shows she feared for him and his father tried to keep him in a place safe

I have personally experienced this especially on Twitter or Threads. These two apps prioritize engagement, doesn't matter what type it may be- positive, negative, neutral, the algorithm will take it as a sign that you'd like to see more of that content, and suggest posts that are similar to the one that you interacted with. Tiktok I think is a bit smarter with how they've designed their algorithm, as whenever I leave a negative comment on a negative post that I dont agree with, it doesn't show me more of that content, but rather people also hating on that negative content. It's a lot smarter in the ways that it shows posts to it's users, it's able to read when a user has a positive or negative response and is able to adjust accordingly.

This oversight is noticeable when someone "bots" their streams or their likes and comments. These are key metadata points that algorithms focus on, so taking advantage of the system can generate a big reward. Social media sites do try to regulate this, but individual content creators have an ethical duty to prevent it.

Individual analysis looks at a person’s intentions and choices, while systemic analysis looks at how rules or algorithms can create biased outcomes even when no single individual intends them. Recommendation systems matter ethically because they can systematically amplify harm or inequality at scale, so fixing problems often requires changing the system.

It seems like most social media platforms nowadays prioritize reactivity and anger in creating algorithms. A lot of platforms show me discourse or controversial videos in order to maximize engagement, but these social media structures appear to be affecting real life behavior, especially of children.

This section clearly explains the difference between individual and systemic responsibility, and the sentencing example makes the idea of systemic bias very concrete and easy to understand. I especially like how recommendation algorithms are framed as systems that can produce harmful outcomes even without bad intent from individual designers or users. The contrast between blaming users and addressing structural problems is effective, and the tweets help connect theory to real-world discourse. Overall, this is a strong and thought-provoking explanation of why ethical analysis of algorithms needs to go beyond individual behavior.

Reviewer #1 (Public review):

Summary:

In this manuscript, Zhang et al. demonstrate that depletion of the 18S rRNA m6A methyltransferase Mettl5 compromises translation fidelity and consequently increases neoantigen generation, thereby uncovering an unexpected role for Mettl5 in tumor immunity. Mettl5-KO tumors exhibit enhanced CD8⁺ T-cell infiltration and show improved responses to immune checkpoint blockade. Mechanistically, loss of Mettl5 perturbs the local structure of 18S rRNA and disrupts the ribosome's ability to perform accurate translation. Subsequent ribosome profiling and mass spectrometry analyses provide compelling evidence that Mettl5 functions as a previously unrecognized regulator of translation to participate in tumor immune evasion.

Strengths:

This study presents a comprehensive set of experimental data supporting a mechanistic link between rRNA modification, translation fidelity, and neoantigen generation. The observed synergistic effect of Mettl5 depletion and anti-PD-1 therapy highlights the potential translational relevance of targeting rRNA modifications in cancer immunotherapy.

Weaknesses:

(1) In light of the principal function of Mettl5, which is to methylate 18S rRNA within the small ribosomal subunit, the authors focus primarily on translation fidelity, largely associated with elongation, but provide limited exploration of potential effects on translation initiation. Loss of Mettl5 may alter the initiation landscape, potentially promoting alternative or noncanonical initiation events (e.g., initiation at CUG codons), which could also contribute to the observed neoantigen repertoire changes. Further investigation into initiation-level alterations would strengthen the mechanistic interpretation.

(2) Given the broad involvement of rRNA methyltransferases in ribosome function, the authors should incorporate a parallel analysis using another enzyme (e.g., Zcchc4 or Nsun5) as a negative control. Such an experiment is essential to demonstrate that the tumor immunity phenotype observed is specific to Mettl5 rather than a general consequence of perturbing rRNA modification.

Out of the large variety of social media apps, I believe Tiktok is the most renowned for its algorithm and the methods in which it recommends videos to users. Just the other day I was in a situation where I wanted to watch videos on silent, and the tiktok I came across was captioned something relating to the audio that was playing, but because I couldn't hear it I went to the comments for some clues. To my surprise I was met with other users who were in the same situation as me- commenting things such as: "So we're all watching Tiktok on mute rn?" or "I'm never on mute, how does tiktok know that everyone whos seeing this video is watching it on mute???" I found it pretty crazy and I was kind of freaked out because it took such a niche scenario for me to be in and put a video on my page that perfectly matched what me and hundreds of thousands of other people were doing. It wasn't like I had just said some words around my phone and the app decided to show me a video related to those words, this was a situation where I didn't think my phone would have any idea that I was on silent. ****

These are all instances of metadata. This is extremely useful to push engaging content, but there is less control on the quality of content. I've noticed many reels that are just short clips of movies, or barely noticeable reaction content that contribute nothing. Maybe this is due to the greater resource load it takes to filter for content, or its harder to decide for someone what types of content they should watch.

Reviewer #2 (Public review):

In their manuscript "TGF-β drives the conversion of conventional NK cells into uterine tissue-resident NK cells to support murine pregnancy", Yokoyama and colleagues investigate the role of Tgfbr2 expression by NK cells in the formation of tissue-resident uterine NK cells and subsequent importance in murine pregnancy. By transferring congenic splenic conventional NK cells into pregnant mice, they show conversion of circulating NK cells into uterine ivCD45 negative tissue-resident NK cells. When interfering with the formation of uterine trNK cells, spiral artery remodelling was impaired, fetal resorption rates were increased, and litter sizes were reduced.

Generally, this is a research topic of high interest, yet the manuscript is lacking detailed mechanistic insights, and some questions remain open. At the current state, the data represent an interesting characterisation of the Tgfbr2-fl/fl Ncr1-Cre mice in pregnancy, but considering (a) the recent publication by the group (Reference 17) on the role of Eomes+ cNK cells during pregnancy, (b) the previously described role of Tgfbr2 and autocrine TGFb expression for uterine NK cell differentiation in virgin mice (also cited by the authors), and (c) the well-known relevance of uterine NK cells during pregnancy, additional experiments addressing the specific role of Tgfb during pregnancy would help to improve novelty and significance of the manuscript. To this end, the following aspects should be discussed and, where applicable, experimentally addressed by the authors:

(1) The authors suggest cNK extravasation and local differentiation into iv- trNK.

Can it be estimated how much this process contributes to the trNK pool vs. a potential local proliferation of already existing trNK? How do absolute numbers of CD49a+ Eomes+ trNK change during pregnancies? (In Figure 1A, the cell numbers of CD49a+ Eomes+ trNK seem to go down dramatically between gd 6.5 and 14.5). The plot in 1B could also include absolute numbers of ILC1s and trNKs. Would recruited cNK cells compensate for a potential loss of CD49a+ Eomes+ trNK?

(2) Figure 1C: 2.5

Mio cNK cells have been transferred, but only very few cells can be detected within the uterus (concatenated FACS plot shown). What may represent the limit to generate uterine trNK out of cNK? Is the niche supporting cNK-trNK differentiation limited? Is it only a specific subset of (splenic) cNK capable of differentiating into trNK? Is gd 0.5 the optimal timepoint for the transfer? Is there continuous recruitment of cNK into the uterus and differentiation into trNK, or is it enhanced at specific timepoints of pregnancy? Could there be local proliferation of cNK-derived trNK? This could be studied by proliferation dye dilution of WT cNK cells in this transfer-setup.

(3) The authors should consider inducible Tgfbr2 deletion (e.g. with Tamoxifen-inducible Cre) to enable development of the uterine NK compartment in virgin mice and only ablate trNK differentiation during pregnancy. This could help to estimate the turnover of cNK into trNK, or to understand if constant cNK recruitment is required to form the uterine trNK compartment during pregnancy.

(4) Did the authors consider transfer of Tgfbr2-floxed Ncr1-Cre cNK in the same setup as in Fig. 1C? This experiment could confirm the requirement of Tgfbr-dependent signalling for cNK to trNK conversion during pregnancy versus effects of Tgfb signals on trNK numbers in the uterus at steady state (before pregnancy).

(5) Figures 2D/E

The authors should state that ILC1s are reduced in the virgin uterus of female Tgfbr2-floxed or Tgfb1-floxed Ncr1-Cre mice and cite the relevant work (the Ref #29 discussed in this context did not show that?). It would be helpful to include an analysis of all three uterine ILC subsets in steady state. This could help to answer the question if the cNK cell changes are pregnancy-specific or a general phenomenon in Tgfbr2-floxed Ncr1-Cre mice.

(6) Figure 2E

Please phrase more carefully about the "concomitant increase" of cNKs, since this increase is much less pronounced compared to the very strong reduction (absence) of trNKs in Tgfbr2-floxed Ncr1-Cre mice. Do the authors suggest that cNKs are halted at this stage and cannot differentiate into trNK, based on these data?

(7) Figure 3/4

Can the reduced litter size and the abnormal spiral artery formation be rescued by transfer of WT cNK into Tgfbr2-floxed Ncr1-Cre mice?

True, I wonder if all those different versions of teachings stayed or were things taken away because they ended up coming to an agreement on teaching certain topics?

This is good the challenging and development of new narratives and memories can help increase its validity. But it may also base those memories on our current society.

This line is the essay’s thesis in miniature: what gets taught as “history” shapes citizenship, belonging, and power. The author is telling you to read historiography as a political struggle over national identity.

This part shares how exercising after having breakfast it helps individuals with T2D to able to balance their blood sugar spikes rather than exercising before breakfast.

This is like the “view source” bit for this 90/90/90 thing

I do partly agree with this, it must be overwhelming, but if you don't want people to watch your content, why do you do it? If someone puts themselves on the internet they must think about the possible consequences. It feels like you always want what you aren't or can't have. It is like the TikTok says, a double edged sword.

This is actually kind of bold. He is basically saying forced religion corrupts everyone. It makes me wonder though if he believes reason and persuasion are the only just tools in religion, why does he not apply that same logic to race and slavery? He trusts reason here, but not when it threatens the social system he benefits from.

He literally dismisses slavery and oppression as explanations and jumps straight to “nature.” Even after admitting differences in education and situation, he still concludes it is biological. That feels like he decided the answer before looking at the evidence.

He admits Black people have “ten thousand recollections” of injuries, meaning he knows slavery is violent and traumatic. But instead of seeing emancipation with justice as the solution, he assumes racial war is inevitable. It's like he's using fear to justify separation instead of equality.

The word “suffered” makes it sound passive, like their extinction just happened. But who caused that? Colonization was not accidental. It feels like he is mourning the loss of tribes while ignoring the role Europeans played in that loss.

He says women have “natural equality,” but then still supports a government run by men only. So does he actually believe women are equal, or just theoretically equal in some abstract way? This feels like another contradiction between his ideals and reality.

How do he say “all humans are morally equal” and then immediately rank them? That feels like he's redefining equality to fit what he already believes. This is already the Enlightenment contradiction before even getting to his actual argument.

I remember a case from the real world where this resulted in a terrible outcome. Because YouTube kept recommending similar videos (these were from ISIS) the user eventually became a recruit, because of the echo chamber created by the algorithm. In this case though it was hard to put blame on an algorithm because of the argument of free choice to click away. But who carries responsibility in these cases?

This made me realize that recommendation algorithms don’t just shape what we see, but also how people behave online. Once creators know what gets rewarded, content starts being designed for the algorithm rather than for actual users. Over time, that seems like it could slowly change the tone and quality of the platform itself.

This is particularly why I have issues with things such as spotify wrapped type content for apps (I do not have spotify, but a ton of other apps have followed this event). At the end of 2024 I was given a ton of memory type events that showed photos and posts I made throughout the year, and it showed photos such as my childhood pets of nearly 13 years being put down, screenshots of my family members suicide notes (there were multiple of these from deceased family members), and photos of me engaging in substance abuse behaviors. While I don't listen to music, I have had friends say that they form extreme emotional connections with certain songs, and that sometimes associations involve specific songs with breakups or abuse, which then get showed as frequently played in their wrapped even if it was only during a 1 month span. A lot of this is automatically shown to users. I think users should be able to opt-out of these type of events, since 1. a user may not want to be tracked in that way and 2. it could bring up painful memories. Additionally, this opt-out should not be hidden (deceptive patterns/dark patterns), it should be clear and easily accessible.

This passage explains Henry’s Law by using a carbonated drink as an example. When the can is sealed, high pressure keeps more carbon dioxide dissolved in the liquid at equilibrium. Once opened, the pressure drops, gas escapes, and a new equilibrium forms with less dissolved gas, causing the drink to go flat.

