui pénalise leur carrière.
• Difficultés des Modes d'Accueil : Le manque et la répartition inégale des places en crèche et chez les assistantes maternelles obligent souvent les mères à compenser les dysfonctionnements du système. "Près de 20% des parents n’obtiennent pas de mode d’accueil, plus de 160 000 ne reprennent pas le travail faute de solution de garde pour leur enfant", les mères étant la "variable d'ajustement".

2. Conséquences Lourdes pour les Mères : Coût Humain, Économique et Social

• Pénalité Parentale au Travail : La parentalité a un "impact négatif de la parentalité sur le parcours professionnel des femmes", alors qu'elle n'a "aucun effet ou presque sur l’évolution professionnelle des hommes". "90% des inégalités de revenu entre les femmes et les hommes sont directement dues à la 'pénalité parentale' que subissent les femmes". Dix ans après l'arrivée du premier enfant, le revenu moyen des femmes chute d'environ 38%.
• Discrimination : Plus de six femmes sur dix estiment qu’être mère est un frein à la carrière. 27% des femmes déclarant être discriminées au travail estiment que cette discrimination est liée à la grossesse ou au congé maternité.
• Vulnérabilité des Mères Solos : Les mères solos (82% des familles monoparentales) sont particulièrement touchées. Elles subissent une "triple pénalité croisée : leur genre, leur situation professionnelle […], leur situation familiale", les exposant aux emplois précaires et mal rémunérés, et augmentant leur risque de pauvreté. "Près d’une mère seule sur cinq est pauvre alors qu’elle a un emploi".
• Risque d'Épuisement et Santé Mentale : La charge disproportionnée entraîne un "risque réel d’épuisement pour les mères". L'isolement peut favoriser la dépression post-partum, qui touche environ 20% des femmes et est la "première cause de la mortalité maternelle dans l’année qui suit la naissance de l’enfant".
• Coût Économique Élevé : Outre la perte de revenus due aux congés maternité et à la réduction d'activité, la séparation a un "lourd coût pour les mères". Une femme séparée sur trois "bascule sous le seuil de pauvreté l’année de la séparation", son niveau de vie baissant d'environ 20% (contre 7% pour les hommes). 39% des enfants vivant en famille monoparentale sont en situation de pauvreté.

3. Propositions pour une Parentalité Égalitaire

Les rapporteures formulent 44 recommandations pour transformer les politiques d'accompagnement à la parentalité, axées sur l'égalité :

Éducation et Information :

• Mettre en place des "cours d’activités domestiques" à l'école ou au collège pour inculquer des compétences à tous les enfants.
• Lancer des "campagnes nationales contre les stéréotypes de genre" sur la parentalité.
• Adopter une "terminologie neutre" (ex: "école pré-élémentaire" au lieu d'"école maternelle", "prestation pour naissance et soin du mineur" au lieu de "congé maternité/paternité").
• Renforcer l'information des parents sur les dispositifs d'accompagnement.

Prise en Compte au Travail :

• Intégrer la parentalité dans la "responsabilité sociétale des entreprises (RSE)" et généraliser la "Charte de la parentalité" aux entreprises de plus de 50 salariés.
• Modifier le Code du travail pour inclure explicitement la parentalité dans les "négociations d’entreprises relatives à l’égalité professionnelle".
• Intégrer des critères sur la parentalité dans le futur "index égalité professionnelle".
• Accorder des "autorisations d’absence" (4 demi-journées/an) aux parents pour les moments clés de la scolarité de leurs enfants.
• Accompagnement dès le Désir d'Enfant et Post-Partum :
• Élargir les "consultations pré-conceptionnelles" au projet parental et permettre au second parent d'assister à tous les rendez-vous médicaux obligatoires de la grossesse.
• Consacrer une séance de préparation à la naissance au "projet parental".
• Renforcer le dispositif d'arrêt en cas d'interruption de grossesse et l'étendre aux interruptions volontaires, avec une autorisation d'absence pour le conjoint.
• Faciliter l'accès aux "consultations sur l’allaitement" et renforcer la "formation des praticiens sur la dépression post-partum".
• Prévoir une "consultation facultative et remboursée à 100% avec un psychologue" pour les mères dans les trois mois après la naissance.
• Étendre le "congé de 'proche aidant'" au second parent en soutien à la mère souffrant de dépression post-partum.
• Lutter contre l'isolement des mères en proposant aux parents "d’être mis en relation avec d’autres parents accueillant leur enfant au même moment".

Réforme des Congés et Modes d'Accueil :

• Congé Paternité : Porter "progressivement le congé paternité à seize semaines, soit à égalité avec le congé maternité". Huit semaines seraient obligatoires (4 à la naissance, 4 après le congé maternité de la mère) et huit facultatives et fractionnables. Cette mesure est un "levier clé pour l’égalité entre les parents" et répond à l'aspiration des pères à s'investir davantage.
• Congé Parental : Réformer le congé parental en "renforçant son attractivité financière sans réduire sa portée pour les ménages modestes", et réfléchir à une "reprise progressive" après le congé.
• Modes d'Accueil : Garantir la "lisibilité et la transparence" des modes de garde, "investir pour augmenter et harmoniser l’offre de crèches sur le territoire", et "revaloriser les métiers de la petite enfance".
• Soutien aux Parents d'Adolescents :
• Élargir les missions des "lieux d’accueil enfants-parents" pour qu’ils puissent "recevoir des adolescents".
• Mettre en place des "politiques publiques ciblant spécifiquement les parents d’adolescents".
• Renforcer l’offre en "pédopsychiatrie" et la "médecine scolaire" face à la dégradation de la santé mentale des jeunes.
• Lancer une "campagne d’information nationale sur la santé mentale des enfants et des adolescents".

Accompagnement des Familles Monoparentales :

• Repenser le "mode de calcul des pensions alimentaires" pour prendre en compte le coût réel de l’entretien d’un enfant et permettre au parent bénéficiaire de "défiscaliser la pension alimentaire".
• Instaurer un "abattement sur le montant de la pension alimentaire pris en compte dans les bases ressources des prestations familiales et des aides au logement, à hauteur de l’allocation de soutien familial (ASF)".
• "Déconjugaliser le versement de l’ASF" et "ouvrir les allocations logement (APL) aux deux parents" pour faciliter l’accueil des enfants.
• Ouvrir aux mères solos la "possibilité de transférer des droits de congés vers un proche de leur choix" et "doubler les jours 'enfant malade'".
• Étudier la création d'un "statut des familles monoparentales" avec des droits spécifiques.

Conclusion des Rapporteures

Les rapporteures affirment que malgré des évolutions, les mères restent le "parent principal", ce qui a des conséquences négatives sur leur santé et leur vie professionnelle.

Une "réforme ambitieuse du système des congés", en particulier du congé second parent, est un "moteur d'égalité" essentiel.

S'inspirant des modèles scandinaves et espagnols, la France peut avancer vers une parentalité égalitaire, non seulement pour l'émancipation des femmes, mais aussi comme réponse aux inquiétudes démographiques.

explains the structure of cubic unit cells in crystallography. It describes the three types: simple cubic (atoms at each corner), body-centered cubic (BCC, with an extra atom in the center), and face-centered cubic (FCC, with atoms on each face). Each corner atom is shared among eight adjacent cells, and the structure helps define how atoms are arranged in a solid. @belfrob99

La investigación buscó identificar los principales problemas en el aprendizaje de las matemáticas y, para ello, utilizó la Matriz de Vester como herramienta para organizar y priorizar las dificultades. Me parece interesante cómo esta matriz me permitió identificar la falta de atención en clase y el abuso de recursos repetitivos, estos son los problemas que evidencie y me parecieron más relevantes por esto mismo, los que debían resolverse con prioridad. sus principales ventajas, es la de dar prioridad para la inclusión de los estudiantes, docentes y padres, lo que hace el proceso más justo y colectivo. Sin embargo, también tiene limitaciones: requiere bastante tiempo y organización, y los resultados dependen de quienes participan, lo que puede volverlo un poco subjetivo. Considero que esta herramienta no solo es útil en matemáticas, sino que puede aplicarse a otras áreas en mi caso la educación fisica o incluso a temas de convivencia escolar y gestión institucional. En mi caso, pienso que podría usarla para analizar la baja participación de los estudiantes en clase, pues al identificar las causas más importantes, como el miedo a equivocarse, sería posible diseñar estrategias para mejorar la confianza y lograr que más alumnos participen. En conclusión, la Matriz de Vester es una herramienta práctica que ayuda a actuar de manera más organizada frente a los problemas o situaciones educativas, favoreciendo decisiones más claras y efectivas.

Este artículo aporta a la educación matemática al emplear la Matriz de Vester como una herramienta para identificar y clasificar las dificultades en el aprendizaje. Su principal fortaleza radica en un enfoque metodológico que combina diagnóstico participativo, análisis de causalidad y una propuesta didáctica adaptada al contexto, lo que lo hace especialmente útil y transferible en entornos rurales. Sin embargo, presenta algunas limitaciones, como el número reducido de participantes y la notable influencia de la experiencia del docente investigador, lo que puede afectar la validez de los resultados. Aunque el marco teórico se basa en la fenomenología didáctica de Freudenthal, la reflexión sobre la Matriz de Vester es algo breve y no se compara con otras metodologías. Los resultados confirman problemas ya conocidos en la literatura, como la desatención, la repetición de estrategias, la baja autodisciplina y las limitaciones socioeconómicas. Sin embargo, su verdadero valor radica en la forma organizada y participativa de abordar estos problemas para guiar la práctica docente. Por último, la secuencia didáctica contextual es un acierto, ya que genera motivación y aprendizajes significativos en los estudiantes, aunque aún queda por analizar su impacto a largo plazo.

Este artículo aporta a la educación matemática al emplear la Matriz de Vester como una herramienta para identificar y clasificar las dificultades en el aprendizaje. Su principal fortaleza radica en un enfoque metodológico que combina diagnóstico participativo, análisis de causalidad y una propuesta didáctica adaptada al contexto, lo que lo hace especialmente útil y transferible en entornos rurales. Sin embargo, presenta algunas limitaciones, como el número reducido de participantes y la notable influencia de la experiencia del docente investigador, lo que puede afectar la validez de los resultados. Aunque el marco teórico se basa en la fenomenología didáctica de Freudenthal, la reflexión sobre la Matriz de Vester es algo breve y no se compara con otras metodologías. Los resultados confirman problemas ya conocidos en la literatura, como la desatención, la repetición de estrategias, la baja autodisciplina y las limitaciones socioeconómicas. Sin embargo, su verdadero valor radica en la forma organizada y participativa de abordar estos problemas para guiar la práctica docente. Por último, la secuencia didáctica contextual es un acierto, ya que genera motivación y aprendizajes significativos en los estudiantes, aunque aún queda por analizar su impacto a largo plazo.

La investigación buscó identificar los principales problemas en el aprendizaje de las matemáticas y, para ello, utilizó la Matriz de Vester como herramienta para organizar y priorizar las dificultades. Me parece interesante cómo esta matriz me permitió identificar la falta de atención en clase y el abuso de recursos repetitivos, estos son los problemas que evidencie y me parecieron más relevantes por esto mismo, los que debían resolverse con prioridad. sus principales ventajas, es la de dar prioridad para la inclusión de los estudiantes, docentes y padres, lo que hace el proceso más justo y colectivo. Sin embargo, también tiene limitaciones: requiere bastante tiempo y organización, y los resultados dependen de quienes participan, lo que puede volverlo un poco subjetivo. Considero que esta herramienta no solo es útil en matemáticas, sino que puede aplicarse a otras áreas en mi caso la educación fisica o incluso a temas de convivencia escolar y gestión institucional. En mi caso, pienso que podría usarla para analizar la baja participación de los estudiantes en clase, pues al identificar las causas más importantes, como el miedo a equivocarse, sería posible diseñar estrategias para mejorar la confianza y lograr que más alumnos participen. En conclusión, la Matriz de Vester es una herramienta práctica que ayuda a actuar de manera más organizada frente a los problemas o situaciones educativas, favoreciendo decisiones más claras y efectivas.

La investigación buscó identificar los principales problemas en el aprendizaje de las matemáticas y, para ello, utilizó la Matriz de Vester como herramienta para organizar y priorizar las dificultades. Me parece interesante cómo esta matriz me permitió identificar la falta de atención en clase y el abuso de recursos repetitivos, estos son los problemas que evidencie y me parecieron más relevantes por esto mismo, los que debían resolverse con prioridad. sus principales ventajas, es la de dar prioridad para la inclusión de los estudiantes, docentes y padres, lo que hace el proceso más justo y colectivo. Sin embargo, también tiene limitaciones: requiere bastante tiempo y organización, y los resultados dependen de quienes participan, lo que puede volverlo un poco subjetivo. Considero que esta herramienta no solo es útil en matemáticas, sino que puede aplicarse a otras áreas en mi caso la educación fisica o incluso a temas de convivencia escolar y gestión institucional. En mi caso, pienso que podría usarla para analizar la baja participación de los estudiantes en clase, pues al identificar las causas más importantes, como el miedo a equivocarse, sería posible diseñar estrategias para mejorar la confianza y lograr que más alumnos participen. En conclusión, la Matriz de Vester es una herramienta práctica que ayuda a actuar de manera más organizada frente a los problemas o situaciones educativas, favoreciendo decisiones más claras y efectivas.

Brayan Piña Este artículo aporta a la educación matemática al emplear la Matriz de Vester como una herramienta para identificar y clasificar las dificultades en el aprendizaje. Su principal fortaleza radica en un enfoque metodológico que combina diagnóstico participativo, análisis de causalidad y una propuesta didáctica adaptada al contexto, lo que lo hace especialmente útil y transferible en entornos rurales. Sin embargo, presenta algunas limitaciones, como el número reducido de participantes y la notable influencia de la experiencia del docente investigador, lo que puede afectar la validez de los resultados. Aunque el marco teórico se basa en la fenomenología didáctica de Freudenthal, la reflexión sobre la Matriz de Vester es algo breve y no se compara con otras metodologías. Los resultados confirman problemas ya conocidos en la literatura, como la desatención, la repetición de estrategias, la baja autodisciplina y las limitaciones socioeconómicas. Sin embargo, su verdadero valor radica en la forma organizada y participativa de abordar estos problemas para guiar la práctica docente. Por último, la secuencia didáctica contextual es un acierto, ya que genera motivación y aprendizajes significativos en los estudiantes, aunque aún queda por analizar su impacto a largo plazo.

Brings together the idea that play is all around us

This is more for the response for children. It is importantly for health benefits, not really for a joyful activity I think.

Is this more of the interactive side of media like when Dora asked a question to the audience expecting an answer, or just any kind

The idea of where make believe and role play come along

La mujer en esta foto parece cansada y preocupada también. ¿Por qué toda la gente en estas fotos parece sucia? ¿No hay agua durante La Gran Depresion?

En mi opinion, el New Deal es algo muy bien para el nación durante el tiempo pero yo sé que hay personas que no le gusta y piensan que era malo y otros grupos usar lo para justificar alguien malo. No sé que es la verdad pero sabes alguien como esto?

La pelicula que se llama Interstellar usa las entrevistas del "Dust Bowl" y se hacen en perpspectiva para mi. Era un tiempo muy duro y sin el perdón.

,

.

.

Napoleon's weakness as well as the delay of taking over Louisiana caused Napoleon to sell the territory

Critica 1: "Onwuegbuzie y Leech (2005) añaden que actualmente los estudios que utilizan un solo método de investigación se convierten en una amenaza para el adelanto de las Ciencias Sociales".

Se menciona que si un estudio es llevado a cabo bajo una sola metodología de investigación se considera una amenaza para las ciencias sociales. Pero yo creo que si un estudio se lleva a cabo bajo una sola metodología es porque esa metodología es la indicada para ejecutarse y cumplir los objetivos que se requieren.