Reviewer #1 (Public review):

The manuscript titled "The distinct role of human PIT in attention control" by Huang et al. investigates the role of the human posterior inferotemporal cortex (hPIT) in spatial attention. Using fMRI experiments and resting-state connectivity analyses, the authors present compelling evidence that hPIT is not merely an object-processing area, but also functions as an attentional priority map, integrating both top-down and bottom-up attentional processes. This challenges the traditional view that attentional control is localized primarily in frontoparietal networks.

The manuscript is strong and of high potential interest to the cognitive neuroscience community. Below, I raise questions and suggestions to help with the reliability, methodology, and interpretation of the findings.

(1) The authors argue that hPIT satisfies the criteria for a priority map, but a clearer justification would strengthen this claim. For example, how does hPIT meet all four widely recognized criteria, such as spatial selectivity, attentional modulation, feature invariance, and input integration, when compared to classical regions such as LIP or FEF? A more systematic summary of how hPIT meets these benchmarks would be helpful. Additionally, to what extent are the observed attentional modulations in hPIT independent of general task difficulty or behavioral performance?

(2) The authors report that hPIT modulation is invariant to stimulus category, but there appear to be subtle category-related effects in the data. Were the face, scene, and scrambled images matched not only in terms of luminance and spatial frequency, but also in terms of factors such as semantic familiarity and emotional salience? This may influence attentional engagement and bias interpretation.

(3) The result that attentional load modulates hPIT is important and adds depth to the main conclusions. However, some clarifications would help with the interpretation. For example, were there observable individual differences in the strength of attentional modulation? How consistent were these effects across participants?

(4) The resting-state data reveal strong connections between hPIT and both dorsal and ventral attention networks. However, the analysis is correlational. Are there any complementary insights from task-based functional connectivity or latency analyses that support a directional flow of information involving hPIT? In addition, do the authors interpret hPIT primarily as a convergence hub receiving input from both DAN and VAN, or as a potential control node capable of influencing activity in these networks? Also, were there any notable differences between hemispheres in either the connectivity patterns or attentional modulation?

(5) A few additional questions arise regarding the anatomical characteristics of hPIT: How consistent were its location and size across participants? Were there any cases where hPIT could not be reliably defined? Given the proximity of hPIT to FFA and LOp, how was overlap avoided in ROI definition? Were the functional boundaries confirmed using independent contrasts?

Comments on revisions:

The authors have successfully addressed my previous questions and concerns. The public comments above reflect my views on the initial submission and, in my opinion, will remain helpful for general readers. Given this, I do not have additional public comments and will keep my previous public review unchanged.

Reviewer #2 (Public review):

Summary

This study investigates the role of the human posterior inferotemporal cortex (hPIT) in attentional control, proposing that hPIT serves as an attentional priority map that integrates both top-down (endogenous) and bottom-up (exogenous) attentional processes. The authors conducted three types of fMRI experiments and collected resting-state data from 15 participants. In Experiment 1, using three different spatial attention tasks, they identified the hPIT region and demonstrated that this area is modulated by attention across tasks. In Experiment 2, by manipulating the presence or absence of visual stimuli, they showed that hPIT exhibits strong attentional modulation in both conditions, suggesting its involvement in both bottom-up and top-down attention. Experiment 3 examined the sensitivity of hPIT to stimulus features and attentional load, revealing that hPIT is insensitive to stimulus category but responsive to task load - further supporting its role as an attentional priority map. Finally, resting-state functional connectivity analyses showed that hPIT is connected to both dorsal and ventral attention networks, suggesting its potential role as a bridge between the two systems. These findings extend prior work on monkey PITd and provide new insights into the integration of endogenous and exogenous attention.

Strength

(1) The study is innovative in its use of specially designed spatial attention tasks to localize and validate hPIT, and in exploring the region's role in integrating both endogenous and exogenous attention, as prior works focus primarily on its involvement in endogenous attention.

(2) The authors provided very comprehensive experiment designs with clear figures and detailed descriptions.

(3) A broad range of analyses was conducted to support the hypothesis that hPIT functions as an attentional priority map -- including experiments of attentional modulation under both top-down and bottom-up conditions, sensitivity to stimulus features and task load, and resting-state functional connectivity. These analyses showed consistent results.

(4) Multiple appropriate statistical analyses - including t-tests, ANOVAs, and post-hoc tests-were conducted, and the results are clearly reported.

Comments on revisions:

The authors have addressed our comments in their revised manuscript and in their response to the reviewers. We don't have any further suggestions or comments.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

The manuscript titled "The distinct role of human PIT in attention control" by Huang et al. investigates the role of the human posterior inferotemporal cortex (hPIT) in spatial attention. Using fMRI experiments and resting-state connectivity analyses, the authors present compelling evidence that hPIT is not merely an object-processing area, but also functions as an attentional priority map, integrating both top-down and bottom-up attentional processes. This challenges the traditional view that attentional control is localized primarily in frontoparietal networks.

The manuscript is strong and of high potential interest to the cognitive neuroscience community. Below, I raise questions and suggestions to help with the reliability, methodology, and interpretation of the findings.

Thank you for a nice summary of the key points of our study. Below you will find our reply to your questions.

(1) The authors argue that hPIT satisfies the criteria for a priority map, but a clearer justification would strengthen this claim. For example, how does hPIT meet all four widely recognized criteria, such as spatial selectivity, attentional modulation, feature invariance, and input integration, when compared to classical regions such as LIP or FEF? A more systematic summary of how hPIT meets these benchmarks would be helpful. Additionally, to what extent are the observed attentional modulations in hPIT independent of general task difficulty or behavioral performance?

Great suggestions! For the first suggestion, we have included a clearer justification in the discussion part of manuscript (line 405-406). For the second one, all participants received task practice prior to scanning, and task accuracy exceeded 90%, suggesting the tasks were not overly demanding. Although ceiling effects limit the interpretability of behavioral-performance correlations, we argue that higher task demands would likely require greater attentional effort, leading to stronger modulation in hPIT, which aligns with our findings.

(2) The authors report that hPIT modulation is invariant to stimulus category, but there appear to be subtle category-related effects in the data. Were the face, scene, and scrambled images matched not only in terms of luminance and spatial frequency, but also in terms of factors such as semantic familiarity and emotional salience? This may influence attentional engagement and bias interpretation.

The response of hPIT is not sensitive to stimulus category, but attentional modulation in hPIT is slightly stronger to faces than scenes and scrambled images. Although faces used in the task had neutral expressions and the scene pictures were also neutral, we acknowledge that we indeed cannot exclusively eliminate the possibility that potential semantic familiarity or emotional salience may contribute to the subtle category-related effects in the results of experiment 3. This limitation has been noted in the discussion part of manuscript (line 440-442).

(3) The result that attentional load modulates hPIT is important and adds depth to the main conclusions. However, some clarifications would help with the interpretation. For example, were there observable individual differences in the strength of attentional modulation? How consistent were these effects across participants?

Yes, individual differences exist. In the manuscript, we have included individual subject data points in the figure 6B. No data exceeded three standard deviations from the group mean, suggesting that the attentional modulation effects were generally consistent across participants.

(4) The resting-state data reveal strong connections between hPIT and both dorsal and ventral attention networks. However, the analysis is correlational. Are there any complementary insights from task-based functional connectivity or latency analyses that support a directional flow of information involving hPIT? In addition, do the authors interpret hPIT primarily as a convergence hub receiving input from both DAN and VAN, or as a potential control node capable of influencing activity in these networks? Also, were there any notable differences between hemispheres in either the connectivity patterns or attentional modulation?

Though it’s hard to generate directional flow of information from fMRI due to the low temporal resolution. We agree that besides resting-state connection, task-based functional connectivity analyses would have the potential to provide additional information about whether hPIT serves as a convergence node or a control hub. We have conducted task-based functional connectivity analyses, specifically PPI, using data from experiment 2, which revealed task-modulated right hPIT connectivity with FFA, LOp, and TPJ, suggesting hPIT may allocate attentional resources to object-processing regions following priority map generation (line 378-383). Given the limited number of significant PPI results and the inherent constraints of fMRI in capturing fast or transient attention-related interactions, the present data do not allow us to determine the role of hPIT. Future studies combining effective connectivity or causal perturbation methods (e.g., DCM, TMS-fMRI) would be ideal to test whether hPIT acts as a control node influencing activity within DAN and VAN.

We also observed modest hemispheric asymmetries in connectivity—for instance, both left and right hPIT showed stronger connectivity with right-hemisphere attention nodes. This has been described in the results part of manuscript (line 373-377).

(5) A few additional questions arise regarding the anatomical characteristics of hPIT: How consistent were its location and size across participants? Were there any cases where hPIT could not be reliably defined? Given the proximity of hPIT to FFA and LOp, how was overlap avoided in ROI definition? Were the functional boundaries confirmed using independent contrasts?

We can see a relatively consistent size and location of hPIT across subjects in Supplementary Figure 1, where the voxel size and location for individual subjects reported. The consistency also demonstrated by figure 4C.

We avoided overlap with the FFA and LOp by manually delineating the hPIT which is defined by conjunction maps across three tasks and by avoiding overlapping voxels. The FFA was defined using an independent contrast (Exp3 contrast [face-scene]) and the Lop location was defined by anatomical parcellation (Glasser et al., 2016).

Reviewer #2 (Public review):

Summary

This study investigates the role of the human posterior inferotemporal cortex (hPIT) in attentional control, proposing that hPIT serves as an attentional priority map that integrates both top-down (endogenous) and bottom-up (exogenous) attentional processes. The authors conducted three types of fMRI experiments and collected resting-state data from 15 participants. In Experiment 1, using three different spatial attention tasks, they identified the hPIT region and demonstrated that this area is modulated by attention across tasks. In Experiment 2, by manipulating the presence or absence of visual stimuli, they showed that hPIT exhibits strong attentional modulation in both conditions, suggesting its involvement in both bottom-up and top-down attention. Experiment 3 examined the sensitivity of hPIT to stimulus features and attentional load, revealing that hPIT is insensitive to stimulus category but responsive to task load - further supporting its role as an attentional priority map. Finally, resting-state functional connectivity analyses showed that hPIT is connected to both dorsal and ventral attention networks, suggesting its potential role as a bridge between the two systems. These findings extend prior work on monkey PITd and provide new insights into the integration of endogenous and exogenous attention.

Strengths

(1) The study is innovative in its use of specially designed spatial attention tasks to localize and validate hPIT, and in exploring the region's role in integrating both endogenous and exogenous attention, as prior works focus primarily on its involvement in endogenous attention.

(2) The authors provided very comprehensive experiment designs with clear figures and detailed descriptions.

(3) A broad range of analyses was conducted to support the hypothesis that hPIT functions as an attentional priority map -- including experiments of attentional modulation under both top-down and bottom-up conditions, sensitivity to stimulus features and task load, and resting-state functional connectivity. These analyses showed consistent results.

(4) Multiple appropriate statistical analyses - including t-tests, ANOVAs, and post-hoc tests - were conducted, and the results are clearly reported.

Thank you for a nice summary of the key points and strengths of our study.

Weaknesses

(1) The sample size is relatively small (n = 15), and inter-subject variability is big in Figures 5 and 6, as seen in the spread of individual data points and error bars. The analysis of attention-modulated voxel map intersections appears to be influenced by multiple outliers.

We agree that the sample size (n = 15) is not ideal, and we acknowledge that some data points in Figures 5 and 6 appear to be potential outliers. However, according to conventional outlier detection criteria, all data points fell within three standard deviations of the group mean and were therefore retained for analysis.

Moreover, the attention-modulated voxel intersection map shown in Figure 4C is insensitive to outliers, because the intersection plotted is based on the number of subjects

(2) The authors acknowledge important limitations, including the lack of exploration of feature-based attention and the temporal constraints inherent to fMRI.

Yes, we have mentioned these limitations in the discussion.

(3) Prior research has established that regions such as the prefrontal cortex (PFC) and posterior parietal cortex (PPC) are involved in both endogenous and exogenous attention and have been proposed as attentional priority maps. It remains unclear what is uniquely contributed by hPIT, how it functionally interacts with these classical attentional hubs, and whether its role is complementary or redundant. The study would benefit from more direct comparisons with these regions.

In this study, we define the ROI base on intersection across three different types of spatial attention tasks, which is a stricter criterion. And the results didn’t reveal spatial attentional modulation across tasks besides PITd. This could be due to the lack of lateralized responses in PFC/PPC. To evaluate whether a region qualifies as a priority map, we applied four widely accepted criteria (as mentioned in introduction). While dorsal and ventral attention network (DAN and VAN) regions can be considered supportive components of the priority map system, our findings suggest that among the regions tested, only hPIT fully meets all criteria. In Experiment 2, we included regions such as VFC (as part of PFC) and IPS (as part of PPC), and our findings suggest these areas are more involved in top-down attention. In the revision, we have performed additional analysis on PPC (IPS) and PFC (FEF, VFC), shown in Figure S2.