Critica 2: "Brown (2014) afirma que tomar decisiones correctas con respecto al tipo de instrumentos que se van a utilizar en una investigación puede requerir de mucho tiempo y consideración. Por otro lado, a más de decidir qué instrumentos darán a conocer información confiable, es necesario buscar la forma de reunir datos que finalmente sean fáciles y eficientes al momento de recopilarlos y analizarlos. "

Critica 3: "Seidman (1998) recomienda a los investigadores que administren una secuencia de tres entrevistas con los mismos participantes para obtener suficiente información. La primera entrevista se aplica con el propósito de romper el hielo y crear un ambiente de empatía, a la vez que se realiza un rápido barrido de las áreas que serán investigadas posteriormente".

Al momento de realizar 2 o más entrevistas a los mismos participantes, se puede llegar a dar el caso de que los participantes se empiecen a presentar fatiga por el tiempo que lleva respondiendo preguntas, es por esto que esta opción a mí consideración puede que no sea muy eficiente dependiendo el tipo de persona.

Critica 4: " Luego del respectivo análisis de las transcripciones de las dos primeras entrevistas, el propósito de la tercera, según Seidman (1998), es cubrir ciertos temas que no fueron tratados en las sesiones previas"

El realizar una tercera entrevista para abaracra temas que no fueron tocados creo que no es la mejor opción, ya que el tema que no fue abordado se pudiera abordar en las 2 entrevistas anteriores para evitar una sobrecarga en el entrevistado.

Critica 5: "Mackey y Gass (2005) recomiendan a los investigadores novatos o principiantes que antes de conducir un análisis estadístico de datos, se familiaricen con la estadística de su estudio a través de cursos, textos o consultorías con expertos en el área".

En esta parte el autor tiene razón en que los investigadores novatos que trabajan con datos estadisiticos previamente indaguen en la estadistica para una mejor interpretación de datos.

Estoy de acuerdo con lo que plantea Seidman (1998) porque sugiere un proceso progresivo y estructurado que enriquece la calidad de la información recopilada.

Si bien se menciona la metodología mixta como una alternativa integradora, no se profundiza en cómo se articula esta combinación en la práctica. Gheitasi y Lindgren (2015) señalan que los métodos mixtos deben responder a los objetivos del estudio más allá de compromisos filosóficos, lo cual requiere una explicación más detallada que el artículo no proporciona.

Reseña 3

Este texto busca motivar a los que recién se inician en la investigación. Lo bueno es que no solo explica la teoría, sino que también da ejemplos de cómo elegir participantes, qué instrumentos usar, y cómo analizar los datos. Además, insiste mucho en la importancia de la validez y confiabilidad, lo que hace ver que, aunque sea un artículo introductorio nos explica de manera practica su objetivo

reseña 5 el texto está bien para motivar, pero en algunos momentos se repite mucho con lo mismo de la importancia de la validez y la confiabilidad. Está padre que lo digan, pero hubiera estado mejor que mostraran un caso concreto o una mini guía paso a paso. Como que a ratos parece más un repaso de conceptos que un apoyo práctico para principiantes.

Reseña 4 este artículo está bueno porque no se siente tan pesado como otros sobre investigación. Los autores van explicando qué es investigar y cómo se hace, pero de forma bien digerible. Te cuentan lo básico de los métodos cuantitativos, cualitativos y hasta los mixtos, sin tanta palabra rebuscada. Yo lo veo útil para alguien que apenas empieza, porque no te abruma con pura teoría complicada, más bien te va dando una idea general de cómo se arma un trabajo de investigación

Reseña 2

Me parece que el artículo está bien estructurado porque va de lo general a lo específico. Empieza con una reseña histórica, luego explica los tipos de métodos y finalmente habla de aspectos prácticos como los instrumentos de recolección de datos, análisis, validación y presentación de resultados. Se nota que su intención es servir de guía básica para principiantes, y cumple con eso porque está escrito de forma sencilla, sin tanto tecnicismo.

me parece interesante como citan la validez y confiabilidad ya que se entiende que esto da solidez y credibilidad. Esto garantiza que las conclusiones de un estudio no estén basadas en datos dudosos.

E igual puedo observar como es que la investigación no solo es recolectar y llenarnos de número e información, sino que estos datos sean reales

Me parece interesante que incluyan las entrevistas, ya que esto brinda información útil y concreta para quienes estamos iniciando en las investigaciones y esto aporta una visión amplia de las herramientas disponibles.

La constante dependencia a ciertos autores como Dornyei como principales referencias centrales me parece interesante como es que toman a este y otro autor para la constante observación

Miller y Glassner (2016) señalan que las entrevistas pueden ofrecer descripciones auténticas, aunque no se desarrollen en contextos naturales. Este punto es valioso, pero el texto no aborda cómo el diseño de la entrevista (por ejemplo, el lugar, la relación entrevistador-entrevistado) puede influir en la autenticidad de las respuestas.

Dornyei (2007) destaca la popularidad de los cuestionarios por su facilidad y rapidez. Aunque esto es cierto, el texto no problematiza el riesgo de que esta herramienta, por su estructura cerrada, limite la profundidad de las respuestas. Sería útil advertir que, si bien son eficientes, pueden no captar matices importantes en fenómenos complejos

Aunque el artículo se propone ofrecer una “discusión concisa” de conceptos básicos, la densidad de autores citados y la amplitud de temas tratados podrían resultar abrumadoras para investigadores principiantes.

La explicación de Brown (1988) sobre los dos tipos de investigación es clara y pertinente. No obstante, el texto podría haber enriquecido esta sección con ejemplos concretos de cada tipo en el ámbito de la enseñanza

Es de suma importancia dar a conocer los resultados de nuestra investigación de una forma clara y precisa, ya que cualquier persona que lea el documento tiene que entenderlos y comprender a lo que queremos llegar. Los resultados serán significativos para la toma de decisiones en un futuro.

Concuerdo que siempre tenemos que tener a una cierta población o hacer un segmento de mercado para que nuestra investigación sea más eficaz y más detallada

Critica 5.

Creswell (2009) sostiene que, en investigación cuantitativa, el investigador se preocupa de dos temas de validez: la calidad de los resultados de los instrumentos usados y la calidad de las conclusiones obtenidas de los resultados del análisis cuantitativo. Eso es muy cierto ya que al ser datos precisos deben de estar 100% fundamentados y validados para una buena investigación

Seidman, considera contar con tres entrevistas, siento que muchos autores no consideraron mucho el sesgo, teniendo en cuanta el amplio espectro de búsqueda de respuestas. El hecho de que la mayoría tiene una forma de entrevista o recolección de datos muy compleja me hace pensar más que sera una prueba y error y no como tal un descubrimiento, buscar efectividad es bueno, pero hacer pensar que tu modelo es el correcto, es el peor error humano posible.

Creswell y Plano Clark destacan la rigurosidad necesaria en la recolección de datos dentro de la metodología mixta, se corre el riesgo de que el proceso se vuelva demasiado complejo y demandante para el investigador. Coordinar de manera exhaustiva los procedimientos cuantitativos y cualitativos no solo exige más tiempo y recursos, sino que también puede generar dificultades en la integración final de los datos, lo cual pone en cuestión la viabilidad práctica de este enfoque cuando no se cuenta con una planeación clara o con las competencias suficientes.

POST 4. SON TECNICAS DE RECOPILACIÓN DIFERENTE, LA CUALITATIVA ES RECOPILACIÓN DE DATOS DE "OBSERVACIÓN" MÁS NO NÚMERICA COMO LO ES LA TECNICA CUANTITATIVA, QUE CIERTAMENTE ES NÚMERICA Y CONTABLE POR LO QUE PUEDE SER MAS ACERTADA Y VERIDICA

Concuerdo que ambos métodos tienen que ir de la mano ya que buscan el conocimiento y la comprensión de un tema en especifico o buscarle una solución a un problema. Al analizar los datos es contribuir al conocimiento

En lo personal, me parece interesante cómo la investigación social cuantitativa surge imitando el modelo de las ciencias naturales del siglo XIX. Entiendo que en ese momento era lógico querer darle a lo social la misma validez y objetividad que tenían disciplinas como la física

Yo pienso que lo que plantea McKay (2006) es muy cierto: la investigación sí ayuda a los docentes a ser mejores maestros, porque los obliga a cuestionar su práctica y buscar nuevas formas de enseñar. Sin embargo, siento que su visión es un poco limitada, porque reduce el valor de la investigación solo a mejorar la enseñanza. Para mí, investigar no solo sirve para encontrar respuestas u orientaciones, usino también para abrir la mente del docente, motivarlo a innovar y hasta transformar la manera en que se entiende l educación.

Los autores destacan las ventajas de cuestionarios y entrevistas como instrumentos de recolección de datos, pero no profundizan en sus limitaciones. La elección de instrumentos no debería centrarse solo en la accesibilidad o versatilidad, sino también en la calidad y pertinencia de los resultados que se esperan obtener.

2

Habla sobre una distinción más general y conocida de 2 autores diferentes en donde la metodología cuantitativa y cualitativa ya que el método de realizar el análisis de cada una de ellas es abierta y no numérica al contrario del otro. sin embargo, al principio dice que son muy similares

Aunque resaltan la validez de trabajar con muestras pequeñas en la investigación cualitativa, pueden tener limitaciones. Un número reducido de participantes puede introducir sesgos o dejar fuera perspectivas relevantes del fenómeno estudiado. Además, la falta de criterios claros y uniformes sobre el tamaño muestral en estudios cualitativos puede afectar y la comparabilidad entre investigaciones.

POST 3, PIENSO DIFERENTE A LORD RUTHERFORD YA QUE SI BIEN LOS DATOS CUANTITATIVOS SON HASTA CIERTA PARTE EXACTOS, NO QUIERE DECIR QUE LOS OTROS TIPOS DE INVESTIGACIÓN NO LO SEAN O QUE SEAN MENOS IMPORTANTES, YA QUE SIRVEN PARA DIFERENTES TIPOS DE INVESTIGACIONES Y TODAS SON BUENAS DEPENDEINDO DONDE LAS DESENVUELVAS

Critica 2.

De acuerdo con Mackey y Gass (2005) y sus recomendaciones de un análisis de datos, nos fomentan que es recomendable estar relacionados y creo es algo muy importante ya que se debe saber interpretar cuantitativamente cada dato

El hecho de que en 1995 apenas se hizo el diferenciador entre lo cuantitativo y cualitativo, es muy poco razonable, pero se entiende que no querían hacerlo, ya que en todo momento estaba sumergida por el area científica.

POST 2. AÚN EN EL METODO CIENTIFICO PUEDE HABER SESGO PORQUE LOS INVESTIGADORES OSMOS SERES HUMANOS, TENEMOS CREENCIAS, PREFERENCIAS Y SOMO SECEPTIBLES, ASI QUE NO ES 100% CONFIABLE O VERIDICO

Justificación: Tradicionalmente, la investigación, como actividad científica, ha sido considerada como una tarea exclusiva de grupos académicos de élite; sin embargo, los requerimientos de la sociedad y la Academia actual han originado una mayor necesidad de llevar a cabo estudios científicos y reportarlos en publicaciones académicas.

Rutherford tenía cierta razón al resaltar la importancia de medir en ciencia, pero su afirmación resulta parcial y no del todo correcta. Un conocimiento no es “pobre” por no ser numérico; simplemente responde a otra lógica. Lo cuantitativo aporta precisión y ser objetivo, mientras lo cualitativo aporta el sentido del porque

Post #2 Estoy totalmente de acuerdo con el pensamiento de Bernal (2013) que una investigación cualitativa no tiene que ser generalizada, bien dicen cada cabeza es un mundo y cada quien puede verlo desde un panorama o punto de vista diferente e incluso otros investigadores o lectores encuentren similitudes y apliquen lo aprendido en contextos parecidos.

El artículo de Rodas Pacheco y , aunque útil como introducción, presenta varias limitaciones. En primer lugar, su tratamiento de la metodología es demasiado superficial y se limita a definiciones generales sin ofrecer ejemplos concretos de aplicación en investigaciones reales. Además, no desarrolla con suficiente detalle los procedimientos estadísticos en el enfoque cuantitativo ni las técnicas de codificación en el cualitativo, Finalmente, al ser un texto más descriptivo que analítico, corre el riesgo de repetir información ya ampliamente disponible en manuales básicos de metodología, sin aportar innovaciones significativas al campo.

Aunque el método mixto busca integrar lo mejor de ambos enfoques, su aplicación puede volverse compleja y demandante; requiere de un alto nivel de preparación metodológica y claridad

Metodología Katia Amaya y Alonso Martínez

Cada aportación es de suma importancia y siempre se deben respetar cada una de estas, siempre y cuando sean coherentes, importantes y validas.

1

el autor hace referencia que el método científico está basado diciendo que cualquier conocimiento que no puede ser medido numéricamente era un conocimiento pobre y difiero en que las ciencias sociales antes de ser reconocidas ya podían ser sustentables.

segundo motivo: Motivar Alonso Martínez y Katia Amaya

Justificacion del tema: Tradicionalmente, la investigación, como actividad científica, ha sido considerada como una tarea exclusiva de grupos académicos de élite; sin embargo, los requerimientos de la sociedad y la Academia actual han originado una mayor necesidad de llevar a cabo estudios científicos y reportarlos en publicaciones académicas.

primer objetivo: proveer Katia Amaya y Alonso Martinez

POST 1, DIFIERO TOTALMENTE EN LO QE SE MANIFIESTA YA QUE UNA PERSONA COMÚN PUEDE SER BUENA SI SE DEDICA A INVESTIGAR Y COCNOCER A PROFUNDIDAD DEL TEMA

Critica 1.

Al aplicar las entrevistas según Seidman (1998) establece que es bueno implementar 3 entrevistas para romper el hielo, yo creo que no necesariamente tengan q ser 3, sino una introducción con una buena comunicación tipo platica y después la entrevista que es la va a permitir al investigador poder involucrarse más que es como lo dice el autor y un desenlace muy breve dando a entender que se logró el objetivo de la entrevista

justificación de la investigación Alonso Martínez y Katia Amaya

Justificación: Recolección, análisis, exploración y representación de datos; al igual que la interpretación y validación de resultados.

Metodología: Una reseña histórica, y un análisis sucinto de los distintos métodos de investigación y los componentes de estos procesos, a saber: participantes; recolección, análisis, exploración y representación de datos; al igual que la interpretación y validación de resultados.

Esta es la justificación de la investigación : Tradicionalmente, la investigación, como actividad científica, ha sido considerada como una tarea exclusiva de grupos académicos de élite; sin embargo, los requerimientos de la sociedad y la Academia actual han originado una mayor necesidad de llevar a cabo estudios científicos y reportarlos en publicaciones académicas.

recolección, análisis, exploración y representación de datos; al igual que la interpretación y validación de resultados.

Tradicionalmente, la investigación, como actividad científica, ha sido considerada como una tarea exclusiva de grupos académicos de élite; sin embargo, los requerimientos de la sociedad y la Academia actual han originado una mayor necesidad de llevar a cabo estudios científicos y reportarlos en publicaciones académicas.

Metodología

Una reseña histórica, y un análisis sucinto de los distintos métodos de investigación y los componentes de estos procesos, a saber: participantes; recolección, análisis, exploración y representación de datos; al igual que la interpretación y validación de resultados.

Esta es la justificación de la investigación. Tradicionalmente, la investigación, como actividad científica, ha sido considerada como una tarea exclusiva de grupos académicos de élite; sin embargo, los requerimientos de la sociedad y la Academia actual han originado una mayor necesidad de llevar a cabo estudios científicos y reportarlos en publicaciones académicas.

Justificación Tradicionalmente, la investigación, como actividad científica, ha sido considerada como una tarea exclusiva de grupos académicos de élite; sin embargo, los requerimientos de la sociedad y la Academia actual han originado una mayor necesidad de llevar a cabo estudios científicos y reportarlos en publicaciones académicas.

Justificación.