(4) The functional connectivity analysis is only performed on resting-state data, and this approach does not capture context-dependent interactions. Task-based data analysis can provide stronger evidence.

We acknowledge that resting-state FC is limited in assessing task-specific communication. To further investigate the role of hPIT, we have conducted PPI analysis, which revealed task-modulated right hPIT connectivity in attention allocation (line 378-383).

(5) The study does not report whether attentional modulation in hPIT is consistent across the two hemispheres. A comparison of hemispheric effects could provide important insight into lateralization and inter-individual variability, especially given the bilateral localization of hPIT.

We thank the reviewer for this suggestion. hPIT was localized bilaterally using the same intersection-based method in Experiment 1. We have now performed additional analysis and found hemispheric differences in hPIT attentional modulation (Experiment 2). Besides, we also found in Experiment 3, the difference of load modulation (averaged across stimulus categories) in left and right hPIT was not significant. These results have been reported in the results part of manuscript (line 347-351).

The part about personal space caught my attention because it shows we all kinda have an invisible bubble around us even if we do not think about it. When someone stands too close or too far it just feels off and changes how we see the interaction. The classroom example made sense because the professor reacted differently to each student just based on distance, not what they said. That shows communication is not only words, it is also space and body position. It explains why someone can feel awkward or rude even when they did not actually say anything bad. So distance affects how we judge people without us realizing it.

The part about self-disclosure being reciprocal stood out to me because it explains why some conversations feel normal and others feel awkward. When one person shares something kinda personal, the other person usually feels like they should share something too so it doesn’t feel one sided. If only one person keeps opening up while the other stays basic, the relationship can’t really get closer. This shows why relationships do not become deep right away and instead happen step by step. People basically test the waters before they trust someone more. So closeness comes from both people matching each other’s openness, not just talking a lot.

Burgoon’s definition of personal space shows that the distance we keep from others is not accidental but shaped by both cultural norms and personal comfort levels. What stood out to me is the idea that personal space is a balance between our need for closeness and our need for privacy, which connects directly to Expectancy Violations Theory. This helps explain why the same behavior, like standing too close, can feel friendly to one person but uncomfortable or invasive to another. Hall’s concept of proxemics adds to this by emphasizing that many of these spatial expectations operate without us even realizing it, making violations more noticeable and impactful when they occur.

This reignites with me a lot. When i see growth in myself i tend to think more positive. It makes me feel good when i see my growth and then when others see it as well. From writing to doing small task to keep up with my daily life. I dont think that one person is born with a skill set that another person cant do them selves. Unless theres a medical reasoning wht they can not perform the same act. I can write a book just as good as Stephen King- ONLY if I worked hard at it. building the skills that he has. Yes, some people might be able to do things easier than me. However, I possess my own set of skills that come easier to me than others as well.

I use to use short cuts when texting or writing something on social media. We do write all the time however I also lacked the knowledge to write a proper professional email without sounding like someone talking to their friends. Writing and basic communication skills are a must.

I need to make sure that when taking notes, I don't rely on just what I see but make sure to also record the things I see, feel, hear, smell, etc. It is important to include the senses when writing a detailed description so I can create a visual representation for those who are not at the site I am recording.

This tells me that I need to make sure I communicate thoroughly with my participants for this research and make sure it is okay for me to use what they are saying. I want to make sure they feel comfortable being included in this study, as well as how I plan to use their words/quotes.

To apply this, I need to focus on the "why" and "what". I also need to focus on the meaning behind people's actions and why they chose to interact that way. For example, if I go to the movie theater to observe a medical documentary, I'm not going to write down that people are getting popcorn, but instead focus on if the person getting the popcorn interacts with anyone else, are they doing it fast, etc.

If the ocean mixes around every 1000 years but the major ions stay for millions of years, does that basically mean the ocean has just had way more than enough time to fully mix everything evenly? Or could anything realistically throw that balance off?

I didn't realize water is most dense at 4 degrees Celsius instead of right when it freezes. That honestly seems like a small detail, but it actually makes a huge difference because it's the reason lakes freeze from the top down instead of turning into a giant block of ice. It's kind of wild that something this specific ends up protecting entire aquatic ecosystems.

Picking a good topic will catch the audiences attention.

Religion is no longer a belief, but a state device.

I knew what are tariffs but I didn't know how they worked. The car example makes a lot of sense to me because I live close by the Port Hueneme Base. They ship a lot of cars like Volvo, BMW, and Land Rover. I always see them drive through the street and wonder about how tariffs affect these new cars. Now, I'm more informed about tariffs.

main argument

This shows how breaking down a claim and looking at all perspectives can reveal information or counterclaims.

This video (which I've seen before) makes me consider how hierarchies get baked not just into the code of the digital world, but the very infrastructure. Its been well documented that the fact that cameras were designed by people with lighter skin made it hard for black people (especially in the age of film rather than digital photography) to find cameras and filmstock that captured their skintones. I wonder if something similar happens with the digital environment, if the lack of coders and designers from the Global South leads to assumptions in digital infrastructure that goes unnoticed and unchallenged.

defintion

When I read this paragraph, it feels like the writer is showing how one problem led to another until things got serious. Instead of just saying “a traffic light was needed,” the paragraph walks me through all the small issues that built up to that decision. It’s basically connecting the dots so I can see why the final outcome actually makes sense.

I wholeheartedly agree with this perspective, as students in the classroom may come from diverse backgrounds and possess their own unique personalities. Therefore, adapting to each student's preferred teaching style is something many educators in teaching positions continually strive for. I believe that only by fully respecting each student's individuality can we maximize the potential to inspire their creativity.

2003 DTI Smart Award for Innovation Feasability study:

"Personalized Mobile Conputing"

all about Personal Knowledge Work interchangeably on Desktop and Mobile Devices (Palm and Windows Mobile)

Doing DocBook Structure without the complexity of MarkDown

but morphic MarkIn Notation

HyperText without the complexiies of XML/XSD

Extensible Augmented Morphic Writing

Rendering was slow, 10 years ahead of the capabilities

The hidden goal was to eventually go from Peronal Learning Networks

towards Personal first, Autonomous InterPersonal InerPlanetary Mutual Learning Networks

Indy Learning Commons

only list sources that you cited

This section shows that virality isn’t just about how many people see something, it’s also about how others reshape it, sometimes in ways that support the creator’s original purpose. Features like TikTok Duets can amplify a post by letting others add on in a way that stays aligned with the initial intent, but that alignment isn’t guaranteed.

• Task Expansion & Role Blurring: AI lowers the barrier to entry for complex tasks, leading employees to take on work outside their core expertise. Product managers and designers are now writing code, while researchers take on engineering tasks.
• Specialist Burden: This expansion creates a "cleanup" tax. For example, senior engineers now spend significant time reviewing, debugging, and mentoring colleagues who produce "vibe-coded" AI outputs, often through informal and unmanaged channels like Slack.
• The "Ambient Work" Phenomenon: Because AI interactions feel conversational and "easy," work has become ambient. Employees find themselves prompting AI during lunch, between meetings, or late at night, eliminating natural mental downtime.
• Intensified Multitasking: Workers are running multiple AI agents in parallel while simultaneously performing manual tasks. This creates a high sense of "momentum" but leads to extreme cognitive load and constant attention-switching.
• The Productivity Trap: AI acts as a "partner" that makes revived or deferred tasks feel doable. This creates a flywheel where people don't work less; they simply take on more volume, leading to "unsustainable intensity" that managers often mistake for genuine productivity.
• Sustainability Risks: The researchers warn that while AI feels like "play" initially, it eventually leads to cognitive fatigue, impaired decision-making, and burnout as the quiet increase in workload becomes overwhelming.

Hacker News Discussion

• Cognitive Fatigue: Users highlighted that "AI fatigue" is distinct from normal work tiredness. It stems from the "constant vigilance" required to audit AI output and the lack of a "flow state" due to unpredictable waiting times for generations.
• Executive Function Strain: Commenters noted that managing autonomous agents is more exhausting than manual work. One user compared it to Level 3 autonomous driving—you aren't driving, but you must remain "fully hands-on" to ensure the AI doesn't touch the wrong files or hallucinate.
• The Jevons Paradox: Several participants pointed out that as the "cost" of work decreases due to AI, the demand for work increases proportionally. Instead of saving time, workers are expected to triple their output, which leaves them more stressed than before.
• Management Expectations: A common theme was that leadership often mandates AI usage and pre-supposes productivity gains, leaving no room for cases where AI makes work slower or lower quality. This forces employees to "perform" productivity while working longer hours.
• Vibe Coding vs. Engineering: There is a heated debate between those who see "vibe coding" (prompt-heavy development) as a massive efficiency gain and veterans who argue it produces "average code" that becomes a maintenance nightmare in large, legacy codebases.

So this study shows that there is no puberty change fore ages 12-17? The concern is puberty onset, not general maturation, meaning it is not applicable?

So hint of modern day anti-federalism but also what the hell happened, why did missouri become conservative

I think it is quite interesting how there are so many undeciphered writing systems just like this one. Goes to show how advanced their civilization may have been at the time. I can’t even fathom how a new language or writing system would be interpreted in today's day and age.

Council has unique position in terms of input -> individual member leaders see that the yhave input legitimacy by virtue of being NATIONALLY elected

And yet as a GROUP, the Council is not elected and lacks input legitimacy -> making decisions in interests of multiple parties.

Again, though, Schmidt's argument is that the TYPE of rhetoric is never examined in White / Kreuder-Sonner.

Okay, so legitimacy is not just CONSENT of governed but also the EFFECTIVENESS of government's solutions to the crisis (i.e., who cares if he overturned the election, he got rid of all the immigrants!)

"defined in terms of governing activities, legitimacy is linked to policy effectiveness and performance for the common good (output legitimacy); citizen participation and representation along with political elites’ responsiveness to citizens’ concerns (input legitimacy) (Scharpf, Citation1999, Ch. 1); and the quality of governance procedures (throughput legitimacy), including the efficacy of the policymaking processes, the accountability to relevant forums of those engaged in making the decisions, the transparency of their actions and access to information, along with their openness and inclusiveness with regard to civil society "

OKAY -> so is building off of Kreuder-Sonner / White -> says that not only do supranational entities have four LEVELS of emergency politics, all of which are united by the same script, but have to JUSTIFY their seizure of power / deviation from the norm THROUGH THREE DIFFERENT WAYS: 1. Coercive power OVER ides / discourse (i.e., controlling the narrative over their increased power) 2. Structural / institutional power IN ideas / discourse (idk) 3. Persuasive power THROUGH ideas via discourse (i.e., convincing people that increased power in this situation is good)

Will look at the RHETORIC of executive actors at times of EU crisis in orderto demonstarte this (i.e., what terms / langauge they use to justify the extraordinary power measures). Specifically Covid / Eurozone.

"actors involved in emergency politics may appeal to three different forms of legitimacy while making use of three different kinds of rhetorical power in their efforts to legitimize and normalize their actions"

The results of the study concluded that LKM increased positive emotions in people, which then lead to an increase in developing positive resources and overall satisfaction with their life.

via Todd Young https://www.facebook.com/groups/TypewriterCollectors/posts/10163194812329678/

As technology grows into different variations, it causes our brains to rely on technology more.

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Reply to the reviewers

General statements.

We thank the reviewers for their positive response and useful suggestions on our manuscript. They recognize the ‘proof of concept’ nature of the work and the importance of extending the number of human mutation-specific DMD mouse models from one to five for preclinical research. We feel that the quality of the manuscript has been improved upon implementation of the reviewer’s suggestions.

Reviewer 1.

OPTIONAL - From the point of view of the reviewer, it seems plausible to use CRISP/Cas9 to "clean up" the original hDMDmdx mouse line by selectively removing one of the YACs forming the tail-to-tail tandem in the mouse genome. Once such single copy mouse line is generated (and proven viable?) any subsequent rearrangement of the hDMD transgene would prove much less challenging. Such mouse line would also better represent human model where only one DMD copy is carried on the X chromosome.

The reviewer gives the optional suggestion that the generation of these models could have been combined with the removal of one of the copies of the YAC to extend the use of the new models to CRISPR-based therapies. This is correct, but we note that when the data on the removal of a copy of the YAC were published, our new models were already generated and in different stages of QC, colony building and analysis. The procedure described by Chey et al could be used on our new models, but this would require additional time and funding and is therefore outside the scope of this manuscript.

The labels in figure 2B and 3A would benefit from showing the PCR fragment lengths as well as the sizes of obtained hDMD exon deletions. On could also include an additional figure panel demonstrating the principle of ASO-induced exon skipping

Reviewer #1 also has a minor comment regarding the exact deletions in figure 2B and 3A. For fig. 2B he/she suggests to include the sizes of the PCR fragments next to the gel. Especially for the gel regarding PCR1, which detects the deleted YAC copy, this will not be very informative as this can be (and is) different for different clones depending on the NHEJ-mediated repair in the specific clone. Adding sizes is only interesting for each specific clone, and adding them all will make a very messy figure. The important message from this gel is the presence of any fragment, as the undeleted copy is not amplified under the conditions used. For the gel of PCR2 the opposite is the case, here the PCR fragment shown is simply the undeleted YAC copy, and here we are only interested in the absence of the PCR fragment.

We thank the reviewer for the suggestion of adding the deletion sizes to fig 3A. This made us realize that an additional table with the details of the mutant alleles in all models had been omitted, and we apologize for this error. With the revised version we include details on the size of the deletions and their genomic coordinates (in the human genome as it is in the human YAC) of each of the new models (revised Sup. Table 1). We trust that adding these details will clarify this reviewer’s minor comment.

The reviewer requests to include an additional figure panel demonstrating the principle of ASO-induced exon skipping. We have now added this to the revised version of the manuscript (new fig. 5).

The study is fairly limited in scope and will be of primary interest to those working in the DMD field.

We are aware of 9 clinical trials for exon 51 and 53 studies that are ongoing or were recently stopped. For four of these compounds companies have a license to our hDMDdel52/mdx mouse model, and one of these studies has been published. An additional 7 clinical trials are planned or ongoing for exon 44, 45 and 50 skipping for which the newly developed models are being or can be used for preclinical studies.

Reviewer 2.

To further strengthen the rigor of the study, it would be valuable to include an analysis of potential off-target effects of CRISPR editing, particularly given that double targeting of two YAC copies was required. This is especially important for germline edits, as off-target mutations could introduce confounding phenotypes in the resulting mice. Demonstrating minimal or absent off-target activity would increase confidence in the specificity and safety of the generated models.

There has indeed been one major study suggesting a large number of CRISPR-induced off-target mutations in mouse models. However, this publication was rapidly questioned by multiple groups for having used the wrong control animals and the original publication was retracted (https://doi.org/10.1038/nmeth0518-394a). Another study at that time, using the correct controls, did not find mutations that could be attributed to CRISPR-induced off-target mutations. A more recent study analysed founder animals from transgenic projects using 163 different guide RNAs and concluded ‘In total, only 4.9% (8/163) of guides tested have detectable off-target activity, at a rate of 0.2 Cas9 off-target mutations per founder analysed. In comparison, we observe __~1,100 unique variants in each mouse regardless of genome exposure to Cas9 __indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice.’ In short, the background mutation rate in mice is much higher than the Cas9 off-target mutation rate. In addition to this, we only used guide RNAs that did not have any predicted off-target sites (according to the CRISPOR tool; https://crispor.gi.ucsc.edu/crispor.py) on the same chromosome or in protein coding sequences, so that any undetected off-target mutation will rapidly be lost in the subsequent breeding. We also would like to refer the reviewer to the ‘referee cross-commenting remark’ from reviewer #3 on this topic.

The validation of the dystrophic phenotype is generally convincing. However, the authors should clarify how "human dystrophin" is detected in the deletion models. Since only part of the dystrophin gene in these mice is humanized (the remainder is murine), it is important to specify, also in the results, which antibody was used and which epitope/exon it recognizes. If the antibody targets a deleted exon in a given model, this could lead to misinterpretation of the dystrophin signal. Providing this clarification would ensure the conclusions regarding dystrophin expression are fully supported.

This question is based on the incorrect assumption that only part of the DMD gene in these models is humanized. As described in the original publication on the YAC transgenics the complete human gene is in the YAC. Here, we deleted a particular exon from this complete human DMD gene. In combination with the mdx allele, these mice lack the full-length mouse and human dystrophin isoforms expressed in muscle. As mentioned in the materials section, the human dystrophin protein was detected with the Mandys 106 antibody (recognizing exon 43; amino acids 2063-2078), which only has reactivity with human dystrophin according to the product specification of Sigma Aldrich. We confirmed this for wild type mouse tissue, showing no dystrophin for this antibody. In fig 4 we confirm lack of human dystrophin in the deletion models using this antibody. The mouse and human dystrophin protein was detected with the AB154168 antibody of Abcam (recognizing the last 100 amino acids of the C-terminal part of the protein), which has reactivity with both mouse and human. So neither antibody did target a deleted exon. For the exon skipping validation, solely the Abcam antibody was used, as none of the deleted or skipped exons was recognized by this antibody. Information regarding the targeted protein region has now been added to the materials section.

Additionally, to further strengthen the characterization of the muscular dystrophy phenotype, the authors could quantify muscle fibre size and the percentage of centrally nucleated fibres, both of which are widely accepted quantitative markers of ongoing degeneration/regeneration in DMD models.

and

The validation of exon skipping in the new hDMD deletion models is convincing at the molecular level. However, since the ASOs were injected into both gastrocnemius and triceps muscles, it would be helpful to include at least a brief characterization of the triceps, even in supplementary data, as different muscles can show slightly different pathology and responses. Additionally, while the molecular readouts (RT-PCR and Western blot) demonstrate restoration of dystrophin expression, including simple histological analysis, such as H&E staining, could further support functional improvement and reinforce the physiological relevance of exon skipping in these models.

The proof-of-principle nature of the current manuscript is focused on restoration of dystrophin expression shortly after ASO treatment, and the current sample sizes (n=3 mice per strain) are too limited for actual quantification of histopathological improvements. Furthermore, the timespan between the intramuscular injection and tissue collection (2 weeks) does not allow sufficient time for histopathological improvements to develop. Notably, a large natural history analysis of all these new models is currently ongoing, which includes a large variety of in vivo functional outcome measures and provides a full description of the histopathological aspects of these mice. The proposed characterization of the triceps is now included as supplementary data of the manuscript (Sup. Fig 1).

Reviewer 3.

This reviewer starts with pointing out some typos, or requested rephrasing to sentences for clarification. We appreciate this and have addressed this in the revised version of the manuscript.

Generation of the models: it is not clear why the authors generated line 44 in ES cells, then switched to direct gene editing in zygotes. Was this due to advent of electroporation of zygotes at the time? This may need clarification beyond the sentence "Encouraged by the specificity of our new prescreen workflow and the efficiency of correct targeting of human exon 44 in ES cells, we generated additional models ... directly in mouse zygotes".

The simple answer to this is that we were (pleasantly) surprised ourselves by the efficiency we got in the ES cells (which was based on the previous experience generating the del52 model). For animal welfare reason we prefer to generate models via ES cells if we expect a long and cumbersome quality control process and / or very low efficiency, as ES cells allow us to do this QC before the actual animals are generated, thus reducing the number of animals generated during the model generation phase. Expecting very low efficiency, we originally picked 10 x 96 well plates of clones for this del44 targeting, but after pre-screening the first two plates (192 clones), we realized this was an enormous overkill in clones, and the additional 8 plates were not analysed. With this much higher than expected efficiency, and the power of the two-step pre-screen described in the manuscript, we decided to try the next model (the del45) directly in zygotes. This was found efficient enough to also do the last two models directly in zygotes. We can only speculate on the much higher efficiency than observed for the del52 targeting. Clearly the fact that we knew of the double integration this time allowed us to develop the successful 2-step pre-screen. Another difference is that the del52 model was generated using TALENs as genome editors, whereas now we could use CRISPR/Cas9.

Antisense oligonucleotide treatment: there is no description of the design of the ASOs beyond their sequence in suppl. Table 4. How were they designed? Moreover, they have been injected at two different doses (i.e., 50ul for Exon 51 & 53; 100ul for Exon 44 & 45). What is the rational for this? There is no justification in the manuscript.

The requested additional details on ASO design and dosing have been added to the materials section of the revised manuscript. The reviewer also pointed out that fig 4 includes both a protein sample diluted to 10% of protein of both a C57BL/6J and hDMD/mdx control mouse, and requested a justification for this. We included samples of both wildtype strains to confirm species reactivity of the dystrophin antibodies used, with the AB145168 antibody being specific for both mouse and human protein (showing a dystrophin band in both wildtype samples), and the Mandys106 antibody being specific to only human protein (showing a dystrophin band in the hDMD/mdx control only).

Phenotypic validation of the new models: a description of the mdx line with C57BL/6J mice is mentioned. Is this why Fig.4 includes "10% Bl6" and "10% hDMD/mdx"? If so, this should be clarified in the text (or deleted from the figure). The authors mentioned "As expected, the gastrocnemius of healthy hDMD/mdx mice expressed dystrophin of human origin at wildtype levels". Why would this be expected? If 2 copies of the gene, including the human promoter, are integrated, why would one expect a wildtype level of expression? In fact, in the original paper describing the hDMD/mdx model ('t Hoen et al. 2008), the human transcripts are expressed at 2 to 4-fold higher than their endogenous counterparts (which is in line with the integration of 2 copies).

It is true, as he/she points out, that qRT-PCR data in the original YAC transgenic publication showed double expression of the human transcript, consistent with the double integration. However, fig. 3b in the same paper shows that at the protein level the expression of human DMD is comparable to the mouse protein. We don’t know the reason for the discrepancy between transcript and protein levels in this model, but in the current manuscript we are referring to this protein expression.

A quantification of the expression levels on Figure 4 should be done (normalized to actinin) to resolve this. The size of the Marker should also be added on Figure 4.

We feel that proper quantification can only be done with the utilization of a standard curve. As we expected no, or trace levels of dystrophin in the deletion models, we only included wildtype samples diluted to 10% of wildtype protein. This prevents us from accurate quantification of the trace dystrophin levels observed in the del45 and del51 models. However, as can be appreciated from fig 4, expression is very minimal. We added information on the marker in the materials section, and indicated the size (85 kDa) in the figure legend.

Finally, the authors observed histological hallmarks of the disease in the new models (i.e., muscle degeneration and fibrosis). Although obvious on the images, it may be useful to add indications (e.g., arrows) on the images for readers non familiar with DMD.

We added information on the marker in the materials section, and indicated the size (85 kDa) in the figure legend. Lastly, we also added the requested arrows to the pictures of fig. 4B to allow distinction between different histopathological hallmarks, and refer to these in the figure legend.

Prescreen PCR of hDMD/mdx ES cells (Fig. 2): the authors mentioned that "The PCR conditions were chosen for not being able to amplify the undeleted allele." What does this mean? Was the elongation time reduced? As per the text, the theoretical size of a WT band is around 1.6kb. Yet, on the gel, bands higher than 1kb are visible for some clones.

This is indeed based on the extension time of the PCR reaction shown in PCR 1 from fig 2B, amplified with primers upstream and downstream of the deleted region (see fig 1 and 2A). However, the approx. 1.6 kb fragment the reviewer refers to is the undeleted-specific amplification shown in Fig 2B PCR 2, which is the result of a primer outside and a primer inside the deleted region (fig 1and 2A). Amplification of the undeleted copy with the primers used in PCR 1 would give a fragment of 3902 nt. The deletion of exon 44 in the final model is 3584 nt, which details will be shown in the excel file that was erroneously omitted (see our response to reviewer #1), with the PCR 1 product of the deleted copy in the clone used for the mouse model being 318 nt. It is straight-forward to select an extension time that would be insufficient for a 3.9 kb fragment, but which can amplify fragments that are shorter due to the deletion. Even in a clone with a single copy of exon 44 deleted, one would not expect to see the 3902 nt fragment due to preferential amplification of the much shorter mutant band. This has now been clarified in the legend of figure 2 of the revised version of the manuscript.