Tradicionalmente, la investigación, como actividad científica, ha sido considerada como una tarea exclusiva de grupos académicos de élite; sin embargo, los requerimientos de la sociedad y la Academia actual han originado una mayor necesidad de llevar a cabo estudios científicos y reportarlos en publicaciones académicas.

Metodología Una reseña histórica, y un análisis de métodos de investigación y los componentes de estos procesos, recolección de análisis, exploración y representación de datos.

Esta es la justificacion Tradicionalmente, la investigación, como actividad científica, ha sido considerada como una tarea exclusiva de grupos académicos de élite; sin embargo, los requerimientos de la sociedad y la Academia actual han originado una mayor necesidad de llevar a cabo estudios científicos y reportarlos en publicaciones académicas.

Metodología Una reseña histórica, y un análisis de métodos de investigación y los componentes de estos procesos, recolección de análisis, exploración y representación de datos.

Metodología

Una reseña histórica, y un análisis sucinto de los distintos métodos de investigación y los componentes de estos procesos, a saber: participantes; recolección, análisis, exploración y representación de datos; al igual que la interpretación y validación de resultados.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

Reply to the reviewers

We thank the reviewers for their careful assessment and enthusiastic appreciation of our work.

__Reviewer #1 (Evidence, reproducibility and clarity (Required)): __In this article, Thomas et al. use a super-resolution approach in living cells to track proteins involved in the fusion event of sexual reproduction. They study the spatial organization and dynamics of the actin fusion focus, a key structure in cell-cell fusion in Schizosaccharomyces pombe. The researchers have adapted a high-precision centroid mapping method using three-color live-cell epifluorescence imaging to map the dynamic architecture of the fusion focus during yeast mating. The approach relies on tracking the centroid of fluorescence signals for proteins of interest, spatially referenced to Myo52-mScarlet-I (as a robust marker) and temporally referenced using a weakly fluorescent cytosolic protein (mRaspberry), which redistributes strongly upon fusion. The trajectories of five key proteins, including markers of polarity, cytoskeleton, exocytosis and membrane fusion, were compared to Myo52 over a 75-minute window spanning fusion. Their observations indicate that secretory vesicles maintain a constant distance from the plasma membrane whereas the actin network compacts. Most importantly, they discovered a positive feedback mechanism in which myosin V (Myo52) transports Fus1 formin along pre-existing actin filaments, thereby enhancing aster compaction.

This article is well written, the arguments are convincing and the assertions are balanced. The centroid tracking method has been clearly and solidly controlled. Overall, this is a solid addition to our understanding of cytoskeletal organization in cell fusion.

Major comments: No major comment.

Minor comments: _ Page 8 authors wrote "Upon depletion of Myo52, Ypt3 did not accumulate at the fusion focus (Figure 3C). A thin, wide localization at the fusion site was occasionally observed (Figure 3C, Movies S3)" : Is there a quantification of this accumulation in the mutant?

We will provide the requested quantification. The localization is very faint, so we are not sure that quantification will capture this faithfully, but we will try.

_ The framerate of movies could be improved for reader comfort: For example, movie S6 lasts 0.5 sec.

We agree that movies S3 and S6 frame rates could be improved. We will provide them with slower frame rate.

Reviewer #1 (Significance (Required)):

This study represents a conceptual and technical breakthrough in our understanding of cytoskeletal organization during cell-cell fusion. The authors introduce a high-precision, three-color live-cell centroid mapping method capable of resolving the spatio-temporal dynamics of protein complexes at the nanometer scale in living yeast cells. This methodological innovation enables systematic and quantitative mapping of the dynamic architecture of proteins at the cell fusion site, making it a powerful live-cell imaging approach. However, it is important to keep in mind that the increased precision achieved through averaging comes at the expense of overlooking atypical or outlier behaviors. The authors discovered a myosin V-dependent mechanism for the recruitment of formin that leads to actin aster compaction. The identification of Myo52 (myosin V) as a transporter of Fus1 (formin) to the fusion focus adds a new layer to our understanding of how polarized actin structures are generated and maintained during developmentally regulated processes such as mating.

Previous studies have shown the importance of formins and myosins during fusion, but this paper provides a quantitative and dynamic mapping that demonstrates how Myo52 modulates Fus1 positioning in living cells. This provides a better understanding of actin organization, beyond what has been demonstrated by fixed-cell imaging or genetic perturbation.

Audience: Cell biologists working on actin dynamics, cell-cell fusion and intracellular transport. Scientists involved in live-cell imaging, single particle tracking and cytoskeleton modeling.

I have expertise in live-cell microscopy, image analysis, fungal growth machinery and actin organization.

We thank the reviewer for their appreciation of our work.

__Reviewer #2 (Evidence, reproducibility and clarity (Required)): __ A three-color imaging approach to use centroid tracking is employed to determine the high resolution position over time of tagged actin fusion focus proteins during mating in fission yeast. In particular, the position of different protein components (tagged in a 3rd color) were determined in relation to the position (and axis) of the molecular motor Myo52, which is tagged with two different colors in the mating cells. Furthermore, time is normalized by the rapid diffusion of a weak fluorescent protein probe (mRaspberry) from one cell to the other upon fusion pore opening. From this approach multiple important mechanistic insights were determined for the compaction of fusion focus proteins during mating, including the general compaction of different components as fusion proceeds with different proteins having specific stereotypical behaviors that indicate underlying molecular insights. For example, secretory vesicles remain a constant distance from the plasma membrane, whereas the formin Fus1 rapidly accumulates at the fusion focus in a Myo52-dependent manner.

I have minor suggestions/points: (1) Figure 1, for clarity it would be helpful if the cells shown in B were in the same orientation as the cartoon cells shown in A. Similarly, it would be helpful to have the orientation shown in D the same as the data that is subsequently presented in the rest of the manuscript (such as Figure 2) where time is on the X axis and distance (position) is on the Y axis.

We have turned each image in panel B by 180° to match the cartoon in A. For panel D, we are not sure what the reviewer would like. This panel shows the coordinates of each Myo52 position, whereas Figure 2 shows oriented distance (on the Y axis) over time (on the X axis). Perhaps the reviewer suggests that we should display panel D with a rotation onto the Y axis rather than the X axis. We feel that this would not bring more clarity and prefer to keep it as is.

(2) Figure 2, for clarity useful to introduce how the position of Myo52 changes over time with respect to the fusion site (plasma membrane) earlier, and then come back to the positions of different proteins with respect to Myo52 shown in 2E. Currently the authors discuss this point after introducing Figure 2E, but better for the reader to have this in mind beforehand.

We have added a sentence at the start of the section describing Figure 2, pointing out that the static appearance of Myo52 is due to it being used as reference, but that in reality, it moves relative to the plasma membrane: “Because Myo52 is the reference, its trace is flat, even though in reality Myo52 also moves relative to other proteins and the plasma membrane (see Figure 2E)”. This change is already in the text.

(3) First sentence of page 8 "..., peaked at fusion time and sharply dropped post-fusion (Figure S3)." Figure S3 should be cited so that the reader knows where this data is presented.

Thanks, we have added the missing figure reference to the text.

(4) Figure 3D-H, why is Exo70 used as a marker for vesicles instead of Ypt3 for these experiments? Exo70 seems to have a more confusing localization than Ypt3 (3C vs 3D), which seems to complicate interpretations.

There are two main reasons for this choice. First, the GFP-Ypt3 fluorescence intensity is lower than that of Exo70-GFP, which makes analysis more difficult and less reliable. Second, in contrast to Exo70-GFP where the endogenous gene is tagged at the native genomic locus, GFP-Ypt3 is expressed as additional copy in addition to endogenous untagged Ypt3. Although GFP-Ypt3 was reported to be fully functional as it can complement the lethality of a ypt3 temperature sensitive mutant (Cheng et al, MBoC 2002), its expression levels are non-native and we do not have a strain in which ypt3 is tagged at the 5’ end at the native genomic locus. For these reasons, we preferred to examine in detail the localization of Exo70. We do not think it complicates interpretations. Exo70 faithfully decorates vesicles and exhibits the same localization as Ypt3 in WT cells (see Figure 2D) and in myo52-AID (see Figure 3C-D). We realize that our text was a bit confusing as we opposed the localization of Exo70 and Ypt3, when all we wanted to state was that the Exo70-GFP signal is stronger. We have corrected this in the text.

(5) Page 10, end of first paragraph, "We conclude...and promotes separation of Myo52 from the vesicles." This is an interesting hypothesis/interpretation that is consistent with the spatial-temporal organization of vesicles and the compacting fusion focus, but the underlying molecular mechanism has not be concluded.

This is an interpretation that is in line with our data. Firm conclusion that the organization of the actin fusion focus imposes a steric barrier to bulk vesicle entry will require in vitro reconstitution of an actin aster driven by formin-myosin V feedback and addition of myosin V vesicle-like cargo, which can be a target for future studies. To make clear that it is an interpretation and not a definitive statement, we have added “likely” to the sentence, as in: “We conclude that the distal position of vesicles in WT cells is a likely steric consequence of the architecture of the fusion focus, which restricts space at the center of the actin aster and promotes separation of Myo52 from the vesicles”.

(6) Figure 5F and 5G, the results are confusing and should be discussed further. Depletion of Myo52 decreases Fus1 long-range movements, indicating that Fus1 is being transported by Myo52 (5F). Similarly, the Fus1 actin assembly mutant greatly decreases Fus1 long-range movements and prevents Myo52 binding (5G), perhaps indicating that Fus1-mediated actin assembly is important. It seems the author's interpretations are oversimplified.

We show that Myo52 is critical for Fus1 long-range movements, as stated by the reviewer. We also show that Fus1-mediated actin assembly is important. The question is in what way.

One possibility is that FH2-mediated actin assembly powers the movement, which in this case represents the displacement of the formin due to actin monomer addition on the polymerizing filament. A second possibility is that actin filaments assembled by Fus1 somehow help Myo52 move Fus1. This could be for instance because Fus1-assembled actin filaments are preferred tracks for Myo52-mediated movements, or because they allow Myo52 to accumulate in the vicinity of Fus1, enhancing their chance encounter and thus the number of long-range movements (on any actin track). Based on the analysis of the K1112A point mutant in Fus1 FH2 domain, our data cannot discriminate between these three different options, which is why we concluded that the mutant allele does not allow us to make a firm conclusion. However, the Myo52-dependence clearly shows that a large fraction of the movements requires the myosin V. We have clarified the end of the paragraph in the following way: “Therefore, analysis of the K1112A mutant phenotype does not allow us to clearly distinguish between Fus1-powered from Myo52-powered movements. Future work will be required to test whether, in addition to myosin V-dependent transport, Fus1-mediated actin polymerization also directly contributes to Fus1 long-range movements.”

(7) Figure 6, why not measure the fluorescence intensity of Fus1 as a proxy for the number of Fus1 molecules (rather than the width of the Fus1 signal), which seems to be the more straight-forward analysis?

The aim of the measurement was to test whether Myo52 and Fus1 activity help focalize the formin at the fusion site, not whether these are required for localization in this region. This is why we are measuring the lateral spread of the signal (its width) rather than the fluorescence intensity of the signal. We know from previous work that Fus1 localizes to the shmoo tip independently of myosin V (Dudin et al, JCB 2015), and we also show this in Figure 6. However, the precise distribution of Fus1 is wider in absence of the myosins.

We can and will measure intensities to test whether there is also a quantitative difference in the number of molecules at the shmoo tip.

(8) Figure 7, the authors should note (and perhaps discuss) any evidence as to whether activation of Fus1 to facilitate actin assembly depends upon Fus1 dissociating from Myo52 or whether Fus1 can be activated while still associated with Myo52, as both circumstances are included in the figure.

This is an interesting point. We have no experimental evidence for or against Fus1 dissociating from Myo52 to assemble actin. However, it is known that formins rotate along the actin filament double helix as they assemble it, a movement that seems poorly compatible with processive transport by myosin V. In Figure 7, we do not particularly want to imply that Myo52 associates with Fus1 linked or not with an actin filament. The figure serves to illustrate the focusing mechanism of myosin V transporting a formin, which is more evident when we draw the formin attached to a filament end. We have now added a sentence in the figure legend to clarify this point: “Note that it is unknown whether Myo52 transports Fus1 associated or not with an actin filament.”

(9) Figure 7, the color of secretory vesicles should be the same in A and B.

This is now corrected.

Reviewer #2 (Significance (Required)):

This is an impactful and high quality manuscript that describes an elegant experimental strategy with important insights determined. The experimental imaging strategy (and analysis), as well as the insight into the pombe mating fusion focus and its comparison to other cytoskeletal compaction events will be of broad scientific interest.

We thank the reviewer for their appreciation of our work.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Summary:

Fission yeast cell-cell fusion during mating is mediated by an actin-based structure called the 'fusion focus', which orchestrates actin polymerization by the mating-specific formin, Fus1, to direct polarized secretion towards the mating site. In the current study, Thomas and colleagues quantitatively map the spatial distribution of proteins mediating cell-cell fusion using a three-color fluorescence imaging methodology in the fission yeast Schizosaccharomyces pombe. Using Myo52 (Type V myosin) as a fluorescence reference point, the authors discover that proteins known to localize to the fusion focus have distinct spatial distributions and accumulation profiles at the mating site. Myo52 and Fus1 form a complex in vivo detected by co-immunoprecipitation and each contribute to directing secretory vesicles to the fusion focus. Previous work from this group has shown that the intrinsically disordered region (IDR) of Fus1 plays a critical role in forming the fusion focus. Here, the authors swap out the IDR of fission yeast Fus1 for the IDR of an unrelated mammalian protein, coincidentally called 'fused in sarcoma' (FUS). They express the Fus1∆IDR-FUSLC-27R chimera in mitotically dividing fission yeast cells, where Fus1 is not normally expressed, and discover that the Fus1∆IDR-FUSLC-27R chimera can travel with Myo52 on actively polymerizing actin cables. Additionally, they show that acute loss of Myo52 or Fus1 function, using Auxin-Inducible Degradation (AID) tags and point mutations, impair the normal compaction of the fusion focus, suggesting that direct interaction and coordination of Fus1 and Myo52 helps shape this structure.

Major Comments:

(1) In the Results section for Figure 2, the authors claim that actin filaments become shorter and more cross-linked they move away from the fusion site during mating, and suggest that this may be due to the presence of Myo51. However, the evidence to support this claim is not made clear. Is it supported by high-resolution electron microscopy of the actin filaments, or some other results? This needs to be clarified.

Sorry if our text was unclear. The basis for the claim that actin filaments become shorter comes from our observation that the average position of tropomyosin and Myo51, both of which decorate actin filaments, is progressively closer to both Fus1 and the plasma membrane. Thus, the actin structure protrudes less into the cytosol as fusion progresses. The basis for claiming that Myo51 promotes actin filament crosslinking comes mainly from previously published papers, which had shown that 1) Myo51 forms complexes with the Rng8 and Rng9 proteins (Wang et al, JCB 2014), and 2) the Myo51-Rng8/9 not only binds actin through Myo51 head domain but also binds tropomyosin-decorated actin through the Rng8/9 moiety (Tang et al, JCB 2016; reference 27 in our manuscript). We had also previously shown that these proteins are necessary for compaction of the fusion focus (Dudin et al, PLoS Genetics 2017; reference 28 in our manuscript). Except for measuring the width of Fus1 distribution in myo51∆ mutants, which confirms previous findings, we did not re-investigate here the function of Myo51.

We have now re-written this paragraph to present the previous data more clearly: “The distal localization of Myo51 was mirrored by that of tropomyosin Cdc8, which decorates linear actin filaments (Figure 2B) (Hatano et al, 2022). The distal position of the bulk of Myo51-decorated actin filaments was confirmed using Airyscan super-resolution microscopy (Figure 2B, right). Thus, the average position of actin filaments and decreasing distance to Myo52 indicates they initially extend a few hundred nanometers into the cytosol and become progressively shorter as fusion proceeds. Previous work had shown that Myo51 cross-links and slides Cdc8-decorated actin filaments relative to each other (Tang et al, 2016) and that both proteins contribute to compaction of the fusion focus in the lateral dimension along the cell-cell contact area (perpendicular to the fusion axis) (Dudin et al, 2017). We confirmed this function by measuring the lateral distribution of Fus1 along the cell-cell contact area (perpendicular to the fusion axis), which was indeed wider in myo51∆ than WT cells (see below Figure 6A-B).”