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Referee #1

Evidence, reproducibility and clarity

Summary:

The study describes the creation and preliminary validation of four humanized mouse DMD models. The authors utilized the pre-existing hDMDmdx mouse line as a platform to generate clinically relevant models that carry the deletion of exons 44, 45, 51 and 52 of the hDMD transgene using CRISPR/Cas9 technology. This proved somewhat challenging as the hDMD YAC transgene present in the original hDMDmdx line is inserted as a tail-to-tail tandem in the mouse genome. The initial mouse line carrying deletion of exon 44 was performed using a combination of CRISPR/Cas9 and ES cell technologies whereas the remaining mouse lines were generated by applying CRISPR technology directly of hDMCmdx zygotes. In order to identify and select ES cell lines and animals carrying the desired deletion patterns, the authors devised a two steps PCR-based selection strategy followed by a copy number PCR for individual hDMD exons. Once the desired mouse lines were obtained, the authors performed Western blots and histological staining to prove the loss of the hDMD protein expression and the appearance of associated DMD muscle phenotypes. Finally, in vivo experiment was carried out where intramuscular injection of exon-specific ASOs lead to exon skipping and partial restoration of the expression of the truncated but potentially functional hDMD protein variants. The experiment was carried out solely as a proof-of-concept and was terminated before any therapeutic effect of the ASOs could be potentially observed. Nevertheless, the authors argue (correctly) that such models can prove useful in future development of treatment strategies for DMD.

Major comments:

The study has clear aims and is well described. The performed experiments support the final conclusions presented in the paper. OPTIONAL - From the point of view of the reviewer, it seems plausible to use CRISP/Cas9 to "clean up" the original hDMDmdx mouse line by selectively removing one of the YACs forming the tail-to-tail tandem in the mouse genome. Once such single copy mouse line is generated (and proven viable?) any subsequent rearrangement of the hDMD transgene would prove much less challenging. Such mouse line would also better represent human model where only one DMD copy is carried on the X chromosome.

Minor comments:

The labels in figure 2B and 3A would benefit from showing the PCR fragment lengths as well as the sizes of obtained hDMD exon deletions. On could also include an additional figure panel demonstrating the principle of ASO-induced exon skipping

Significance

General Assessment:

The study is fairly limited in scope and will be of primary interest to those working in the DMD field. The new patient-derived hDMD exon deletions will allow testing and validation of human therapeutic moieties in mouse models but as such the study does not advance our knowledge about DMD or transgenic mouse model generation.

Advance:

Perhaps the only novelty is a very diligent genotyping approach aimed at identifying lines where both exons in the tail-to-tail hDMD tandem have been deleted. Given the extensive work put into this approach, the author may have missed an opportunity to reengineer the original hDMDmdx mouse line (see OPTIONAL) to generate a mouse line where any future modifications of the hDMD allele would be much more accessible to both CRISPR-mediate NHEJ and HDR approaches.

Audience:

The study is fairly limited in scope and will be of primary interest to those working in the DMD field.

Reviewer's expertise focuses on CRISPR/Cas9 technologies and transgenic mouse model generation.

Everyone gets one

I am interested what has happened since then

Because she seemed elitist, it became more personality politics than with Obama

But his must be true when it flips too

lol

Again midwest is a microcosm

The midwest can be moderate but why?

We can attribute the success to the south, but I wonder what political opinions in the north were defining

I wonder if the midwest should be used more as a thermometer than a important region.

Because Reagan would make big changes to this ultimately

Holy stray

This is exactly what my comment earlier was talking about. I could see how using AI-generated images in therapy could useful, but just putting them out in the open is not something I think is a good idea. However, I definitely could see a response being that so few people are in therapy for pedophilic disorder, so we'd really only be impacting those who've already committed offenses and are being rehabilitated.

I could see how AI-generated CSA could be used in a therapeutic setting to do this. I feel like maybe psychologists could be figuring out how the person's mind is responding to the images and try to change the response so that they are no longer aroused by the images. I think they are probably already doing something similar to this but this could allow for more understanding maybe. This also could come with it's own set of issues I'd imagine.

This seems like a good idea, but (1) the people creating CSA could probably figure out a way to embed this same watermark if they are already able to hide from law enforcement online and (2) it seems wild that we would use AI to solve a problem (real CSA) and then use it on itself to solve a new problem it has created (determining fake vs. real CSA). What if, instead of addressing a problem by creating a new problem, we just addressed the initial problem.

This is very scary. Both in terms of sexually explicit images but also in terms of basic images. We won't be able to trust anything we see online.

"less harmful"... but they acknowledge it's still harmful

I'm not very familiar with this aspect of labor relations, but I have a feeling a better understanding of it will aid my project even if it doesn't directly correlate to my research question.

A train that traverses Canada from coast to coast.

This research helped me logically understand the benefits of my meditation routine. When you meditate, you can observe how frequently your thoughts shift and how often random ideas pop up. But that process helps us step back from the issues at hand and see clarity on what to do next.

It's hard to read these things. I know just how true all of this is, but thinking of humans in terms of profit margins just doesn't sit right with me.

It's worth noting this wasn't just the British. At least in some ways, they tried to slow colonial expansion, but the newly minted United States wouldn't have such reservations. Or, I guess it's better to say they would have reservations... lots of them.

I struggle a bit with the spiritual tone taken earlier in this article but the case study helped me contextualize the points being made here. It reminds me of an article we read in my Foundations class about librarians as hospitality workers. I see the same thing here, where archivists working with dying donors have certain duties and responsibilities centered on providing care, comfort, and clarity to the people seeking their help. Emphasizing the human element of these interactions and preparing for them as best you can is important so that you can properly serve. I don't know that anyone can fully brace themself for the actual emotions these situations create, but building a framework and giving advice is much more useful than ignoring the topic altogether.

Reviewer #1 (Public review):

Summary:

The article investigates how the Japanese macaque makes gait transitions between quadruped and biped gaits. It presents a compelling neuromechanical simulation that replicates the transition and an interesting analysis based on an inverted pendulum that can explain why some transition strategies are successful and others are not.

Strengths:

I enjoyed reading this article. I think it presents an interesting study and elegant modeling approaches (musculoskeletal + inverted pendulum). The study is well conducted, and the results are interesting. I particularly liked how the success of gait transitions could be predicted based on the inverted pendulum and its saddle node stability. I think it makes a useful and interesting contribution to the state of the art.

Weaknesses:

The article is already in great shape, but could be improved a bit by:

(1) Strengthening the comparison to animal data. In particular, videos of the real animal should be included + snapshots of their gaits (quadruped, biped, and transitions).

(2) Exploring and testing a broader range of conditions. I think it would be very interesting to test gaits and gait transitions on up and down slopes (both with the musculoskeletal model and with the inverted pendulum model). This could be used to make predictions on how the real animal adapts to those conditions. Ideally, this should be tested on the animal as well. I think this could increase (even more) the impact of this work.

(3) Better explaining several aspects of the PSO optimization.

(4) (Ideally) performing a sensitivity analysis on the optimized parameters (e.g. variations of +-5, 10, 20%) in order to determine their respective importance and how much their instantiated values have influenced the results.

(5) Running a spell checker, as there are quite a few typos.

Reviewer #2 (Public review):

Summary:

This article presents a neuromusculoskeletal (NMS) model of the Japanese Macaque. This model is added with a neural feedforward controller based on CPG and synergy that allows for reproducing quadrupedal and bipedal gait as well as the transition between quadrupedal and bipedal gait. The model and controller were validated using experimental data. Results were also compared to an inverted pendulum model to show that the transition between quadrupedal and bipedal in macaque is using this kind of representation for transition and stability. Overall, the article is very interesting, but it sometimes lacks clarity.

Strengths:

The results of the model present impressive results for quadrupedal, bipedal, and transition, validated by experimental data. NMS controllers based on feedforward controllers are very difficult to fine-tune.

Weaknesses:

(1) The movement regulator is not clear and should be better explained. At first, it seems that it is just a new CPG/synergy (feedforward) added, but in the methods, it seems to be a feedback controller.

(2) It is also not clear what is meant by discretizing the weight for the trigger limb from 0 to 1 (page 8).

(3) The controller is mainly using a feedforward controller, allowing only anticipatory movement. Animals are also using a reflex-based feedback controller. A controller with feedback/reflex could reduce failed attempts in training and better represent the transition.

(4) There are small typos throughout the article that should be corrected.

Reviewer #3 (Public review):

Summary:

The purpose of this study was to test the hypothesis that the inverted pendulum mechanism contributes to the gait transition from quadrupedal to bipedal gait in Japanese macaques. The author uses a neuromusculoskeletal model to generate different motor tasks by varying motor command parameters during forward dynamics simulations. After simulations were done, the authors used dynamical system analysis of the inverted pendulum model to reveal the underlying principles of gait transition control. The authors showed that successful gait transition from quadrupedal to bipedal gait mostly depends on increased step length of a hindlimb.

Strengths:

This study is important not only for understanding gait transition, but also to understand stability control of bipedal gaits. Another advantage of this study is that it allows us to estimate the effect of one control mechanism and find its effect and limits. In animal studies, we also have a combination of compensatory stability control mechanisms.

Weaknesses:

Any simulation is not perfect, so discrepancies from experimental data are expected. A 2D model is used, but the advantage of using a 3D model is not clear, and it is much more complicated.

Every teacher i have had since now has been promptly against Wikipedia so i find this very interesting. Wikipedia is easy to edit in theory but as easy as it can be changed it can also be changed back.

This wasn't a national or governmental policy or process, but I discovered an unfair policy at my high school graduation. I went to a public school, and there was a large population of my high school that came from immigrated families. Well, our school was having graduation at the Air Force Academy, and having the ceremony here wouldn't allow any person without a green card to come. This caused a lot of trouble with my friends' families because a lot of their grandparents didn't have green cards, so before a petition and protest caused the school to change venues, they wouldn't have been able to see their kids/grandkids graduate.

I feel like it is definitely important to at times challenge norms and environments. At the same time though it can cause more conflicts than actually help. I feel like as a leader you aren't only thinking about yourself, but whos below you as well. Sometimes you have to pick your battles instead of just challenging something without thinking what can do.

I made a connection between private troubles and public issues. In past classes, social problems were portrayed as individual struggles, but this text refers to them a greater social issues caused by mass institutions. This connects with person-in-environment and ecological systems theory, saying that personal well being can't be separated by structural context or big social institutions.

Author response:

eLife Assessment

This study provides a valuable contribution to understanding how negative affect influences food-choice decision making in bulimia nervosa, using a mechanistic approach with a drift diffusion model (DDM) to examine the weighting of tastiness and healthiness attributes. The solid evidence is supported by a robust crossover design and rigorous statistical methods, although concerns about the interpretation of group differences across neutral and negative conditions limit the interpretability of the results.

We are grateful for this improved assessment. Below, we provide detailed responses that we believe address the noted concerns about interpreting group differences across conditions. If these clarifications resolve the interpretability concerns, we would be grateful if the editors would consider updating the eLife assessment accordingly.

Public Reviews:

Reviewer #1 (Public review):

Summary:

Using a computational modeling approach based on the Drift and Diffusion Model (DDM) introduced by Ratcliff and McKoon in 2008, the article by Shevlin and colleagues investigates whether there are differences between neutral and negative emotional states in:

(1) The timings of the integration in food choices of the perceived healthiness and tastiness of food options in individuals with bulimia nervosa (BN) and healthy participants

(2)The weighting of the perceived healthiness and tastiness of these options.

Strengths:

By looking at the mechanistic part of the decision process, the approach has potential to improve the understanding of pathological food choices.

Weaknesses:

I thank the author for reviewing their manuscript.

However, I still have major concerns.

The authors say that they removed any causal claims in their revised version of the manuscript. The sentence before the last one of the abstract still says "bias for high-fat foods predicted more frequent subjective binge episodes over three months". This is a causal claim that I already highlighted in my previous review, specifically for that sentence (see my second sentence of my major point 2 of my previous review).

We appreciate the Reviewer's continued attention to causal language. We acknowledge that our use of the term 'predicted', though intended to refer to statistical prediction in a regression model, could be misinterpreted as implying causation. We have therefore revised this sentence to read: 'bias for high-fat foods was associated with more frequent subjective binge episodes over three months’.

I also noticed that a comment that I added was not sent to the authors. In this comment I was highlighting that in Figure 2 of Galibri et al., I was uncertain about a difference between neutral and negative inductions of the average negative rating after the induction in the BN group (i.e. comparing the negative rating after negative induction in BN to the negative rating after neutral induction in BN). Figure 2 of Galibri et al. looks to me that:

(1) The BN participants were more negative before the induction when they came to the neutral session than when they came to the negative session.

(2) The BN participants looked almost negatively similar (taking into account the error bars reported) after the induction in both sessions

These observations are of high importance because they may support the fact that BN patients were likely in a similar negative state to run the food decision task in both conditions (negative and neutral). Therefore, the lack of difference in food choices in BN patients is unsurprising and nothing could be concluded from the DDM analyses. Moreover, the strong negative ratings of BN patients in the neutral condition as compared to healthy participants together with almost similar negative ratings after the two inductions contradict the authors' last sentence of their abstract.

I appreciate that the authors reproduced an analysis of their initial paper regarding the negative ratings (i.e. Table S1). It partly answers my aforementioned point but does not address the fact that BN may have been in a similar negative state in both conditions (neutral and negative) when running the food decision task: if BN patients were similarly negative after both induction (neutral and negative), nothing can be concluded from their differences in their results obtained from the DDM. As the authors put it, "not all loss-ofcontrol eating occurs in the context of negative state", I add that far from all negative states lead to a loss-of-control eating in BN patients. This grounds all my aforementioned remarks and my remarks of my first review.

A solution for that is to run a paired t-test in BN patients only comparing the score after the induction in the two conditions (neutral and negative) reported in Figure 2 of their initial article.

We appreciate the reviewer’s concern. We understand how the visual representation in Figure 2, which displays between-subject error bars, might suggest similar post-induction affect levels. However, the within-subject paired comparison (which appropriately accounts for individual differences in baseline affect) reveals a significant difference, which we detail below.

While BN participants did report higher baseline negative affect than the HC group prior to the mood inductions, this does not negate the effectiveness of the manipulation. The critical comparison is the within-subject change from pre- to post-induction (detailed below) which shows that negative affect was significantly higher after the negative induction than the neutral induction.

As we reported in the Supplementary Information (Table S1), our initial analyses of self-reported affect ratings used a linear mixed-effects model with group (HC = 0, BN = 1), condition (Neutral = 0, Negative = 1), and time (pre-induction = 0, post-induction = 1) as fixed effects, including all interactions, and random intercepts for participants. This approach accounts for individual differences in baseline affect.

However, to address the reviewer's concerns, we conducted two simple effects analyses using estimated marginal means. As the reviewer suggested, we directly compared post-induction affect between conditions within the BN group (described in the second analysis below). In the first analysis, we examined the diagnosis × time interaction within each condition separately. In the Negative condition, individuals with BN demonstrated a substantial increase in negative affect from pre- to post-induction (mean difference = 20.36, t = 4.84, p < 0.0001, Cohen’s d = 0.97). In the second analysis, we examined the condition × time interaction within each group separately. Among the BN group, we found that reported affect was significantly higher following the negative mood induction than after the neutral affect induction (mean difference = -17.40, t = -4.13, p = 0.0003, Cohen’s d = 0.83). This difference in post-induction negative affect between conditions within the BN group represents a meaningful and statistically robust difference in affective states. These within-group effects confirm that the negative mood induction was (1) effective in the BN group and (2) produced significantly greater negative affect than the neutral mood induction.

These findings confirm that participants completed the food decision task under meaningfully different affective states, supporting the interpretability of the subsequent DDM analyses. We now report these analyses in the Supplementary Information.

I appreciate the analysis that the authors added with the restrictive subscale of the EDE-Q.

That this analysis does not show any association with the parameters of interest does not show that there is a difference in the link between self reported restrictions and self reported binges. Only such a difference would allow us to claim that the results the authors report may be related to binges.

We thank the reviewer for raising this important point about specificity. To address this concern, we examined the correlation between self-reported binge frequency (both subjective binge episodes and objective binge episodes over the past three months) and EDE-Q Restraint subscale in our BN sample.

The correlation between these measures were modest and non-significant (subjective binge frequency: Spearman’s p = 0.21, p = 0.306; objective binge frequency: Spearman’s p = 0.05, p = 0.806), indicating that both binge frequency measures and dietary restraint were relatively independent dimensions of eating pathology in our sample. This dissociation supports the specificity of our findings: the fact that our DDM parameters were associated with binge frequency but not with dietary restraint suggests that the affect-induced changes in decisionmaking we observed are specifically related to binge-eating behavior rather than reflecting a correlate of dietary restraint. We now report this analysis in the Supplementary Information.

I appreciate the wording of the answer of the authors to my third point: "the results suggest that individuals whose task behavior is more reactive to negative affect tend to be the most symptomatic, but the results do not allow us to determine whether this reactivity causes the symptoms". This sentence is crystal clear and sums very well the limits of the associations the authors report with binge eating frequency. However, I do not see this sentence in the manuscript. I think the manuscript would benefit substantially from adding it.

We thank the reviewer for the suggestion. We have added the following sentences that convey this information to the end of the third paragraph of the discussion:

“These results suggest that individuals whose task behavior is more reactive to negative affect tend to be the most symptomatic. However, our correlational design does not allow us to determine whether this reactivity causes the symptoms.”

Statistical analyses:

If I understood well the mixed models performed, analyses of supplementary tables S1 and S27 to S32 are considering all measures as independent which means that the considered score of each condition (neutral vs negative) and each time (before vs after induction) which have been rated by the same participants are independent. Such type of analyses does not take into account the potential correlation between the 4 scores of a given participant. As a consequence, results may lead to false positives that a linear mixed model does not address. The appropriate analysis would be to run adapted statistical tests pairing the data without running any mixed model.

We appreciate the reviewer's attention to the statistical approach. However, we respectfully note that mixed-effects models do account for within-subject correlations, contrary to the reviewer’s interpretation.

The linear mixed-effects model we employed explicitly accounts for the correlation among repeated measures from the same participant through the random intercept term. This random effect structure models the non-independence of observations within participants, allowing for correlated errors within individuals while assuming independence between individuals. This is a standard and appropriate approach for analyzing repeated-measures data (Bates et al., 2015).

The mixed-effects model is, in fact, more appropriate than separate paired t-tests for our design because it:

(1) Simultaneously models all fixed effects (group, condition, time) and their interactions in a single unified framework;

(2) Properly partitions variance into within-subject and between-subject components;

(3) Provides greater statistical power and more precise estimates by using all available data simultaneously; and

(4) Allows for direct testing of three-way interactions that cannot be assessed through pairwise comparisons alone.

Paired tests (e.g., t-tests), as the reviewer suggests, would require multiple separate analyses and would not allow us to test our primary hypotheses about group × condition × time interactions. The mixed-effects approach provides a more comprehensive and statistically rigorous analysis of our repeated-measures design. To clarify this even further in the manuscript, we have added the following in our methods when describing our model, “participant-level random intercepts were included to account for within-subject correlations across repeated measurements.”

Notes:

It is not because specific methods like correlating self reported measures over long periods with almost instantaneous behaviors (like tasks) have been used extensively in studies that these methods are adapted to answer a given scientific question. Measures aggregated over long periods miss the variations in instantaneous behaviors over these periods.

We acknowledge the reviewer’s concern about the temporal mismatch between our session-level task measures and the 3-month aggregated symptom reports. This is a valid limitation of crosssectional designs, and we agree that examining how task performance fluctuates in relation to real-time symptom variation would provide richer insights into the potential dynamics of these relationships.

We agree that we cannot capture how daily changes in task performance relate to momentary symptom occurrence. In response to previous rounds of helpful reviews, we added this limitation to the Discussion section, noting that future research employing ecological momentary assessment (EMA) or daily diary methods could examine whether the decision-making processes we identified also fluctuate in relation to real-time symptom occurrence.

We note that our finding that affect-induced changes in decision-making parameters were associated with subjective binge frequency suggests that this laboratory-measured reactivity may reflect a stable individual difference that manifests across contexts and time periods. While our current study provides initial evidence that individual differences in affect-related decisionmaking are associated with symptom severity, we acknowledge that longitudinal designs with repeated assessments would strengthen causal and temporal inferences.

Reviewer #2 (Public review):

Summary:

Binge eating is often preceded by heightened negative affect, but the specific processes underlying this link are not well-understood. The purpose of this manuscript was to examine whether affect state (neutral or negative mood) impacts food choice decisionmaking processes that may increase the likelihood of binge eating in individuals with bulimia nervosa (BN). The researchers used a randomized crossover design in women with BN (n=25) and controls (n=21), in which participants underwent a negative or neutral mood induction prior to completing a food-choice task. The researchers found that despite no differences in food choices in the negative and neutral conditions, women with BN demonstrated a stronger bias toward considering the 'tastiness' before the 'healthiness' of the food after the negative mood induction.

Strengths:

The topic is important and clinically relevant, and the methods are sound. The use of computational modeling to understand nuances in decision-making processes and how that might relate to eating disorder symptom severity is a strength of the study.

Weaknesses:

Sample size was relatively small, and participants were all women with BN, which limits generalizability of findings to the larger population of individuals who engage in binge eating. It is likely that the negative affect manipulation was weak and may not have been potent enough to change behavior. These limitations are adequately noted in the discussion.

We are grateful to Reviewer #2 for their careful and supportive review of our manuscript. We appreciate their recognition that computational modeling can reveal nuanced alterations in decision-making processes that may not be apparent in overt behavioral choices. Their balanced assessment of both the strengths and limitations of our work has been helpful in contextualizing our findings appropriately. We have carefully considered their comments regarding sample size and the potential limitations of our mood induction procedure, both of which we discuss in detail in the manuscript's limitations section.

Reviewer #3 (Public review):

Summary:

The study uses the food choice task, a well-established method in eating disorder research, particularly in anorexia nervosa. However, it introduces a novel analytical approach-the diffusion decision model-to deconstruct food choices and assess the influence of negative affect on how and when tastiness and healthiness are considered in decision-making among individuals with bulimia nervosa and healthy controls.

Strengths:

The introduction provides a comprehensive review of the literature, and the study design appears robust. It incorporates separate sessions for neutral and negative affect conditions and counterbalances tastiness and healthiness ratings. The statistical methods are rigorous, employing multiple testing corrections.

A key finding-that negative affect induction biases individuals with bulimia nervosa toward prioritizing tastiness over healthiness-offers an intriguing perspective on how negative affect may drive binge eating behaviors.

Weaknesses:

A notable limitation is the absence of a sample size calculation, which, combined with the relatively small sample, may have contributed to null findings. Additionally, while the affect induction method is validated, it is less effective than alternatives such as image or film-based stimuli (Dana et al., 2020), potentially influencing the results.

We are grateful to Reviewer #3 for their thoughtful evaluation of our work. We appreciate their recognition that the diffusion decision model provides a novel analytical lens for understanding how negative affect influences the dynamics of food-related decision-making in bulimia nervosa. Their balanced assessment of both the methodological strengths of our design (counterbalancing, rigorous statistical corrections) and its limitations (sample size, mood induction efficacy) has been valuable in ensuring we appropriately contextualize our findings and their implications. Specifically, we have taken their comments regarding sample size and the relative efficacy of different mood induction methods seriously, and we address these important methodological considerations in our discussion of the study's limitations.

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

The authors have addressed my previous comments, and I do not have any additional suggestions for improvement.

We thank the reviewer for their time, effort, and insightful feedback.

Reviewer #3 (Recommendations for the authors):

The authors have adequately addressed my feedback. I have no further comments.

We thank the reviewer for their time, effort, and insightful feedback.

Reviewer #1 (Public review):

Summary:

Using a computational modeling approach based on the Drift and Diffusion Model (DDM) introduced by Ratcliff and McKoon in 2008, the article by Shevlin and colleagues investigates whether there are differences between neutral and negative emotional states in:

(1) The timings of the integration in food choices of the perceived healthiness and tastiness of food options in individuals with bulimia nervosa (BN) and healthy participants (2) The weighting of the perceived healthiness and tastiness of these options.

Strengths:

By looking at the mechanistic part of the decision process, the approach has potential to improve the understanding of pathological food choices.

Weaknesses:

I thank the authors for revising their manuscript.

I still notice that the authors did not go through their manuscript to look for wordings refering to a prediction interpretation of their results while I already highlighted the inappropriateness of this wording in my two first rounds of reviews: e.g. there is still "we used zero-inflated negative binomial models to predict the three-month frequency" and I can find other statements like this. The design of their study does not allow such claims.

The authors answered my major concern regarding the experimental induction towards a negative or a neutral state before running the food decision task. My concern is: BN patients already seemed to be already in a high negative state before undergoing the neutral induction, while these patients are in a lower negative state before undergoing the negative induction. It is therefore not surprising that patients seem to report a similar level of negative state after the two inductions (according to the figure of the authors' previous article). Of note is that the additional analysis the authors ran within the BN group only provides a significant result: this result shows that there has been an induction but does not rule out that patients were in the exact same magnitude of negative state to perform the task as the figure in their previously published article suggests it. The major issue is to show that:

(1) As compared to the neutral induction, there has been a higher variation in negative state after as compared to before the negative induction.