(2) In Figure 4, the authors comment that disrupting Fus1 results in more disperse Myo52 spatial distribution at the fusion focus, raising the possibility that Myo52 normally becomes focused by moving on the actin filaments assembled by Fus1. This can be tested by asking whether latrunculin treatment phenocopies the 'more dispersed' Myo52 localization seen in fus1∆ cells? If Myo52 is focused instead by its direct interaction with Fus1, the latrunculin treatment should not cause the same phenotype.

This is in principle a good idea, though it is technically challenging because pharmacological treatment of cell pairs in fusion is difficult to do without disturbing pheromone gradients which are critical throughout the fusion process (see Dudin et al, Genes and Dev 2016). We will try the experiment but are unsure about the likelihood of technical success.

We note however that a similar experiment was done previously on Fus1 overexpressed in mitotic cells (Billault-Chaumartin et al, Curr Biol 2022; Fig 1D). Here, Fus1 also forms a focus and latrunculin A treatment leads to Myo52 dispersion while keeping the Fus1 focus, which is in line with our proposal that Myo52 becomes focused by moving on Fus1-assembled actin filaments. Similarly, we showed in Figure 5B that Latrunculin A treatment of mitotic cells expressing Fus1∆IDR-FUSLC-27R also results in Myo52, but not Fus1 dispersion.

(3) The Fus1∆IDR-FUSLC-27R chimera used in Figure 5 is an interesting construct to examine actin-based transport of formins in cells. I was curious if the authors could provide the rates of movement for Myo52 and for Fus1∆IDR-FUSLC-27R, both before and after acute depletion of Myo52. It would be interesting to see if loss of Myo52 alters the rate of movement, or instead the movement stems from formin-mediated actin polymerization.

We will measure these rates.

(4) Also, Myo52 is known to interact with the mitotic formin For3. Does For3 colocalize with Myo52 and Fus1∆IDR-FUSLC-27R along actin cables?

This is an interesting question for which we do not have an answer. For technical reasons, we do not have the tools to co-image For3 with Fus1∆IDR-FUSLC-27R because both are tagged with GFP. We feel that this question goes beyond the scope of this paper.

(5) If Fus1∆IDR-FUSLC-27R is active, does having ectopic formin activity in mitotic cells affect actin cable architecture? This could be assessed by comparing phalloidin staining for wildtype and Fus1∆IDR-FUSLC-27R cells.

We are not sure what the purpose of this experiment is, or how informative it would be. If it is to evaluate whether Fus1∆IDR-FUSLC-27R is active, our current data already demonstrates this. Indeed, Fus1∆IDR-FUSLC-27R recruits Myo52 in a F-actin and FH2 domain-dependent manner (shown in Figure 5B and 5G), which demonstrates that Fus1∆IDR-FUSLC-27R FH2 domain is active. Even though Fus1∆IDR-FUSLC-27R assembles actin, we predict that its effect on general actin organization will be weak. Indeed, it is expressed under endogenous fus1 promoter, leading to very low expression levels during mitotic growth, such that only a subset of cells exhibit a Fus1 focus. Furthermore, most of these Fus1 foci are at or close to cell poles, where linear actin cables are assembled by For3, such that they may not have a strong disturbing effect. Because analysis of actin cable organization by phalloidin staining is difficult (due to the more strongly staining actin patches), cells with clear change in organization predicted to be rare in the population, and the gain in knowledge not transformative, we are not keen to do this experiment.

Minor Comments:

Prior studies are referenced appropriately. Text and figures are clear and accurate. My only suggestion would be Figure 1E-H could be moved to the supplemental material, due to their extremely technical nature. I believe this would help the broad audience focus on the experimental design mapped out in Figure 1A-D.

We are relatively neutral about this. If this suggestion is supported by the Editor, we can move these panels to supplement.

Reviewer #3 (Significance (Required)):

Significance: This study provides an improved imaging method for detecting the spatial distributions of proteins below 100 nm, providing new insights about how a relatively small cellular structure is organized. The use of three-color cell imaging to accurately measure accumulation rates of molecular components of the fusion focus provides new insight into the development of this structure and its roles in mating. This method could be applied to other multi-protein structures found in different cell types. This work uses rigorously genetic tools such as knockout, knockdown and point mutants to dissect the roles of the formin Fus1 and Type V myosin Myo52 in creating a proper fusion focus. The study could be improved by biochemical assays to test whether Myo52 and Fus1 directly interact, since the interaction is only shown by co-immunoprecipitation from extracts, which may reflect an indirect interaction.

Indeed, future studies should dissect the Fus1-Myo52 interaction, to determine whether it is direct and identify mutants that impair it.

I believe this work advances the cell-mating field by providing others with a spatial and temporal map of conserved factors arriving to the mating site. Additionally, they identified a way to study a mating specific protein in mitotically dividing cells, offering future questions to address.

This study should appeal to a range of basic scientists interested in cell biology, the cytoskeleton, and model organisms. The three-colored quantitative imaging could be applied to defining the architecture of many other cellular structures in different systems. Myosin and actin scientists will be interested in how this work expands the interplay of these two fields.

I am a cell biologist with expertise in live cell imaging, genetics and biochemistry.

We thank the reviewer for their appreciation of our work.

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

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

A three-color imaging approach to use centroid tracking is employed to determine the high resolution position over time of tagged actin fusion focus proteins during mating in fission yeast. In particular, the position of different protein components (tagged in a 3rd color) were determined in relation to the position (and axis) of the molecular motor Myo52, which is tagged with two different colors in the mating cells. Furthermore, time is normalized by the rapid diffusion of a weak fluorescent protein probe (mRaspberry) from one cell to the other upon fusion pore opening. From this approach multiple important mechanistic insights were determined for the compaction of fusion focus proteins during mating, including the general compaction of different components as fusion proceeds with different proteins having specific stereotypical behaviors that indicate underlying molecular insights. For example, secretory vesicles remain a constant distance from the plasma membrane, whereas the formin Fus1 rapidly accumulates at the fusion focus in a Myo52-dependent manner.

I have minor suggestions/points:

(1) Figure 1, for clarity it would be helpful if the cells shown in B were in the same orientation as the cartoon cells shown in A. Similarly, it would be helpful to have the orientation shown in D the same as the data that is subsequently presented in the rest of the manuscript (such as Figure 2) where time is on the X axis and distance (position) is on the Y axis.

(2) Figure 2, for clarity useful to introduce how the position of Myo52 changes over time with respect to the fusion site (plasma membrane) earlier, and then come back to the positions of different proteins with respect to Myo52 shown in 2E. Currently the authors discuss this point after introducing Figure 2E, but better for the reader to have this in mind beforehand.

(3) First sentence of page 8 "..., peaked at fusion time and sharply dropped post-fusion (Figure S3)." Figure S3 should be cited so that the reader knows where this data is presented.

(4) Figure 3D-H, why is Exo70 used as a marker for vesicles instead of Ypt3 for these experiments? Exo70 seems to have a more confusing localization than Ypt3 (3C vs 3D), which seems to complicate interpretations.

(5) Page 10, end of first paragraph, "We conclude...and promotes separation of Myo52 from the vesicles." This is an interesting hypothesis/interpretation that is consistent with the spatial-temporal organization of vesicles and the compacting fusion focus, but the underlying molecular mechanism has not be concluded.

(6) Figure 5F and 5G, the results are confusing and should be discussed further. Depletion of Myo52 decreases Fus1 long-range movements, indicating that Fus1 is being transported by Myo52 (5F). Similarly, the Fus1 actin assembly mutant greatly decreases Fus1 long-range movements and prevents Myo52 binding (5G), perhaps indicating that Fus1-mediated actin assembly is important. It seems the author's interpretations are oversimplified.

(7) Figure 6, why not measure the fluorescence intensity of Fus1 as a proxy for the number of Fus1 molecules (rather than the width of the Fus1 signal), which seems to be the more straight-forward analysis?

(8) Figure 7, the authors should note (and perhaps discuss) any evidence as to whether activation of Fus1 to facilitate actin assembly depends upon Fus1 dissociating from Myo52 or whether Fus1 can be activated while still associated with Myo52, as both circumstances are included in the figure.

(9) Figure 7, the color of secretory vesicles should be the same in A and B.

Significance

This is an impactful and high quality manuscript that describes an elegant experimental strategy with important insights determined. The experimental imaging strategy (and analysis), as well as the insight into the pombe mating fusion focus and its comparison to other cytoskeletal compaction events will be of broad scientific nterest.

Author response:

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

Reviewer #1 (Public review):

Summary:

Parise presents another instantiation of the Multisensory Correlation Detector model that can now accept stimulus-level inputs. This is a valuable development as it removes researcher involvement in the characterization/labeling of features and allows analysis of complex stimuli with a high degree of nuance that was previously unconsidered (i.e., spatial/spectral distributions across time). The author demonstrates the power of the model by fitting data from dozens of previous experiments, including multiple species, tasks, behavioral modalities, and pharmacological interventions.

Thanks for the kind words!

Strengths:

One of the model's biggest strengths, in my opinion, is its ability to extract complex spatiotemporal co-relationships from multisensory stimuli. These relationships have typically been manually computed or assigned based on stimulus condition and often distilled to a single dimension or even a single number (e.g., "-50 ms asynchrony"). Thus, many models of multisensory integration depend heavily on human preprocessing of stimuli, and these models miss out on complex dynamics of stimuli; the lead modality distribution apparent in Figures 3b and c is provocative. I can imagine the model revealing interesting characteristics of the facial distribution of correlation during continuous audiovisual speech that have up to this point been largely described as "present" and almost solely focused on the lip area.

Another aspect that makes the MCD stand out among other models is the biological inspiration and generalizability across domains. The model was developed to describe a separate process - motion perception - and in a much simpler organism - Drosophila. It could then describe a very basic neural computation that has been conserved across phylogeny (which is further demonstrated in the ability to predict rat, primate, and human data) and brain area. This aspect makes the model likely able to account for much more than what has already been demonstrated with only a few tweaks akin to the modifications described in this and previous articles from Parise.

What allows this potential is that, as Parise and colleagues have demonstrated in those papers since our (re)introduction of the model in 2016, the MCD model is modular - both in its ability to interface with different inputs/outputs and its ability to chain MCD units in a way that can analyze spatial, spectral, or any other arbitrary dimension of a stimulus. This fact leaves wide open the possibilities for types of data, stimuli, and tasks a simplistic, neutrally inspired model can account for.

And so it's unsurprising (but impressive!) that Parise has demonstrated the model's ability here to account for such a wide range of empirical data from numerous tasks (synchrony/temporal order judgement, localization, detection, etc.) and behavior types (manual/saccade responses, gaze, etc.) using only the stimulus and a few free parameters. This ability is another of the model's main strengths that I think deserves some emphasis: it represents a kind of validation of those experiments, especially in the context of cross-experiment predictions (but see some criticism of that below).

Finally, what is perhaps most impressive to me is that the MCD (and the accompanying decision model) does all this with very few (sometimes zero) free parameters. This highlights the utility of the model and the plausibility of its underlying architecture, but also helps to prevent extreme overfitting if fit correctly (but see a related concern below).

We sincerely thank the reviewer for their thoughtful and generous comments. We are especially pleased that the core strengths of the model—its stimulus-computable architecture, biological grounding, modularity, and cross-domain applicability—were clearly recognized. As the reviewer rightly notes, removing researcher-defined abstractions and working directly from naturalistic stimuli opens the door to uncovering previously overlooked dynamics in complex multisensory signals, such as the spatial and temporal richness of audiovisual speech.

We also appreciate the recognition of the model’s origins in a simple organism and its generalization across species and behaviors. This phylogenetic continuity reinforces our view that the MCD captures a fundamental computation with wide-ranging implications. Finally, we are grateful for the reviewer’s emphasis on the model’s predictive power across tasks and datasets with few or no free parameters—a property we see as key to both its parsimony and explanatory utility.

We have highlighted these points more explicitly in the revised manuscript, and we thank the reviewer for their generous and insightful endorsement of the work.

Weaknesses:

There is an insufficient level of detail in the methods about model fitting. As a result, it's unclear what data the models were fitted and validated on. Were models fit individually or on average group data? Each condition separately? Is the model predictive of unseen data? Was the model cross-validated? Relatedly, the manuscript mentions a randomization test, but the shuffled data produces model responses that are still highly correlated to behavior despite shuffling. Could it be that any stimulus that varies in AV onset asynchrony can produce a psychometric curve that matches any other task with asynchrony judgements baked into the task? Does this mean all SJ or TOJ tasks produce correlated psychometric curves? Or more generally, is Pearson's correlation insensitive to subtle changes here, considering psychometric curves are typically sigmoidal? Curves can be non-overlapping and still highly correlated if one is, for example, scaled differently. Would an error term such as mean-squared or root mean-squared error be more sensitive to subtle changes in psychometric curves? Alternatively, perhaps if the models aren't cross-validated, the high correlation values are due to overfitting?

The reviewer is right: the current version of the manuscript only provides limited information about parameter fitting. In the revised version of the manuscript, we included a parameter estimation and generalizability section that includes all information requested by the reviewer.

To test whether using the MSE instead of Pearson correlation led to a similar estimated set of parameter values, we repeated the fitting using the MSE. The parameter estimated with this method (TauV, TauA, TauBim) closely followed those estimated using Pearson correlation (TauV, TauA, TauBim). Given the similarity of these results, we have chosen not to include further figures, however this analysis is now included in the new section (pages 23-24).

Regarding the permutation test, it is expected that different stimuli produce analogous psychometric functions: after all, all studies relied on stimuli containing identical manipulation of lags. As a result, MCD population responses tend to be similar across experiments. Therefore, it is not a surprise that the permuted distribution of MCD-data correlation in Supplementary Figure 1K has a mean as high as 0.97. However, what is important is to demonstrate that the non-permuted dataset has an even higher goodness of fit. Supplementary Figure 1K demonstrates that none of the permuted stimuli could outperform the non-permuted dataset; the mean of the non-permuted distribution is 4.7 (standard deviations) above the mean of the already high  permuted distribution.

We believe the new section, along with the present response, fully addresses the legitimate concerns of the reviewer.

While the model boasts incredible versatility across tasks and stimulus configurations, fitting behavioral data well doesn't mean we've captured the underlying neural processes, and thus, we need to be careful when interpreting results. For example, the model produces temporal parameters fitting rat behavior that are 4x faster than when fitting human data. This difference in slope and a difference at the tails were interpreted as differences in perceptual sensitivity related to general processing speeds of the rat, presumably related to brain/body size differences. While rats no doubt have these differences in neural processing speed/integration windows, it seems reasonable that a lot of the differences in human and rat psychometric functions could be explained by the (over)training and motivation of rats to perform on every trial for a reward - increasing attention/sensitivity (slope) - and a tendency to make mistakes (compression evident at the tails). Was there an attempt to fit these data with a lapse parameter built into the decisional model as was done in Equation 21? Likewise, the fitted parameters for the pharmacological manipulations during the SJ task indicated differences in the decisional (but not the perceptual) process and the article makes the claim that "all pharmacologically-induced changes in audiovisual time perception" can be attributed to decisional processes "with no need to postulate changes in low-level temporal processing." However, those papers discuss actual sensory effects of pharmacological manipulation, with one specifically reporting changes to response timing. Moreover, and again contrary to the conclusions drawn from model fits to those data, both papers also report a change in psychometric slope/JND in the TOJ task after pharmacological manipulation, which would presumably be reflected in changes to the perceptual (but not the decisional) parameters.