(2) The magnitude of the negative state after the negative induction is higher than the magnitude of the negative state after the neutral induction.

The first point shows that the induction worked. The second point shows that the participants are in two distinct states. Without showing the second point, it may be possible that one induction increases the negative state of participants to the same level as the one of the second induction that has not increased anything.

Within this context, how is it possible to associate, in patients, a difference in the DDM between the two sessions to a negative state (which is one of the main focus of the article) rather than to another parameter that has not been captured? A similar situation would be in an experiment studying the consequence of stress, a stressfull induction over relaxed participants attending the lab has high chances to raise the level of stress of those participants to the same level as the one that the same participants would experience after a neutral induction when these participants attend the lab with an already high level of stress. In that case, would it be approrpiate to claim that a difference at a task performed after the induction would be related to stress while the participants would be at the same level of stress when performing the task despite the fact that the induction worked ?

In the experiment performed by the authors, the additional analysis to perform would be a paired sample t-test (or the appropriate non-parametric test) to check whether the magnitude of negative state of BN patients was different between the negative and neutral conditions after the induction only. If not, associating the difference at the DDM with negative states in BN is highly misleading.

I read carefully the authors' answer related to mixed models: they claim that mixed models take into account correlations within their repeated data. The specification of the structure of the covariance matrix allows to control only partly for that. I notice that the authors did not specify the structure of that matrix: the article they refer to to justify the appropriatness of their analyses is not adapted. The specification of the structure of the covariance matrix needs to address, in a mixed model, the difference in handling 4 repeated data per participants that cannot be paired as compared to 4 repeated data that can be paired (two per session with one before and one after the neutral or negative priming sessions, if I count right). Of note is that a covariance structure that is left free of constraint for the fit of the model does not capture appropriately the pairing of the data: it has all chances to capture the covariance in a different way. And a covariance structure that has constraints has more chances to lead to a model that cannot be estimated because of an absence of convergence of the algorithms.

By the way, a single two-sample t-test (or a Mann-Whitney test if appropriate), and not a set of multiple paired-sample t-test as the authors suggest, would answer the goal of the authors to test for what they call the three-way interaction in their comment. This test would be performed between the two groups of participants (BN/controls) with the computation for each participant separately: (assessment after neutral induction-assessment before neutral induction)-(assessment after negative induction-assessment before negative induction). This analysis answers points 1, 2 and 4 they raise together with my point of controlling for the paired data. I would have agreed with their choice of a mixed model if they had an unbalanced dataset within each participant.

Reviewer #2 (Public review):

Summary:

Binge eating is often preceded by heightened negative affect, but the specific processes underlying this link are not well-understood. The purpose of this manuscript was to examine whether affect state (neutral or negative mood) impacts food choice decision-making processes that may increase likelihood of binge eating in individuals with bulimia nervosa (BN). The researchers used a randomized crossover design in women with BN (n=25) and controls (n=21), in which participants underwent a negative or neutral mood induction prior to completing a food-choice task. The researchers found that despite no differences in food choices in the negative and neutral conditions, women with BN demonstrated a stronger bias toward considering the 'tastiness' before the 'healthiness' of the food after the negative mood induction.

Strengths:

The topic is important and clinically relevant and methods are sound. The use of computational modeling to understand nuances in decision-making processes and how that might relate to eating disorder symptom severity is a strength of the study.

Weaknesses:

Sample size was relatively small, and participants were all women with BN, which limits generalizability of findings to the larger population of individuals who engage in binge eating. It is likely that the negative affect manipulation was weak and may not have been potent enough to change behavior. These limitations are adequately noted in the discussion.

Reviewer #1 (Public review):

Summary:

Hoverflies are known for a striking sexual dimorphism in eye morphology and early visual system physiology. Surprisingly, the male and female flight behaviors show only subtle differences. Nicholas et al. investigate the sensori-motor transformation of sexually dimorphic visual information to flight steering commands via descending neurons. The authors combined intra- and extracellular recordings, neuroanatomy, and behavioral analysis. They convincingly demonstrate that descending neurons show sexual dimorphisms - in particular at high optic flow velocities - while wing steering responses seem relatively monomorphic. The study highlights a very interesting discrepancy between neuronal and behavioral response properties.

More specifically, the authors focused on two types of descending neurons that receive inputs from well-characterized wide-field sensitive tangential cells: OFS DN1, which receives inputs from so-called HS cells, and OFS DN2, which receives input from a set of VS cells. Their likely counterparts in Drosophila connect to the neck, wing, and haltere neuropils. The authors characterized the visual response properties of these two neuronal classes in both male and female hoverflies and identified several interesting differences. They then presented the same set of stimuli, tracked wing beat amplitude, and analyzed the sum and the difference of right and left wing beat amplitude as a readout of lift or thrust, and yaw turning, respectively. Behavioral responses showed little to no sexual dimorphism, despite the observed neuronal differences.

Strengths:

I find the question very interesting and the results both convincing and intriguing. A fundamental goal in neuroscience is to link neuronal responses and behavior. The current study highlights that the transformations - even at the level of descending neurons to motoneurons - are complex and less straightforward than one might expect.

Weaknesses:

The authors investigated two types of descending neurons, but it was not clear to me how many other descending neurons are thought to be involved in wing steering responses to wide-field motion. I would suggest providing a more in-depth overview of what is known about hoverflies and Drosophila, since the conclusions drawn from the study would be different if these two types were the only descending neurons involved, as opposed to representing a subset of the neurons conveying visual information to the wing neuropil.

Both neuronal classes have counterparts in Drosophila that also innervate neck motor regions. The authors filled the hoverfly DNs in intracellular recordings to characterize their arborization in the ventral nerve cord. In my opinion, these anatomical data could be further exploited and discussed a bit more: is the innervation in hoverflies also consistent with connecting to the neck and haltere motor regions? Are there any obvious differences and similarities to the Drosophila neurons mentioned by the authors? If the arborization also supports a role in neck movements, the authors could discuss whether they would expect any sexual dimorphism in head movements.

Reviewer #2 (Public review):

Summary:

Many fly species exhibit male-specific visual behaviors during courtship, while little is known about the circuit underlying the dimorphic visuomotor transformations. Nicholas et al focus on two types of visual descending neurons (DNs) in hoverflies, a species in which only males exhibit high-speed pursuit of conspecifics. They combined electrophysiology and behavior analysis to identify these DNs and characterize their response to a variety of visual stimuli in both male and female flies. The results show that the neurons in both sexes have similar receptive fields but exhibit speed-dependent dimorphic responses to different optic flow stimuli.

Strengths:

Hoverflies, though not a common model system, show very interesting dimorphic behaviors and provide a unique and valuable entry point to explore the brain organization behind sexual dimorphism. The findings here are not only interesting on their own right but will also likely inspire those working in other systems, particularly Drosophila.

The authors employed rigorous morphology, electrophysiology, and behavior methods to deliver a comprehensive characterization of the neurons in question. The precision of the measurements allowed for identifying a subtle and nuanced neuronal dimorphism and set a standard for future work in this area.

Weaknesses:

Cell-typing using receptive field preferred directions (RFPDs): if I understood correctly, this classification method mostly relies on the LPDs near the center of the receptive field (median within the contour in Fig.1). I have two concerns here. First, this method is great if we are certain there are only two types of visual DNs as described in the manuscript. But how certain is this? Given the importance of vision in flight control, I would expect many DNs that transmit optic flow information to the motor center. I'd also like to point out that there are other lobula plate tangential cells (LPTCs) than HS and VS cells, which are much less studied and could potentially contribute to dimorphic behaviors. Second, this method feels somewhat impoverished given the richness of the data. The authors have nicely mapped out the directional tuning for almost the entire visual field. Instead of reducing this measurement to 2 values (center and direction), I was wondering if there is a better method to fully utilize the data at hand to get a better characterization of these DNs. As the authors are aware, local features alone can be ambiguous in characterizing optic flows. What's more, taking into account more global features can be useful for discovering potentially new cell types.

Line 131, it wasn't clear to me why full-screen stimuli were used for comparison here, instead of the full receptive field maps. Male flies exhibit sexual dimorphic behaviors only during courtship, which would suggest that small-sized visual stimuli (mimicking an intruder or female conspecific) would be better suited to elicit dimorphic neuronal responses. A similar comment applies to the later results as well. Based on the receptive field mapping in Figure 1, I'm under the impression that these 2 DN types are more suited to detect wide-field optic flows, those induced by self-motion as mentioned in the manuscript. The results are still very interesting, but it's good to make this point clear early on to help set appropriate expectations. Conversely, this would also suggest that there are other visual DN types that are responsible for the courtship-related sexually dimorphic behaviors.

Author response:

eLife Assessment

Hoverflies are known for their sexually dimorphic visual systems and exquisite flight behaviors. This valuable study reports how two types of visual descending neurons differ between males and females in their motion- and speed-dependent responses, yet surprisingly, the behavior they control lacks any sexual dimorphism. The results convincingly support these findings, which will be of interest for studies of visuomotor transformations and network-level brain organization.

This statement perfectly recapitulates our findings.

Public Reviews:

Reviewer #1 (Public review):  

Summary: 

Hoverflies are known for a striking sexual dimorphism in eye morphology and early visual system physiology. Surprisingly, the male and female flight behaviors show only subtle differences. Nicholas et al. investigate the sensori-motor transformation of sexually dimorphic visual information to flight steering commands via descending neurons. The authors combined intra- and extracellular recordings, neuroanatomy, and behavioral analysis. They convincingly demonstrate that descending neurons show sexual dimorphisms - in particular at high optic flow velocities - while wing steering responses seem relatively monomorphic. The study highlights a very interesting discrepancy between neuronal and behavioral response properties.

Thank you for this summary. Most of the statement perfectly recapitulates the main findings of our paper. However, we want to emphasize that some hoverfly flight behaviors are strongly sexually dimorphic, especially those related to courtship and mating. Indeed, only male hoverflies pursue targets at high speed, chase away territorial intruders, and pursue females for mating. However, other flight behaviours, such as those related to optomotor responses and flights between flowers when feeding, are not sexually dimorphic. We will amend the Introduction to make the difference between flight behaviors clear.

More specifically, the authors focused on two types of descending neurons that receive inputs from well-characterized wide-field sensitive tangential cells: OFS DN1, which receives inputs from so-called HS cells, and OFS DN2, which receives input from a set of VS cells. Their likely counterparts in Drosophila connect to the neck, wing, and haltere neuropils. The authors characterized the visual response properties of these two neuronal classes in both male and female hoverflies and identified several interesting differences. They then presented the same set of stimuli, tracked wing beat amplitude, and analyzed the sum and the difference of right and left wing beat amplitude as a readout of lift or thrust, and yaw turning, respectively. Behavioral responses showed little to no sexual dimorphism, despite the observed neuronal differences.

Thank you for this very nice summary of our work. We want to clarify that LPTC input to DN1 and DN2 has not been shown directly in hoverflies using e.g. dye coupling, or dual recordings. Instead, the presumed HS and VS input is inferred from morphological and physiological DN evidence, and comparisons to similar data in Drosophila and blowflies. We will amend the Introduction to clarify this. The rest of the paragraph perfectly recapitulates the main findings of our paper.

Strengths:

I find the question very interesting and the results both convincing and intriguing. A fundamental goal in neuroscience is to link neuronal responses and behavior. The current study highlights that the transformations - even at the level of descending neurons to motoneurons - are complex and less straightforward than one might expect.

Thank you.

Weaknesses:

The authors investigated two types of descending neurons, but it was not clear to me how many other descending neurons are thought to be involved in wing steering responses to wide-field motion. I would suggest providing a more in-depth overview of what is known about hoverflies and Drosophila, since the conclusions drawn from the study would be different if these two types were the only descending neurons involved, as opposed to representing a subset of the neurons conveying visual information to the wing neuropil.

This is a great point. There are around 1000 fly DNs, of which many could respond to widefield motion, without being specifically tuned to widefield motion. For example, many looming sensitive neurons also respond to widefield motion, and could therefore be involved in the WBA movements that we measured here. In addition, there are many multimodal neurons that could be involved in optomotor responses in free flight, but these may not have been stimulated when we only provided visual input. Furthermore, many visual neurons are modulated by proprioceptive feedback, which is lacking in immobilized physiology preps. Finally, in blowflies, up to 5 optic flow sensitive DNs have been identified morphologically, and in Drosophila 3 have been identified morphologically and physiologically. In summary, it is more than likely that other neurons project visual widefield motion information to the wing neuropil. We will amend our Introduction and Discussion to make this important point clear to the readers.