Fitting or predicting behaviour does not in itself demonstrate that a model captures the underlying neural computations—though it may offer valuable constraints and insights. In line with this, we were careful not to extrapolate the implications of our simulations to specific neural mechanisms.

Temporal sensitivity is, by definition, a behavioural metric, and—as the reviewer correctly notes—its estimation may reflect a range of contributing factors beyond low-level sensory processing, including attention, motivation, and lapse rates (i.e., stimulus-independent errors). In Equation 21, we introduced a lapse parameter specifically to account for such effects in the context of monkey eye-tracking data. For the rat datasets, however, the inclusion of a lapse term was not required to achieve a close fit to the psychometric data (ρ = 0.981). While it is likely that adding a lapse component would yield a marginally better fit, the absence of single-trial data prevents us from applying model comparison criteria such as AIC or BIC to justify the additional parameter. In light of this, and to avoid unnecessary model complexity, we opted not to include a lapse term in the rat simulations.

With respect to the pharmacological manipulation data, we acknowledge the reviewer’s point that observed changes in slope and bias could plausibly arise from alterations at either the sensory or decisional level—or both. In our model, low-level sensory processing is instantiated by the MCD architecture, which outputs the MCDcorr and MCDlag signals that are then scaled and integrated during decision-making. Importantly, this scaling operation influences the slope of the resulting psychometric functions, such that changes in slope can arise even in the absence of any change to the MCD’s temporal filters. In our simulations, the temporal constants of the MCD units were fixed to the values estimated from the non-pharmacological condition (see parameter estimation section above), and only the decision-related parameters were allowed to vary. From this modelling perspective, the behavioural effects observed in the pharmacological datasets can be explained entirely by changes at the decisional level. However, we do not claim that such an explanation excludes the possibility of genuine sensory-level changes. Rather, we assert that our model can account for the observed data without requiring modifications to early temporal tuning.

To rigorously distinguish sensory from decisional effects, future experiments will need to employ stimuli with richer temporal structure—e.g., temporally modulated sequences of clicks and flashes that vary in frequency, phase, rhythm, or regularity (see Fujisaki & Nishida, 2007; Denison et al., 2012; Parise & Ernst, 2016, 2025; Locke & Landy, 2017; Nidiffer et al., 2018). Such stimuli engage the MCD in a more stimulus-dependent manner, enabling a clearer separation between early sensory encoding and later decision-making processes. Unfortunately, the current rat datasets—based exclusively on single click-flash pairings—lack the complexity needed for such disambiguation. As a result, while our simulations suggest that the observed pharmacologically induced effects can be attributed to changes in decision-level parameters, they do not rule out concurrent sensory-level changes.

In summary, our results indicate that changes in the temporal tuning of MCD units are not necessary to reproduce the observed pharmacological effects on audiovisual timing behaviour. However, we do not assert that such changes are absent or unnecessary in principle. Disentangling sensory and decisional contributions will ultimately require richer datasets and experimental paradigms designed specifically for this purpose. We have now modified the results section (page 6) and the discussion (page 11) to clarify these points.

The case for the utility of a stimulus-computable model is convincing (as I mentioned above), but its framing as mission-critical for understanding multisensory perception is overstated, I think. The line for what is "stimulus computable" is arbitrary and doesn't seem to be followed in the paper. A strict definition might realistically require inputs to be, e.g., the patterns of light and sound waves available to our eyes and ears, while an even more strict definition might (unrealistically) require those stimuli to be physically present and transduced by the model. A reasonable looser definition might allow an "abstract and low-dimensional representation of the stimulus, such as the stimulus envelope (which was used in the paper), to be an input. Ultimately, some preprocessing of a stimulus does not necessarily confound interpretations about (multi)sensory perception. And on the flip side, the stimulus-computable aspect doesn't necessarily give the model supreme insight into perception. For example, the MCD model was "confused" by the stimuli used in our 2018 paper (Nidiffer et al., 2018; Parise & Ernst, 2025). In each of our stimuli (including catch trials), the onset and offset drove strong AV temporal correlations across all stimulus conditions (including catch trials), but were irrelevant to participants performing an amplitude modulation detection task. The to-be-detected amplitude modulations, set at individual thresholds, were not a salient aspect of the physical stimulus, and thus only marginally affected stimulus correlations. The model was of course, able to fit our data by "ignoring" the on/offsets (i.e., requiring human intervention), again highlighting that the model is tapping into a very basic and ubiquitous computational principle of (multi)sensory perception. But it does reveal a limitation of such a stimulus-computable model: that it is (so far) strictly bottom-up.

We appreciate the reviewer’s thoughtful engagement with the concept of stimulus computability. We agree that the term requires careful definition and should not be taken as a guarantee of perceptual insight or neural plausibility. In our work, we define a model as “stimulus-computable” if all its inputs are derived directly from the stimulus, rather than from experimenter-defined summary descriptors such as temporal lag, spatial disparity, or cue reliability. In the context of multisensory integration, this implies that a model must account not only for how cues are combined, but also for how those cues are extracted from raw inputs—such as audio waveforms and visual contrast sequences.

This distinction is central to our modelling philosophy. While ideal observer models often specify how information should be combined once identified, they typically do not address the upstream question of how this information is extracted from sensory input. In that sense, models that are not stimulus-computable leave out a key part of the perceptual pipeline. We do not present stimulus computability as a marker of theoretical superiority, but rather as a modelling constraint that is necessary if one’s aim is to explain how structured sensory input gives rise to perception. This is a view that is also explicitly acknowledged and supported by Reviewer 2.

Framed in Marr’s (1982) terms, non–stimulus-computable models tend to operate at the computational level, defining what the system is doing (e.g., computing a maximum likelihood estimate), whereas stimulus-computable models aim to function at the algorithmic level, specifying how the relevant representations and operations might be implemented. When appropriately constrained by biological plausibility, such models may also inform hypotheses at the implementational level, pointing to potential neural substrates that could instantiate the computation.

Regarding the reviewer’s example illustrating a limitation of the MCD model, we respectfully note that the account appears to be based on a misreading of our prior work. In Parise & Ernst (2025), where we simulated the stimuli from Nidiffer et al. (2018), the MCD model reproduced participants’ behavioural data without any human intervention or adjustment. The model was applied in a fully bottom-up, stimulus-driven manner, and its output aligned with observer responses as-is. We suspect the confusion may stem from analyses shown in Figure 6 - Supplement Figure 5 of Parise & Ernst (2025), where we investigated the lack of a frequency-doubling effect in the Nidiffer et al. data. However, those analyses were based solely on the Pearson correlation between auditory and visual stimulus envelopes and did not involve the MCD model. No manual exclusion of onset/offset events was applied, nor was the MCD used in those particular figures. We also note that Parise & Ernst (2025) is a separate, already published study and is not the manuscript currently under review. 

In summary, while we fully agree that stimulus computability does not resolve all the complexities of multisensory perception (see comments below about speech), we maintain that it provides a valuable modelling constraint—one that enables robust, generalisable predictions when appropriately scoped. 

The manuscript rightly chooses to focus a lot of the work on speech, fitting the MCD model to predict behavioral responses to speech. The range of findings from AV speech experiments that the MCD can account for is very convincing. Given the provided context that speech is "often claimed to be processed via dedicated mechanisms in the brain," a statement claiming a "first end-to-end account of multisensory perception," and findings that the MCD model can account for speech behaviors, it seems the reader is meant to infer that energetic correlation detection is a complete account of speech perception. I think this conclusion misses some facets of AV speech perception, such as integration of higher-order, non-redundant/correlated speech features (Campbell, 2008) and also the existence of top-down and predictive processing that aren't (yet!) explained by MCD. For example, one important benefit of AV speech is interactions on linguistic processes - how complementary sensitivity to articulatory features in the auditory and visual systems (Summerfield, 1987) allow constraint of linguistic processes (Peelle & Sommers, 2015; Tye-Murray et al., 2007).

We thank the reviewer for their thoughtful comments, and especially for the kind words describing the range of findings from our AV speech simulations as “very convincing.”

We would like to clarify that it is not our view that speech perception can be reduced to energetic correlation detection. While the MCD model captures low- to mid-level temporal dependencies between auditory and visual signals, we fully agree that a complete account of audiovisual speech perception must also include higher-order processes—including linguistic mechanisms and top-down predictions. These are critical components of AV speech comprehension, and lie beyond the scope of the current model.

Our use of the term “end-to-end” is intended in a narrow operational sense: the model transforms raw audiovisual input (i.e., audio waveforms and video frames) directly into behavioural output (i.e., button press responses), without reliance on abstracted stimulus parameters such as lag, disparity or reliability. It is in this specific technical sense that the MCD offers an end-to-end model. We have revised the manuscript to clarify this usage to avoid any misunderstanding.

In light of the reviewer’s valuable point, we have now edited the Discussion to acknowledge the importance of linguistic processes (page 13) and to clarify what we mean by end-to-end account (page 11). We agree that future work will need to explore how stimulus-computable models such as the MCD can be integrated with broader frameworks of linguistic and predictive processing (e.g., Summerfield, 1987; Campbell, 2008; Peelle & Sommers, 2015; Tye-Murray et al., 2007).

References

Campbell, R. (2008). The processing of audio-visual speech: empirical and neural bases. Philosophical Transactions of the Royal Society B: Biological Sciences, 363(1493), 1001-1010. https://doi.org/10.1098/rstb.2007.2155

Nidiffer, A. R., Diederich, A., Ramachandran, R., & Wallace, M. T. (2018). Multisensory perception reflects individual differences in processing temporal correlations. Scientific Reports 2018 8:1, 8(1), 1-15. https://doi.org/10.1038/s41598-018-32673-y

Parise, C. V, & Ernst, M. O. (2025). Multisensory integration operates on correlated input from unimodal transient channels. ELife, 12. https://doi.org/10.7554/ELIFE.90841

Peelle, J. E., & Sommers, M. S. (2015). Prediction and constraint in audiovisual speech perception. Cortex, 68, 169-181. https://doi.org/10.1016/j.cortex.2015.03.006

Summerfield, Q. (1987). Some preliminaries to a comprehensive account of audio-visual speech perception. In B. Dodd & R. Campbell (Eds.), Hearing by Eye: The Psychology of Lip-Reading (pp. 3-51). Lawrence Erlbaum Associates.

Tye-Murray, N., Sommers, M., & Spehar, B. (2007). Auditory and Visual Lexical Neighborhoods in Audiovisual Speech Perception: Trends in Amplification, 11(4), 233-241. https://doi.org/10.1177/1084713807307409

Reviewer #2 (Public review):

Summary:

Building on previous models of multisensory integration (including their earlier correlation-detection framework used for non-spatial signals), the author introduces a population-level Multisensory Correlation Detector (MCD) that processes raw auditory and visual data. Crucially, it does not rely on abstracted parameters, as is common in normative Bayesian models," but rather works directly on the stimulus itself (i.e., individual pixels and audio samples). By systematically testing the model against a range of experiments spanning human, monkey, and rat data, the authors show that their MCD population approach robustly predicts perception and behavior across species with a relatively small (0-4) number of free parameters.

Strengths:

(1) Unlike prior Bayesian models that used simplified or parameterized inputs, the model here is explicitly computable from full natural stimuli. This resolves a key gap in understanding how the brain might extract "time offsets" or "disparities" from continuously changing audio-visual streams.

(2) The same population MCD architecture captures a remarkable range of multisensory phenomena, from classical illusions (McGurk, ventriloquism) and synchrony judgments, to attentional/gaze behavior driven by audio-visual salience. This generality strongly supports the idea that a single low-level computation (correlation detection) can underlie many distinct multisensory effects.

(3) By tuning model parameters to different temporal rhythms (e.g., faster in rodents, slower in humans), the MCD explains cross-species perceptual data without reconfiguring the underlying architecture.

We thank the reviewer for their positive evaluation of the manuscript, and particularly for highlighting the significance of the model's stimulus-computable architecture and its broad applicability across species and paradigms. Please find our responses to the individual points below.

Weaknesses:

(1) The authors show how a correlation-based model can account for the various multisensory integration effects observed in previous studies. However, a comparison of how the two accounts differ would shed light on the correlation model being an implementation of the Bayesian computations (different levels in Marr's hierarchy) or making testable predictions that can distinguish between the two frameworks. For example, how uncertainty in the cue combined estimate is also the harmonic mean of the unimodal uncertainties is a prediction from the Bayesian model. So, how the MCD framework predicts this reduced uncertainty could be one potential difference (or similarity) to the Bayesian model.

We fully agree with the reviewer that a comparison between the correlation-based MCD model and Bayesian accounts is valuable—particularly for clarifying how the two frameworks differ conceptually and where they may converge.

As noted in the revised manuscript, the key distinction lies in the level of analysis described by Marr (1982). Bayesian models operate at the computational level, describing what the system is aiming to compute (e.g., optimal cue integration). In contrast, the MCD functions at the algorithmic level, offering a biologically plausible mechanism for how such integration might emerge from stimulus-driven representations.

In this context, the MCD provides a concrete, stimulus-grounded account of how perceptual estimates might be constructed—potentially implementing computations with Bayesian-like characteristics (e.g., reduced uncertainty, cue weighting). Thus, the two models are not mutually exclusive but can be seen as complementary: the MCD may offer an algorithmic instantiation of computations that, at the abstract level, resemble Bayesian inference.

We have now updated the manuscript to explicitly highlight this relationship (pages 2 and 11). In the revised manuscript, we also included a new figure (Figure 5) and movie (Supplementary Movie 3), to show how the present approach extends previous Bayesian models for the case of cue integration (i.e., the ventriloquist effect).

(2) The authors show a good match for cue combination involving 2 cues. While Bayesian accounts provide a direction for extension to more cues (also seen empirically, for eg, in Hecht et al. 2008), discussion on how the MCD model extends to more cues would benefit the readers.

We thank the reviewer for this insightful comment: extending the MCD model to include more than two sensory modalities is a natural and valuable next step. Indeed, one of the strengths of the MCD framework lies in its modularity. Let us consider the MCDcorr​ output (Equation 6), which is computed as the pointwise product of transient inputs across modalities. Extending this to include a third modality, such as touch, is straightforward: MCD units would simply multiply the transient channels from all three modalities, effectively acting as trimodal coincidence detectors that respond when all inputs are aligned in time and space.

By contrast, extending MCDlag is less intuitive, due to its reliance on opponency between two subunits (via subtraction). A plausible solution is to compute MCDlag in a pairwise fashion (e.g., AV, VT, AT), capturing relative timing across modality pairs.

Importantly, the bulk of the spatial integration in our framework is carried by MCDcorr, which generalises naturally to more than two modalities. We have now formalised this extension and included a graphical representation in a supplementary section of the revised manuscript.

Likely Impact and Usefulness:

The work offers a compelling unification of multiple multisensory tasks- temporal order judgments, illusions, Bayesian causal inference, and overt visual attention - under a single, fully stimulus-driven framework. Its success with natural stimuli should interest computational neuroscientists, systems neuroscientists, and machine learning scientists. This paper thus makes an important contribution to the field by moving beyond minimalistic lab stimuli, illustrating how raw audio and video can be integrated using elementary correlation analyses.

Reviewer #1 (Recommendations for the authors):

Recommendations:

My biggest concern is a lack of specificity about model fitting, which is assuaged by the inclusion of sufficient detail to replicate the analysis completely or the inclusion of the analysis code. The code availability indicates a script for the population model will be included, but it is unclear if this code will provide the fitting details for the whole of the analysis.

We thank the reviewer for raising this important point. A new methodological section has been added to the manuscript, detailing the model fitting procedures used throughout the study. In addition, the accompanying code repository now includes MATLAB scripts that allow full replication of the spatiotemporal MCD simulations.

Perhaps it could be enlightening to re-evaluate the model with a measure of error rather than correlation? And I think many researchers would be interested in the model's performance on unseen data.

The model has now been re-evaluated using mean squared error (MSE), and the results remain consistent with those obtained using Pearson correlation. Additionally, we have clarified which parts of the study involve testing the model on unseen data (i.e., data not used to fit the temporal constants of the units). These analyses are now included and discussed in the revised fitting section of the manuscript (pages 23-24).

Otherwise, my concerns involve the interpretation of findings, and thus could be satisfied with minor rewording or tempering conclusions.

The manuscript has been revised to address these interpretative concerns, with several conclusions reworded or tempered accordingly. All changes are marked in blue in the revised version.

Miscellanea:

Should b0 in equation 10 be bcrit to match the below text?

Thank you for catching this inconsistency. We have corrected Equation 10 (and also Equation 21) to use the more transparent notation bcrit instead of b0, in line with the accompanying text.

Equation 23, should time be averaged separately? For example, if multiple people are speaking, the average correlation for those frames will be higher than the average correlation across all times.

We thank the reviewer for raising this thoughtful and important point. In response, we have clarified the notation of Equation 23 in the revised manuscript (page 20). Specifically, we now denote the averaging operations explicitly as spatial means and standard deviations across all pixel locations within each frame.

This equation computes the z-score of the MCD correlation value at the current gaze location, normalized relative to the spatial distribution of correlation values in the same frame. That is, all operations are performed at the frame level, not across time. This ensures that temporally distinct events are treated independently and that the final measure reflects relative salience within each moment, not a global average over the stimulus. In other words, the spatial distribution of MCD activity is re-centered and rescaled at each frame, exactly to avoid the type of inflation or confounding the reviewer rightly cautioned against.

Reviewer #2 (Recommendations for the authors):

The authors have done a great job of providing a stimulus computable model of cue combination. I had just a few suggestions to strengthen the theoretical part of the paper:

(1) While the authors have shown a good match between MCD and cue combination, some theoretical justification or equivalence analysis would benefit readers on how the two relate to each other. Something like Zhang et al. 2019 (which is for motion cue combination) would add to the paper.

We agree that it is important to clarify the theoretical relationship between the Multisensory Correlation Detector (MCD) and normative models of cue integration, such as Bayesian combination. In the revised manuscript, we have now modified the introduction and added a paragraph in the Discussion addressing this link more explicitly. In brief, we see the MCD as an algorithmic-level implementation (in Marr’s terms) that may approximate or instantiate aspects of Bayesian inference.

(2) Simulating cue combination for tasks that require integration of more than two cues (visual, auditory, haptic cues) would more strongly relate the correlation model to Bayesian cue combination. If that is a lot of work, at least discussing this would benefit the paper

This point has now been addressed, and a new paragraph discussing the extension of the MCD model to tasks involving more than two sensory modalities has been added to the Discussion section.

DOI: 10.1038/s41597-025-05795-y

Resource: None

Curator: @scibot

SciCrunch record: RRID:SCR_024957

What is this?

DOI: 10.1038/s41597-025-05795-y

Resource: R Project for Statistical Computing (RRID:SCR_001905)

Curator: @scibot

SciCrunch record: RRID:SCR_001905

What is this?

Brayan Piña Este artículo aporta a la educación matemática al emplear la Matriz de Vester como una herramienta para identificar y clasificar las dificultades en el aprendizaje. Su principal fortaleza radica en un enfoque metodológico que combina diagnóstico participativo, análisis de causalidad y una propuesta didáctica adaptada al contexto, lo que lo hace especialmente útil y transferible en entornos rurales. Sin embargo, presenta algunas limitaciones, como el número reducido de participantes y la notable influencia de la experiencia del docente investigador, lo que puede afectar la validez de los resultados. Aunque el marco teórico se basa en la fenomenología didáctica de Freudenthal, la reflexión sobre la Matriz de Vester es algo breve y no se compara con otras metodologías. Los resultados confirman problemas ya conocidos en la literatura, como la desatención, la repetición de estrategias, la baja autodisciplina y las limitaciones socioeconómicas. Sin embargo, su verdadero valor radica en la forma organizada y participativa de abordar estos problemas para guiar la práctica docente. Por último, la secuencia didáctica contextual es un acierto, ya que genera motivación y aprendizajes significativos en los estudiantes, aunque aún queda por analizar su impacto a largo plazo.

WHen heading to the Y-axis remainds constanst.

En las novelas policíacas la inverosimilitud es la regla. Estas novelas invierten lo que se puede observar: los culpables son los menos sospechosos. Son revelados desde lo posible, pero menos verosímil.

Lo anti-verosímil en la novela policíaca

Ley general del suspenso

Nuevo verosímil para Barthes: destrucción del signo de manera regresiva, para cuestionar la representación desde el estudio de lo bello y las experiencias artísticas desde una perspectiva que no se basa en principios religiosos o espirituales, sino en la razón, la experiencia humana y el mundo terrenal.

semióticamente construida por el detalle concreto: relación entre el referente y el significante, sin posibilidad de desarrollar una forma de significado, no hay denotación. El significante remite al referente.

Esto parece un testimonio de la importancia que se le da a las historias "basadas en un hecho real".

Sensación de inestabilidad o desorientación que produce el texto en el lector al desestabilizar el sentido único y monolítico. Este vértigo surge al confrontar un texto que es lo opuesto a un tratado de un solo significado, ya que lo no codificado, lo que queda fuera del discurso normativo, genera esta sensación de profundidad y multiplicidad de interpretaciones.

Finalidad estética de la descripción en Flaubert

Oposición entre la estructura general del relato que es predictiva y la descripción que es puramente sumatoria y no va creando una elección de consecuencia sobre el relato como la primera.

estructura desordenada de la notación insignificante de las descripciones

Descripciones detalladas en la narrativa que, aunque parecen ser meros registros de lo real, en realidad son selecciones deliberadas que construyen el efecto de realidad, ya que el texto remplaza lo real con lo escrito y crea una ilusión de transparencia

Omisiones del análisis estructural sobre lo que se considera relleno de valor funcional indirecto

Figura retórica que consiste en una descripción realista, viva y detallada de algo o alguien, tan vívida que parece ser experimentada en el momento. El término, proveniente del griego hupotúpōsis (que significa "boceto" o "esbozo").

Author response:

Reviewer #1 (Public review):

This study established a C921Y OGT-ID mouse model, systematically demonstrating in mammals the pathological link between O-GlcNAc metabolic imbalance and neurodevelopmental disorders (cortical malformation, microcephaly) as well as behavioral abnormalities (hyperactivity, impulsivity, learning/memory deficits). However, critical flaws in the current findings require resolution to ensure scientific rigor.

The most concerning finding appears in Figure S12. While Supplementary Figure S12 demonstrates decreased OGA expression without significant OGT level changes in C921Y mutants via Western blot/qPCR, previous reports (Florence Authier, et al., Dis Model Mech. 2023) described OGT downregulation in Western blot and an increase in qPCR in the same models. The opposite OGT expression outcomes in supposedly identical mouse models directly challenge the model's reliability. This discrepancy raises serious concerns about either the experimental execution or the interpretation of results. The authors must revalidate the data with rigorous controls or provide a molecular biology-based explanation.

The referee’s assessment is based on a misunderstanding – these are certainly not the same experiment repeated twice with different answers. In the previous report of the OGT-C921Y mutant mice (Florence Authier, et al., Dis Model Mech. 2023), OGT and OGA mRNA/protein expression have been assessed in total brain protein extract from 3 months old male mice. In that study we observed a significant reduction in OGT protein levels while OGT mRNA levels were significantly increased in the mutant compared to WT controls. However, in our the current study (Figure S12), OGA and OGT mRNA/protein expression have been a) restricted to the pre-frontal cortex and b) are from 4 months old male mice, which does not allow a direct comparison of the two studies. In the pre-frontal cortex, OGT protein levels are not changed while OGT mRNA levels are increased (similarly to the total brain data), albeit not significantly. The different outcomes of OGT protein levels in both total brain and prefrontal cortex could suggest regional differences in OGT protein levels/stability as OGT mRNA levels are increased in both cases. Three other brain regions (hippocampus, striatum and cerebellum) have now also been assessed for OGT mRNA/protein expression, supporting such regional differences in OGT protein levels and these data will be included in the new version of the manuscript.

A few additional comments to the author may be helpful to improve the study.

Major

(1) While this study systematically validated multi-dimensional phenotypes (including neuroanatomical abnormalities and behavioral deficits) in OGT C921Y mutant mice, there is a lack of relevant mechanisms and intervention experiments. For example, the absence of targeted intervention studies on key signaling pathways prevents verification of whether proteomics-identified molecular changes directly drive phenotypic manifestations.

We agree with the referee that these experiments would further strenghten the work. They would, however, result in a 1-5 year delay in sharing this work with the scientific and patient communities. We will continue to work along these lines and report separately in the future.

(2) Although MRI detected nodular dysplasia and heterotopia in the cingulate cortex, the cellular basis remains undefined. Spatiotemporal immunofluorescence analysis using neuronal (NeuN), astrocytic (GFAP), and synaptic (Synaptophysin) markers is recommended to identify affected cell populations (e.g., radial glial migration defects or intermediate progenitor differentiation abnormalities).

We are currently performing these experiments so that they can be included in the version of record of this manuscript.

(3) While proteomics revealed dysregulation in pathways including Wnt/β-catenin and mTOR signaling, two critical issues remain unresolved: a) O-GlcNAc glycoproteomic alterations remain unexamined; b) The causal relationship between pathway changes and O-GlcNAc imbalance lacks validation. It is recommended to use co-immunoprecipitation or glycosylation sequencing to confirm whether the relevant proteins undergo O-GlcNAc modification changes, identify specific modification sites, and verify their interactions with OGT.

We agree with the referee that these experiments would further strenghten the work and will perform further experiments to explore whether these pathways are functionally affected. However, it is important to note that the inference that these proteins must themselves be O-GlcNAc modified is incorrect – indeed, O-GlcNAcylation of unknown protein kinase X, E3 ligase/DUB, Y or transcription factor Z could indirectly affect these pathways/proteins.

(4) Given that OGT-ID neuropathology likely originates embryonically, we recommend serial analyses from E14.5 to P7 to examine cellular dynamics during critical corticogenesis phases.

We agree with the referee that these experiments would further strenghten the work. They would, however, result in a significant delay in sharing this work with the scientific and patient communities. We will continue to work along these lines and report separately in the future.

(5) The interpretation of Figure 8A constitutes overinterpretation. Current data fail to conclusively demonstrate impairment of OGT's protein interaction network and lack direct evidence supporting the proposed mechanisms of HCF1 misprocessing or OGA loss.

For clarity, we will remove panel A from Figure 8 in the version of record – this panel was only ever meant to represent a priori hypotheses for OGT-CDG mechanisms, none of which have been either excluded or confirmed.

Reviewer #2 (Public review):

Summary:

The authors are trying to understand why certain mutants of O-GlcNAc transferase (OGT) appear to cause developmental disorders in humans. As an important step towards that goal, the authors generated a mouse model with one of these mutations that disrupts OGT activity. They then go on to test these mice for behavioral differences, finding that the mutant mice exhibit some signs of hyperactivity and differences in learning and memory. They then examine alterations to the structure of the brain and skull, and again find changes in the mutant mice that have been associated with developmental disorders. Finally, they identify proteins that are up- or down-regulated between the two mice as potential mechanisms to explain the observations.

Strengths:

The major strength of this manuscript is the creation of this mouse model, as a key step in beginning to understand how OGT mutants cause developmental disorders. This line will prove important for not only the authors but other investigators as well, enabling the testing of various hypotheses and potentially treatments. The experiments are also rigorously performed, and the conclusions are well supported by the data.

Weaknesses:

The only weakness identified is a lack of mechanistic insight. However, this certainly may come in the future through more targeted experimentation using this mouse model.

We agree with the referee that these experiments would further strenghten the work. They would, however, result in a 1-5 year delay in sharing this work with the scientific and patient communities. We will continue to work along these lines and report separately in the future.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Public Review):

Summary:

Previous studies have shown that treatment with 17α-estradiol (a stereoisomer of the 17β-estradiol) extends lifespan in male mice but not in females. The current study by Li et al, aimed to identify cell-specific clusters and populations in the hypothalamus of aged male rats treated with 17α-estradiol (treated for 6 months). This study identifies genes and pathways affected by 17α-estradiol in the aged hypothalamus.

Strengths:

Using single-nucleus transcriptomic sequencing (snRNA-seq) on the hypothalamus from aged male rats treated with 17α-estradiol they show that 17α-estradiol significantly attenuated age-related increases in cellular metabolism, stress, and decreased synaptic activity in neurons.

Thanks.

Moreover, sc-analysis identified GnRH as one of the key mediators of 17α-estradiol's effects on energy homeostasis. Furthermore, they show that CRH neurons exhibited a senescent phenotype, suggesting a potential side effect of the 17α-estradiol. These conclusions are supported by supervised clustering by neuropeptides, hormones, and their receptors.

Thanks.

Weaknesses:

However, the study has several limitations that reduce the strength of the key claims in the manuscript. In particular:

(1) The study focused only on males and did not include comparisons with females. However, previous studies have shown that 17α-estradiol extends lifespan in a sex-specific manner in mice, affecting males but not females. Without the comparison with the female data, it's difficult to assess its relevance to the lifespan.

This study was originally designed based on previous findings indicating that lifespan extension is only effective in males, leading to the exclusion of females from the analysis. The primary focus of our research was on the transcriptional changes and serum endocrine alterations induced by 17α-estradiol in aged males compared to untreated aged males. We believe that even in the absence of female subjects, the significant effects of 17α-estradiol on metabolism in the hypothalamus, synapses, and endocrine system remain evident, particularly regarding the expression levels of GnRH and testosterone. Notably, lower overall metabolism, increased synaptic activity, and elevated levels of GnRH and testosterone are strong indicators of health and well-being in males, supporting the validity of our primary conclusions. However, including female controls would enhance the depth of our findings. If female controls were incorporated, we propose redesigning the sample groups to include aged male control, aged female control, aged female treated, aged male treated, as well as young male control, young male treated, young female control, and young female treated. We regret that we cannot provide this data in the short term. Nevertheless, we believe this reviewer’s creative idea presents a valuable avenue for future research on this topic. In this study, we emphasize the role of 17α-estradiol in overall metabolism, synaptic function, GnRH, and testosterone in aged males and underscore the importance of supervised clustering of neuropeptide-secreting neurons in the hypothalamus.

(2) It is not known whether 17α-estradiol leads to lifespan extension in male rats similar to male mice. Therefore, it is not possible to conclude that the observed effects in the hypothalamus, are linked to the lifespan extension.

Thanks for the reminding. 17α-estradiol was reported to extend lifespan in male rats similar to male mice (PMID: 33289482). We have added the valuable reference to introduction in the new version.  

(3) The effect of 17α-estradiol on non-neuronal cells such as microglia and astrocytes is not well-described (Figure 1). Previous studies demonstrated that 17α-estradiol reduces microgliosis and astrogliosis in the hypothalamus of aged male mice. Current data suggest that the proportion of oligo, and microglia were increased by the drug treatment, while the proportions of astrocytes were decreased. These data might suggest possible species differences, differences in the treatment regimen, or differences in drug efficiency. This has to be discussed.

We have reviewed reports describing changes in cell numbers following 17α-estradiol treatment in the brain, using the keywords "17α-estradiol," "17alpha-estradiol," and "microglia" or "astrocyte." Only a limited amount of data was obtained. We found one article indicating that 17α-estradiol treatment in Tg (AβPP(swe)/PS1(ΔE9)) model mice resulted in a decreased microglial cell number compared to the placebo (AβPP(swe)/PS1(ΔE9) mice), but this change was not significant when compared to the non-transgenic control (PMID: 21157032). The transgenic AβPP(swe)/PS1(ΔE9) mouse model may differ from our wild-type aging rat model in this context.

Moreover, the calculation of cell numbers was based on visual observation under a microscope across several brain tissue slices. This traditional method often yields controversial results. For example, oligodendrocytes in the corpus callosum, fornix, and spinal cord have been reported to be 20-40% more numerous in males than in females based on microscopic observations (PMID: 16452667). In contrast, another study found no significant difference in the number of oligodendrocytes between sexes when using immunohistochemistry staining (PMID: 18709647). Such discrepancies arising from traditional observational methods are inevitable.

We believe the data presented in this article are reliable because the cell number and cell ratio data were derived from high-throughput cell counting of the entire hypothalamus using single-cell suspension and droplet wrapping (10x Genomics).

(4) A more detailed analysis of glial cell types within the hypothalamus in response to drugs should be provided.

We provided more enrichment analysis data of differentially expressed genes between Y, O, and O.T in microglia and astrocytes in Figure 2—figure supplement 3. In this supplemental data, we found unlike that in neurons, Micro displayed lower levels of synapse-related cellular processes in O.T. compared to O.

(5) The conclusion that CRH neurons are going into senescence is not clearly supported by the data. A more detailed analysis of the hypothalamus such as histological examination to assess cellular senescence markers in CRH neurons, is needed to support this claim.

We also noted the inappropriate claim and have changed "senescent phenotype" to "stressed phenotype" and "abnormal phenotype" in both the abstract and results sections. The stressed phenotype could be induced by heightened functional activity in the cells, potentially indicating higher cellular activity. The GnRH and CRH neurons discussed in this paper may represent such a case, as illustrated by the observed high serum GnRH, testosterone, and cortisol levels. This revision suggestion is highly valuable and constructive for our understanding of the unique physiological characteristics revealed by these data.

Reviewer #2 (Public Review):

Summary:

Li et al. investigated the potential anti-ageing role of 17α-Estradiol on the hypothalamus of aged rats. To achieve this, they employed a very sophisticated method for single-cell genomic analysis that allowed them to analyze effects on various groups of neurons and non-neuronal cells. They were able to sub-categorize neurons according to their capacity to produce specific neurotransmitters, receptors, or hormones. They found that 17α-Estradiol treatment led to an improvement in several factors related to metabolism and synaptic transmission by bringing the expression levels of many of the genes of these pathways closer or to the same levels as those of young rats, reversing the ageing effect. Interestingly, among all neuronal groups, the proportion of Oxytocin-expressing neurons seems to be the one most significantly changing after treatment with 17α-Estradiol, suggesting an important role of these neurons in mediating its anti-ageing effects. This was also supported by an increase in circulating levels of oxytocin. It was also found that gene expression of corticotropin-releasing hormone neurons was significantly impacted by 17α-Estradiol even though it was not different between aged and young rats, suggesting that these neurons could be responsible for side effects related to this treatment. This article revealed some potential targets that should be further investigated in future studies regarding the role of 17α-Estradiol treatment in aged males.

Strengths:

(1) Single-nucleus mRNA sequencing is a very powerful method for gene expression analysis and clustering. The supervised clustering of neurons was very helpful in revealing otherwise invisible differences between neuronal groups and helped identify specific neuronal populations as targets.

Thanks.

(2) There is a variety of functions used that allow the differential analysis of a very complex type of data. This led to a better comparison between the different groups on many levels.

Thanks.

(3) There were some physiological parameters measured such as circulating hormone levels that helped the interpretation of the effects of the changes in hypothalamic gene expression

Thanks.

Weaknesses

(1) One main control group is missing from the study, the young males treated with 17α-Estradiol.

Given that the treatment period lasts six months, which extends beyond the young male rats' age range, we aimed to investigate the perturbation of 17α-Estradiol on the normal aging process. Including data from young males could potentially obscure the treatment's effects in aged males due to age effects, though similar effects between young and aged animals may exist. Long-term treatment of hormone may exert more developmental effects on the young than the old. Consequently, we decided to exclude this group from our initial sample design. We apologize for this omission.

(2) Even though the technical approach is a sophisticated one, analyzing the whole rat hypothalamus instead of specific nuclei or subregions makes the study weaker.

The precise targets of 17α-Estradiol within the hypothalamus remain unresolved. Selecting a specific nucleus for study is challenging. The supervised clustering method described in this manuscript allows us to identify the more sensitive neuron subtypes influenced by 17α-Estradiol and aging across the entire hypothalamus, without the need to isolate specific nuclei in a disturbed hypothalamic environment.

(3) Although the authors claim to have several findings, the data fail to support these claims. You may mean the claim as the senescent phenotype in Crh neuron induced by 17a-estradiol.

Thanks. We have changed the "senescent phenotype" to "stressed phenotype" in the abstract and results to avoid such claim. The stressed phenotype may be induced by heightened functional activity in the cells, potentially indicating higher cellular activity.

(4) The study is about improving ageing but no physiological data from the study demonstrated such a claim with the exception of the testes histology which was not properly analyzed and was not even significantly different between the groups.

The primary objective of this study is to elucidate the effects of 17α-Estradiol on the endocrine system in the aging hypothalamus; exploring anti-aging effects is not the main focus. From the characteristics of the aging hypothalamus, we know that down-regulated GnRH and testosterone levels, along with elevated mTOR signaling, are indicators of aging in these organs from previous publications (PMID: 37886966, PMID: 37048056, PMID: 22884327). The contrasting signaling networks related to metabolism and synaptic processes significantly differentiate young and aging hypothalami, and 17α-Estradiol helps rebalance these networks, suggesting its potential anti-aging effects.

(5) Overall, the study remains descriptive with no physiological data to demonstrate that any of the effects on hypothalamic gene expression are related to metabolic, synaptic, or other functions.

The study focuses on investigating cellular responses and endocrine changes in the aging hypothalamus induced by 17α-estradiol, utilizing single-nucleus RNA sequencing (snRNA-seq) and a novel data mining methodology to analyze various neuron subtypes. It is important to note that this study does not mainly aim to explore the anti-aging effects. Consequently, we have revised the claim in the abstract from “the effects of 17α-estradiol in anti-aging in neurons” to “the effects of 17α-estradiol on aging neurons.” We observed that the lower overall metabolism and increased expression levels of cellular processes in the synapses align with findings previously reported regarding 17α-estradiol. To address the lack of physiological data and the challenges in measuring multiple endocrine factors due to their volatile nature, we employed several bidirectional Mendelian analyses of various genome-wide association study (GWAS) data related to these serum endocrine factors to identify their mutual causal effects.

Reviewing Editor Comment:

Based on the Public Reviews and Recommendations for Authors, the Reviewers strongly recommend that revisions include an experimental demonstration of the physiological effects of the treatment on ageing in rats as well as the CRH-senescence link. Additional analysis of the glia would greatly strengthen the study, as would inclusion of females and young male controls. The important point was also raised that the work linking 17a-estradiol was performed in mice, and the link with lifespan in rats is not known. Discussion of this point is recommended.

We thank the reviewers for their constructive feedback. Regarding the recommendations in the Public Reviews and Recommendations for Authors:

a)  Physiological effects & CRH-senescence link:

We acknowledge that 17α-estradiol has been reported to extend lifespan in male rats, consistent with findings in male mice (PMID: 33289482). This point has now been noted in the Introduction. We regret that further experimental validation of the treatment's physiological effects on aging in rats was beyond the scope of this study.

b) Phenotype terminology:

In response to concerns about the "senescent" characterization of CRH neurons, we have revised this terminology to "stressed phenotype" throughout the abstract and results. While we were unable to conduct additional experiments to confirm senescence markers, this revised description better reflects the heightened cellular activity observed (as evidenced by elevated serum GnRH and testosterone levels), without implying confirmed senescence.

c) Glial cell analysis:

To address questions about glial cell function during treatment, we have added new enrichment analysis data of differentially expressed genes in microglia and astrocytes from young (Y), old (O), and old treated (O.T) groups in Figure 2—figure supplement 3. This analysis reveals that microglia exhibit contrasting synaptic-related cellular processes compared to total neurons.

d) Female and young controls:

We sincerely apologize for the absence of female subjects and young male controls in the current study. The reviewers' suggestion to examine the male-specific effects of 17α-estradiol using female controls represents an excellent direction for future research, which we plan to pursue in upcoming studies.

Reviewer #2 (Recommendations For The Authors):

General comments:

(1) The manuscript is very hard to read. Proofreading and editing by software or a professional seems necessary. The words "enhanced", "extensive" etc. are not always used in the right way.

Thanks for the suggestion. We have revised the proofreading and editing. The words "enhanced" and "extensive" were also revised in most sentences.

(2) The numbers of animals and samples are not well explained. Is it 9 rats overall or per group? If there are 8 testes samples per group, should we assume that there were 4 rats per group? The pooling of the hypothalamic how was it done? Were all the hypothalamic from each group pooled together? A small table with the animals per group and the samples would help.

We appreciate your reminder regarding the initial mistake in our manuscript preparation. In the preliminary submission, we reported 9 rats based solely on sequencing data and data mining. The revised version (v1) now includes additional experimental data, with an effective total of 12 animals (4 per group). Unfortunately, we overlooked updating this information in the v1 submission. We have since added detailed information in the Materials and Methods sections: Animals, Treatment and Tissues, and snRNA-seq Data Processing, Batch Effect Correction, and Cell Subset Annotation.

(3) The Clustering is wrong. There are genes in there that do not fall into any of the 3 categories: Neurotransmitters, Receptors, Hormones.

We acknowledge the error in gene clustering and have implemented the following corrections:

(a) The description has been updated to state: 'Vast majority of these subtypes were clustered by neuropeptides, hormones, and their receptors among all neurons.'

(b) Genes not belonging to these three categories have been substantially removed.

(c) The neuropeptide category (now including several growth hormones) has been expanded to 104 genes, while their corresponding receptors (including several sex hormone receptors) now comprise 105 genes.

(4) The coloring of groups in the graphs is inconsistent. It must be more homogeneous to make it easier to identify.

We have changed the colors of groups in Fig. 1D to make the color of cell clusters consistent in Fig. 1A-D.

(5) The groups c1-c4 are not well explained. How did the authors come up with these?

We have added more descriptions of c1-c4 in materials and methods in the new version.

(6) In most cases it's not clear if the authors are talking about cell numbers that express a certain mRNA, the level of expression of a certain mRNA, or both. They need to do a better job using more precise descriptions instead of using general terms such as "signatures", "expression profiles", "affected neurons" etc. It is very hard to understand if the number of neurons is compared between the groups or the gene expression.

We have changed the "signatures" to "gene signatures" to make it more accurate in meaning. The "affected neurons" were also changed to "sensitive neurons". But sorry that we were not able to find better alternatives to the "expression profiles".

(7) Sometimes there are claims made without justification or a reference. For example, the claim about the senescence of CRH neurons due to the upregulation of mitochondrial genes and downregulation of adherence junction genes (lines 326-328) should be supported by a reference or own findings.

The "senescence" here is not appropriate. We have changed it to "stressed phenotype" or "aberrant changes" in abstract and results.

(8) Young males treated with Estradiol as a control group is necessary and it is missing.

Your suggestion is appreciated; however, the treatment duration for aged mice (O.T) was set at 6 months, while the young mice were only 4 months old. This disparity makes it challenging to align treatment timelines for the young animals. The primary aim of this study is to investigate the perturbation of 17α-estradiol on the aging process, and any distinct effects due to age effect observed in young males might complicate our understanding of its role in aged males, though similar endocrine effects may exist in the young animals. Long-term treatment of hormone may exert more developmental effects on the young than the old. Therefore, we made the decision to exclude the young samples in our initial study design. We apologize for any confusion this may have caused.

Specific Comments:

Line 28: "elevated stresses and decreased synaptic activity": Please make this clearer. Can't claim changes in synaptic activity by gene expression.

We have changed it to "the expression level of pathways involved in synapse"

Line 32: "increased Oxytocin": serum Oxytocin.

We have added the “serum”.

Line 52 - 54: Any studies from rats?

Thanks. In rats there is also reported that 17α-estradiol has similar metabolic roles as that in mice (PMID: 33289482) and we have added it to the refences. It’s very useful for this manuscript.

Line 62 - 65: It wasn't investigated thoroughly in this paper so why was it suggested in the introduction?

We have deleted this sentence as being suggested.

Line 70: "synaptic activity" Same as line 28.

We have changed it to "pathways involved in synaptic activity".

Line 79: Why were aged rats caged alone and young by two? Could that introduce hypothalamic gene expression effects?

The young males were bred together in peace. But the aged males will fight and should be kept alone.

Lines 78, 99, 109-110: It is not clear how many animals per group were used and how many samples per group were used separately and/or grouped. Please be more specific.

We have added these information to Materials and methods/Animals, treatment and tissues and Materials and methods/snRNA-seq data processing, batch effect correction, and cell subset annotation.

Line 205: "in O" please add "versus young.".

We have changed accordingly.

Line 207: replace "were" with "was"

We have alternatively changed the "proportion" to "proportions".

Line 208: replace "that" with "compared to" and after "in O.T." add "compared to?"

We have changed accordingly.

Line 223: "O.T." compared to what? Figure?

We have changed it accordingly.

Line 227: Figure?

We have added (Figure 1E) accordingly.

Line 229: "synaptic activity" Same as line 28.

We have revised it.

Line 235: "synaptic activity" and "neuropeptide secretion" Same as line 28.

We have revised it.

Line 256:" interfered" please revise.

We changed to "exerted".

Line 263: "on the contrary" please revise.

We have changed "on the contrary" to "opposite".

Line 270: "conversed" did you mean "conserved"?

We have changed "conversed" to "inversed".

Line 296-298: Please explain. Why would these be side effects?

It’s hard to explain, therefore, we deleted the words "side effects".

Line 308: "synaptic activity" Same as line 28.

We have changed it to "expression levels of synapse-related cellular processes".

Line 314: "and sex hormone secretion and signaling"Isn't this expected?

Yes, it is expected. We have added it to the sentence "and, as expected, sex hormone secretion and signaling".

Line 325-328: Why is this senescence? Reference?

We have added “potent” to it.

Line 360-361: This doesn't show elevated synaptic activity.

"elevated synaptic activity" was changed to "The elevated expression of synapse-related pathways"

Line 363-364: "Unfortunately" is not a scientific expression and show bias.

We have changed it to "Notably".

Line 376: Similar as above.

Yes, we have change it to "in contrast".

Lines 382-385: This is speculation. Please move to discussion.

Sorry for that. We think the causal effects derived from MR result is evidence. As such, we have not changed it.

Line 389: Please revise "hormone expressing".

We have changed it accordingly.

Line 401: Isn't this effect expected due to feedback inhibition of the biochemical pathway? Please comment.

The binding capability of 17alpha-estradiol to estrogen receptors and its role in transcriptional activation remain core questions surrounded by controversy. Earlier studies suggest that 17alpha-estradiol exhibits at least 200 times less activity than 17beta-estradiol (PMID: 2249627, PMID: 16024755). However, recent data indicate that 17alpha-estradiol shows comparable genomic binding and transcriptional activation through estrogen receptor α (Esr1) to that of 17beta-estradiol (PMID: 33289482). Additionally, there is evidence that 17alpha-estradiol has anti-estrogenic effects in rats (PMID: 16042770). These findings imply possible feedback inhibition via estrogen receptors. Furthermore, 17alpha-estradiol likely differs from 17beta-estradiol due to its unique metabolic consequences and its potential to slow aging in males, an effect not attributed to 17beta-estradiol. For instance, neurons are also targets of 17alpha-estradiol, with Esr1 not being the sole target (PMID: 38776045). Intriguingly, neurons expressing Ar and Esr1 ranked among the top 20 most perturbed receptor subtypes during aging (O vs Y), but were no longer ranked in this group following treatment (O.T vs Y and O.T vs O comparisons). This indicates that 17α-estradiol administration attenuated age-associated perturbation in these neuronal subtypes, which may be a consequence of potential feedback (Figure 3D). Nevertheless, the precise effective targets of 17alpha-estradiol are still unresolved.

Line 409: This conclusion cannot be made because the effect is not statistically significant. Can say "trend" etc.

Thanks for the recommendation. We have added "potential" in front of the conclusion.

Line 426: "suggesting" please revise.

sorry, it’s a verb.

Lines 426-428: This is speculation. Please move to discussion.

The elevated GnRH levels in O.T., observed through EIA analysis, suggest a deduction regarding the direct causal effects of 17alpha-estradiol on various endocrine factors related to feeding, energy homeostasis, reproduction, osmotic regulation, stress response, and neuronal plasticity through MR analysis. Thus, we have not amended our position. We apologize for any confusion.

Lines 431-432: improved compared to what?

The statement have been revised as " The most striking role of 17α-estradiol treatment revealed in this study showed that HPG axis was substantially improved in the levels of serum Gnrh and testosterone".

Line 435: " Estrogen Receptor Antagonists". Please revise.

Thanks for the recommendation. We have changed it to "estrogen receptor antagonists".

Line 438" "Secrete". Please revise

Sorry, it is "secret".

Lines 439-449: None of this has been demonstrated. Please remove these conclusions.

We appreciate the reviewer's scrutiny regarding lines 439-449. While these statements should not be interpreted as definitive conclusions from our current data, we propose they serve as clinically relevant discussion points worthy of exploration. Our findings demonstrate 17α-estradiol's role in modulating testosterone levels in aged males. This mechanistic insight warrants consideration of its therapeutic potential for age-related hypogonadism - a hypothesis we believe merits discussion given the compound's specific endocrine effects.

Lines 450-457: No females were included in this study. Why? Also, why is this discussed? It is relevant but doesn't belong in this manuscript since it was not studied here.

Testosterone levels are crucial for male health, while estradiol levels are essential for the health and fertility of females. Previous studies have demonstrated that 17α-estradiol does not contribute to lifespan extension in females. Given the effects of 17α-estradiol on males—specifically, its role in promoting testosterone and reducing estradiol levels—we believe it is important to discuss the potential sex-biased effects of 17α-estradiol, as this could inform future investigations. We have refined this section to clarify that these points represent mechanistic hypotheses derived from our male data and existing literature, not conclusions about unstudied female physiology. This framing maintains the discussion's scientific value while respecting the study's scope.

Lines 458-459: This was not demonstrated in this article. Please remove.

We have restricted the claim to "expression level of energy metabolism in hypothalamic neurons".

Line 464: "Promoted lifespan extension" Not demonstrated. Please remove.

At the end of the sentence it was revised as "which may be a contributing factor in promoting lifespan extension".

Line 466: "Showed" No.

The whole sentence was deleted in the new version.

Line 483: "the sex-based effects". Not studied here.

Since the changes in testosterone levels are significant in this dataset and this hormone has a sex-biased nature, we find it worthwhile to suggest this as a topic for future investigation. We have added "which needs further verification in the future" at the end of this sentence.

Document de Briefing : "Savoir ou périr" et les défis de l'éducation en France

Source: Extraits de "Rentrée scolaire : savoir ou réussite, pourquoi l’école tourne à l’envers (avec Bernard Lahire)"

Ce document de briefing synthétise les thèmes principaux, les idées essentielles et les faits importants tirés de l'entretien avec le sociologue Bernard Lahire, en se concentrant sur son ouvrage "Savoir ou périr" et ses réflexions sur le système éducatif français.

1. Le Savoir comme Condition de Survie Collective

Bernard Lahire insiste sur une idée fondamentale : le savoir n'est pas une simple affaire culturelle ou académique, mais une condition intrinsèque à la survie collective de l'humanité.

Il souligne que depuis l'aube de l'humanité, la transmission des expériences et des connaissances a été vitale pour l'adaptation des nouveaux venus et le développement des sociétés.

Citation clé : "on se rend pas compte que le savoir depuis le début de l'expérience de l'humanité euh c'est une des conditions de la survie collective"

Exemple historique : Lahire cite l'échec d'une expédition écossaise en Antarctique en 1845, dont aucun membre n'a survécu faute de savoirs adaptés à l'environnement hostile, contrairement aux Inuits qui y prospéraient.

Application contemporaine : La crise du coronavirus a mis en lumière l'urgence de la recherche et du savoir. Les investissements dans la recherche sur les coronavirus 10-15 ans auparavant, qui avaient été coupés, auraient pu accélérer la réponse.

"la recherche est directement euh concernée par les processus d'adaptation et que si on n'a pas ces savoirs et ben on est mal parti collectivement en fait".

Conséquence : L'oubli de cette vérité fondamentale nous rend "hors sol" et vulnérables aux défis futurs.

2. Le Système Scolaire "Tourne à l'Envers" : L'Obsession de l'Évaluation

Malgré l'importance vitale du savoir, Lahire dénonce un paradoxe français (et plus largement institutionnel) : l'école, censée être un lieu d'apprentissage, est devenue une institution "pilotée par l'évaluation", ce qui la fait "tourner à l'envers".

Dérive institutionnelle : Les institutions, créées avec un objectif précis, finissent souvent par dévier de leur mission initiale. L'école en est un exemple où l'évaluation a pris le pas sur l'apprentissage.

Bâchotage et surcharge des programmes : Lahire critique le "bâchotage" et la "surcharge des programmes", une problématique déjà soulevée par Marc Bloch en 1943. "on remplace le goût de la connaissance par le goût du succès".

Témoignages de grands scientifiques : Il s'appuie sur les expériences de personnalités comme Einstein, qui était "dégoûté de la physique" à force d'ingurgiter des choses par cœur, ou Grothendieck, qui critiquait ses collègues "trop dociles" et manquant d'ambition intellectuelle profonde.

La peur de la faute : Une spécificité française est la "peur de la faute", qui inhibe l'apprentissage des langues étrangères et contredit l'esprit scientifique, où l'erreur est une étape vers la découverte.

"on a tous peur de la faute je sais pas où on l'a attrapé mais évidemment que c'est à l'école que ça s'est passé".

Effets négatifs de la compétition et du stress : La peur et la compétition sont contre-productives pour l'apprentissage et la recherche.

Lahire témoigne de sa propre "boule au ventre" pendant sa scolarité et cite Laurent Lafforgue, lauréat de la médaille Fields, qui n'a presque rien publié pendant 10 ans, soulignant l'importance de laisser du temps aux chercheurs sans pression évaluative excessive.

La nécessité du retour, pas uniquement de l'évaluation : Les élèves ont besoin de retours, d'encouragements et de guidance (comme des tuteurs pour une plante), mais pas d'une évaluation constante et stressante.

3. Les Inégalités Sociales et la Reproduction : "Les enfants ne vivent pas dans le même monde"

Au-delà des problèmes pédagogiques, Lahire met en lumière l'impact profond des inégalités sociales et de la reproduction sociale sur les parcours scolaires, en s'appuyant sur son ouvrage "Enfance de classe".

Différences d'expériences dès le plus jeune âge : L'idée que "les enfants vivent au même moment dans la même société mais pas dans le même monde" illustre que, dès 5-6 ans, des enfants de milieux différents ont déjà des passés, des interactions et des horizons de possibles radicalement distincts.

"l'horizon n'est pas du tout le même les possibilités ne sont pas les mêmes".

Le mythe du "quand on veut on peut" : Lahire réfute fermement cette idée, la qualifiant de "régression scientifique".

Il souligne le "poids très très lourd des déterminismes sociaux d'origine".

Altricialité secondaire et dépendance aux adultes : La longue période de dépendance des enfants vis-à-vis des adultes (altricialité secondaire) a des conséquences majeures.

Les caractéristiques des parents (capitaux culturels, intérêt pour la pédagogie) influencent fortement la capacité des enfants à s'adapter à l'école.

Le rêve républicain de l'égalité : L'égalité n'est pas une réalité, mais un "horizon".

Les sociétés sont inégalitaires, mais l'État et les collectivités ont la responsabilité d'infléchir ces processus en offrant des opportunités culturelles et éducatives à ceux qui en sont le plus éloignés.

L'importance de l'ouverture culturelle : Les activités culturelles (théâtre, musées) sont cruciales pour "donner une chance" aux enfants de milieux défavorisés de s'approprier ces codes et de lutter contre l'autocensure. Sans cela, de nombreux élèves se projettent vers des "études courtes" faute de "background culturel".

4. Le Parcours Personnel de Bernard Lahire et la Critique Politique

Lahire, lui-même issu d'un "milieu ouvrier", a souffert du système scolaire mais a réussi grâce à un ensemble de facteurs (soutien familial, enseignants, chance), réfutant l'idée de sa seule "agentivité".

Réussite non individuelle : Sa réussite est le produit de "toutes les relations que [j'ai] eues avec toutes ces personnes et ces institutions".

Critique de la politique à court terme : Lahire exprime sa "tristesse" et son "dégoût" face à une politique perçue comme "décevante", "hors sol" et focalisée sur des querelles partisanes, plutôt que sur les défis à long terme (comme les enjeux climatiques ou éducatifs) qui nécessitent une vision sur "10000 ans".

Critique des décideurs ignorants des réalités éducatives : Il déplore la nomination de ministres de l'Éducation qui "connaissent très mal leur dossier", citant l'exemple d'Élisabeth Borne affirmant que le projet professionnel devait s'exprimer dès la maternelle.

5. Conséquences Budgétaires et Menaces sur l'Éducation

La situation est aggravée par les contraintes budgétaires, qui menacent directement les initiatives visant à lutter contre les inégalités.

Impact des coupes budgétaires : Les plans d'économie prévus pourraient entraîner une baisse des dotations pour les départements, impactant directement les collèges REP et les activités culturelles essentielles pour ces élèves. "il y a peut-être des activités culturelles qui vont devoir être annulées notamment pour louer un car".

En résumé, Bernard Lahire alerte sur un système éducatif qui a perdu de vue l'essence du savoir, obsédé par l'évaluation et incapable de compenser efficacement les inégalités sociales profondes, le tout aggravé par des décisions politiques court-termistes et un manque de compréhension des enjeux éducatifs.

Hacer diagnóstico diferencia con TDAH o desnutrición

para contrastar lo dicho por varios autores

resaltar la voz experta en el tema

para sustentar algo

para corroborar lo que dice

Acá aparece el lenguaje como generador del mundo. Además de ejercer el poder del lenguaje en la realidad y crear esa gramática mítica que refiere Tolkien necesaria para la subcreación.

La renovación por parte de la fantasía desde esa subcreación tiene que ver con deshacer esa fatiga de la realidad y comodidad visual de su cotidianidad.

La creación fantástica requiere esfuerzo: diferencia con la irrealidad del sueño.

la subcreación en la literatura como una manifestación más elevada por crear un mundo secundario separado del mundo primario y que se hace más poderosa. Parece hacer eco de la máxima de Mallarmé: "hay que elevar la página a la potencia del cielo estrellado".

Noción de lo fantástico.

La mitología como subcreación más que representación. Se trata más de la posibilidad de moldear el mundo que de volver a los temas míticos de origen.

El adjetivo como manifestación de una gramática mítica. Genera un poder de abstracción que se ejerce sobre el mundo exterior a nuestra mente y crear nuevas formas que llevan una fantasía de lo real. Acá es desde donde sitúa Tolkien la idea del hombre (ser humano) como subcreador. El poder de la fantasía es hacer efectiva la voluntad de la visión fantástica.

Contexto de la definición. Características para entender los cuentos de hadas.

Desplazamiento de la noción de poiesis en la obra de arte: de aletheia al operari.

Poiesis en la época moderna

Concepciones griegas

Según esto toda poiesis es acto.

🧠 “La razón se confunde frente al prodigio del amor”

El emperador admite que el amor desafía la lógica. Lo que parece un fenómeno biológico se transforma en un misterio que no puede explicarse con pura razón.

🫀 La carne propia vs. la carne ajena

Nuestro propio cuerpo nos interesa de manera mínima: lo lavamos, lo alimentamos, lo cuidamos para evitar dolor.

Pero, en el amor, el cuerpo ajeno se convierte en objeto de obsesión, de deseo apasionado.

✨ El enigma del deseo

¿Por qué?

Porque ese cuerpo está animado por una individualidad distinta a la nuestra (tiene un alma propia).

Porque exhibe ciertos trazos de belleza, aunque la belleza es tan subjetiva que ni los mejores jueces coinciden en qué la define.

🗝️ Sentido profundo

El texto muestra cómo el amor erosiona la frontera entre lo racional y lo irracional:

La carne deja de ser mera materia y se convierte en misterio encarnado en el otro.

El deseo no surge de la carne en sí, sino del hecho de que esa carne es otredad viviente.

La belleza funciona como chispa, pero es arbitraria y variable.

👉 En resumen: Adriano reconoce el amor como una paradoja —una fuerza que convierte lo banal (un cuerpo) en lo más precioso, por la simple diferencia de que no es el nuestro.

⚰️ Despojamiento como la muerte

El inicio compara el acto del amor (o de la entrega espiritual) con la muerte: desnudez absoluta, pérdida de todo poder, abandono del yo.

🙇 Humildad más que derrota o plegaria

En la derrota uno es humillado por otro.

En la plegaria uno se humilla ante los dioses.

Pero en el amor, la humildad es todavía más radical: es voluntaria. Uno se vacía por decisión propia.

🔄 El retorno de lo humano

Después de ese instante de anulación, vuelve la vida con toda su carga:

Negativas: límites, rechazos.

Responsabilidades: lo que uno debe asumir.

Dones: lo que se recibe y se da.

Tristes confesiones: la vulnerabilidad.

Frágiles ternuras: la delicadeza que sobrevive en lo cotidiano.

🧭 Sentido

El pasaje muestra cómo el amor o la entrega íntima tiene un movimiento doble:

Vaciamiento absoluto (como la muerte).

Retorno a lo complejo y humano (responsabilidad, ternura, confesiones).

Es casi una dialéctica: el amor como aniquilación momentánea seguida por la reconstrucción del tejido vital.

⚰️ Despojamiento como la muerte

El inicio compara el acto del amor (o de la entrega espiritual) con la muerte: desnudez absoluta, pérdida de todo poder, abandono del yo.

🙇 Humildad más que derrota o plegaria

En la derrota uno es humillado por otro.

En la plegaria uno se humilla ante los dioses.

Pero en el amor, la humildad es todavía más radical: es voluntaria. Uno se vacía por decisión propia.

🔄 El retorno de lo humano

Después de ese instante de anulación, vuelve la vida con toda su carga:

Negativas: límites, rechazos.

Responsabilidades: lo que uno debe asumir.

Dones: lo que se recibe y se da.

Tristes confesiones: la vulnerabilidad.

Frágiles ternuras: la delicadeza que sobrevive en lo cotidiano.

🧭 Sentido

El pasaje muestra cómo el amor o la entrega íntima tiene un movimiento doble:

Vaciamiento absoluto (como la muerte).

Retorno a lo complejo y humano (responsabilidad, ternura, confesiones).

Es casi una dialéctica: el amor como aniquilación momentánea seguida por la reconstrucción del tejido vital.

🍷 El bebedor y la razón

Beber no implica necesariamente perder la cabeza: uno puede tomar vino y seguir siendo dueño de sí.

Aquí el vino representa el placer controlado, el exceso manejable.

❤️ El amante y la razón

Pero en el amor, quien se mantiene demasiado lúcido no se entrega del todo.

“No obedece del todo a su dios” = no honra plenamente a Eros.

Amar exige perder algo de control, dejar que la pasión gobierne.

🗝️ Sentido profundo

Yourcenar (a través de Adriano) plantea un contraste:

El vino → placer que se puede modular.

El amor → fuerza divina que pide rendición.

Amar con cálculo frío es casi una contradicción: el dios exige sacrificio de la razón.

👉 Es una idea que viene de la tradición griega: Platón en el Fedro habla de la manía erótica (locura divina del amor) como una forma superior de verdad.

Clima en las finanzas