Both neuronal classes have counterparts in Drosophila that also innervate neck motor regions. The authors filled the hoverfly DNs in intracellular recordings to characterize their arborization in the ventral nerve cord. In my opinion, these anatomical data could be further exploited and discussed a bit more: is the innervation in hoverflies also consistent with connecting to the neck and haltere motor regions? Are there any obvious differences and similarities to the Drosophila neurons mentioned by the authors? If the arborization also supports a role in neck movements, the authors could discuss whether they would expect any sexual dimorphism in head movements.

These are all great points. We did not see any clear arborizations to the frontal nerve, where we would expect to find the neck motor neurons (NMNs). In addition, while we did see fine arborizations throughout the length of the thoracic ganglion, we saw no strong outputs projecting directly to the haltere nerve (HN). In the revised version of the MS we will modify figure 4 (morphological characterization) to clarify.

There are important differences between the morphology of DN1 and DN2 in hoverflies and DNHS1 and DNOVS2 in Drosophila, in terms of their projections in the thoracic ganglion. For example, In Drosophila DNOVS2, there are several fine branches along the length of the neuron in the thoracic ganglia. Similarly, we found fine branches in Eristalis tenax DN2, however, in addition, we found a wide branch projecting to the area of the thoracic ganglion where the prothoracic and pterothoracic nerves likely get their inputs (Figure 4), suggesting that the neuron could contribute to controlling the wings and/or the forelegs (which is why we quantified the WBA). In Drosophila DNHS1, there is a similar fat branch to the prothoracic and pterothoracic nerves, which we also found in Eristalis tenax OFS DN1 (Figure 4). Indeed, while Drosophila DNHS1 and DNOVS2 have quite strikingly different morphology, DN1 and DN2 in Eristalis looked quite similar. We will modify the Results section to make this clear.

In addition, to investigate this further, in the revised version of the MS we will include analysis of the movement of different body parts (including the head) to investigate the presence of any potential sexual dimorphism. Unfortunately, however, this will not include the halteres, as they cannot be seen well in the videos.

Reviewer #2 (Public review):

Summary:

Many fly species exhibit male-specific visual behaviors during courtship, while little is known about the circuit underlying the dimorphic visuomotor transformations. Nicholas et al focus on two types of visual descending neurons (DNs) in hoverflies, a species in which only males exhibit high-speed pursuit of conspecifics. They combined electrophysiology and behavior analysis to identify these DNs and characterize their response to a variety of visual stimuli in both male and female flies. The results show that the neurons in both sexes have similar receptive fields but exhibit speed-dependent dimorphic responses to different optic flow stimuli.

This statement perfectly recapitulates the main findings of our paper. However, as mentioned above, while hoverfly flight behaviors related to courtship and mating are strongly sexually dimorphic, other flight behaviours, such as those related to optomotor responses and flights between flowers when feeding, are not. We will amend the Introduction to make the difference between flight behaviors clear.

Strengths:

Hoverflies, though not a common model system, show very interesting dimorphic behaviors and provide a unique and valuable entry point to explore the brain organization behind sexual dimorphism. The findings here are not only interesting on their own right but will also likely inspire those working in other systems, particularly Drosophila.

Thank you.

The authors employed rigorous morphology, electrophysiology, and behavior methods to deliver a comprehensive characterization of the neurons in question. The precision of the measurements allowed for identifying a subtle and nuanced neuronal dimorphism and set a standard for future work in this area.

Thank you.

Weaknesses:

Cell-typing using receptive field preferred directions (RFPDs): if I understood correctly, this classification method mostly relies on the LPDs near the center of the receptive field (median within the contour in Fig.1). I have two concerns here. First, this method is great if we are certain there are only two types of visual DNs as described in the manuscript. But how certain is this? Given the importance of vision in flight control, I would expect many DNs that transmit optic flow information to the motor center. I'd also like to point out that there are other lobula plate tangential cells (LPTCs) than HS and VS cells, which are much less studied and could potentially contribute to dimorphic behaviors.

This is very true, and an important point. As mentioned above, in blowflies, up to 5 optic flow sensitive DNs have been identified morphologically, however, if these correspond to 5 different physiological types remain unclear. In both blowflies and Drosophila 3 have been identified morphologically and physiologically (DNHS1, DNOVS1, DNOVS2). Importantly, in both blowflies and fruitflies DNOVS1 gives graded responses, and no action potentials, meaning that we would not be able to record from it using extracellular electrophysiology.

We previously used clustering techniques to show that in Eristalis, we can reliably distinguish two types of optic flow sensitive DNs from extracellular electrophysiological data, based on a range of receptive field parameters, and we think that these correspond to DNHS1 and DNOVS2 in Drosophila (Nicholas et al, J Comp Physiol A, 2020, cited in paper). As mentioned above in response to Reviewer 1, this does not mean that there are no other neurons that could respond to widefield optic flow, and which might be involved in the WBA we recorded in the paper. However, the point of this paper was not to conclusively show that there are only two optic flow sensitive descending neurons. The point was to say that there are two quite distinct optic flow sensitive neurons that have similar receptive fields in males and females, while the responses to widefield motion show differences between males and females.

We will modify the Introduction and Discussion to make these important points clear to the Reader, including the discussion of the 45-60 LPTCs that exist in the lobula plate, and what their role might be.

Second, this method feels somewhat impoverished given the richness of the data. The authors have nicely mapped out the directional tuning for almost the entire visual field. Instead of reducing this measurement to 2 values (center and direction), I was wondering if there is a better method to fully utilize the data at hand to get a better characterization of these DNs. As the authors are aware, local features alone can be ambiguous in characterizing optic flows. What's more, taking into account more global features can be useful for discovering potentially new cell types.

This is a great point, and we did an extensive analysis of other receptive field properties in this study (shown in supp fig 1). In addition, and as mentioned above, we have published a clustering analysis across receptive field properties of these neurons (Nicholas et al, J Comp Physiol A, 2020, cited in paper). The point that we attempted to make in this paper was that by using two strikingly simple metrics, we can reliably distinguish which of the two neuron types we are recording from (if we accept that there are two main types that we are likely to record from) simply based on location and overall directional preference. This makes automated analysis very easy and straightforward. Indeed, we now use this routinely to ID what neuron we are recording from, rather than making a human-based assumption.

However, we agree that further in depth analysis is warranted. Therefore, to address this, we will provide additional receptive field analysis and clustering in the revised version of the MS. In addition, we want to highlight that all data is uploaded to DataDryad for anyone interested in doing additional in-depth analyses.

Line 131, it wasn't clear to me why full-screen stimuli were used for comparison here, instead of the full receptive field maps. Male flies exhibit sexual dimorphic behaviors only during courtship, which would suggest that small-sized visual stimuli (mimicking an intruder or female conspecific) would be better suited to elicit dimorphic neuronal responses. A similar comment applies to the later results as well. Based on the receptive field mapping in Figure 1, I'm under the impression that these 2 DN types are more suited to detect wide-field optic flows, those induced by self-motion as mentioned in the manuscript. The results are still very interesting, but it's good to make this point clear early on to help set appropriate expectations. Conversely, this would also suggest that there are other visual DN types that are responsible for the courtship-related sexually dimorphic behaviors.

Thank you for mentioning these important points. Our reasoning for using full-screen stimuli for the analysis on line 131 was that since we used the small sinusoidal gratings for mapping the receptive fields, and to subsequently classify the neurons, it would be unfair to use the same data to investigate potential sexual dimorphism. I.e., we selected neurons that fulfilled certain criteria, and then we cannot rightfully use the same criteria to determine differences. This was not explicitly mentioned in the paper, so we will modify the text to make this clear to the Reader.

However, in Supp Figure 1d/e we show that there are no striking receptive field differences between males and females in terms of receptive field center nor directional preference. In Supp Figure 1f we show that there is no difference between male and female receptive field height and width. We will modify the text to draw the Reader’s attention to this figure, and also mention the additional analysis done in response to the comment above.

As a side note, I personally expected at least DNHS1 to have a smaller receptive field in males, as the hoverfly HSN is strikingly sexually dimorphic (Nordström et al, Curr Biol 2008), and also very sensitive to small objects. However, while optic flow sensitive DNs do respond to small objects (see e.g. the J Comp Physiol paper mentioned above) we did not detect any obvious sexual dimorphism in receptive field properties. Indeed, we think that a different subset of DNs control target pursuit behavior (target selective DNs (TSDNs)). This will be addressed in the modified version of the paper.

eLife Assessment

This valuable study reports results showing how different neurons in the dysgranular retrosplenial cortex code spatial orientation. Specifically, the paper reports that some neurons maintain tuning for a single head direction across multi-compartmental environments, while other neurons are tuned to different head directions that reflect the geometry within each compartment. The study was viewed as likely to expand the field's understanding of directional tuning of neurons, but incomplete evidence was provided to support the conclusions.

Reviewer #1 (Public review):

Summary:

The dysgranular retrosplenial cortex (RSD) and hippocampus both encode information related to an animal's navigation through space. Here, the authors study the different ways in which these two brain regions represent spatial information when animals navigate through interconnected rooms. Most importantly, they find that the RSD contains a small fraction of neurons that encode properties of interconnected rooms by firing in different head directions within each room. This direction is shifted by 180 degrees in 2-room environments, and by 90 degrees in 4-room environments. While it cannot be definitively proven that this encoding is not just related to the presence of exits (doors) in each room, this is a noteworthy finding and will motivate further study in more complex and well-controlled environments to understand this coding scheme in the RSD. The recordings and analyses used to identify these multi-directional cells are mostly solid. Additional conclusions regarding the rotational symmetry across rooms seen in the RSD neurons that do not encode direction (representing the majority of RSD neurons) remain incomplete, given the evidence presented thus far. The differences between RSD and hippocampus encoding of space are clear and consistent with prior observations.

Strengths:

(1) Use of tetrode recordings from the RSD to identify multi-direction cells that only encode one direction in each room, but shift the preferred direction by either 180 or 90 degrees depending on the number of rooms in the environment.

(2) Solid controls to show that this multi-direction encoding is stable over time and across some environmental manipulations.

(3) Convincing evidence that these multi-direction cells can co-exist with single-direction head direction cells in the RSD (as both cell types can be simultaneously recorded).

(4) Convincing evidence for clear differences between directional and spatial encoding in the RSD versus hippocampus, consistent with prior observations.

Weaknesses:

(1) The paper mostly uses the term "retrosplenial cortex", but it is important to clarify that the study is only focused on the dysgranular retrosplenial cortex (RSD; Brodmann Area 30) and not the granular retrosplenial cortex (Brodmann Area 29). These are two distinct regions (despite the similar names), each with distinct connectivity and distinct behavioral encoding and function, so it is important to clarify in the abstract and title that the present study is solely about the RSD to prevent confusion in the literature.

(2) The proportion of each observed cell type is not clearly stated, although it is clear that the multi-directional cells are in the minority. Having the proportion of well-isolated neurons in distinct sessions that encode each type of information (e.g., multi vs single direction encoding) would greatly aid the interpretation of the result and help the field know how common each cell type is in the RSD.

(3) The authors state that "MDCs [multi-directional cells] never exhibited multidirectional activity within a single room" - but many of the single room examples from the 4-room environment (shown in Figures 2E and 2F) reveal multi-peaked directional encoding. This suggests that the multi-direction encoding may be more compatible with encoding some property of the number of exits rather than relative room orientations.

(4) The spatial rotation analyses of non-directional cell analyses are considered incomplete. This is impacted by the slower speed at the doors and hence altered firing rates (as evidenced in spatial rate plots). The population rate is not relevant as the correlational analyses are done on a single cell level. Since some cells fire more with increasing speed and others fire less, that will necessarily result in a population rate map that minimizes firing rate differences near the doorway, where the animals move more slowly. But on a single cell level, that reduced speed is having a big effect, as evidenced by individual rate map examples, and the rooms will need to be rotated to obtain a higher correlation by overlapping the doorway regions. This does not necessarily say anything about spatial coding across the two or four interconnected rooms being rotationally symmetric, and it would appear difficult to draw any conclusions related to spatial encoding from those analyses.

Reviewer #2 (Public review):

Summary:

Laurent et al. perform in vivo electrophysiological recordings in the retrosplenial cortex of rats foraging in multi-compartment environments with either identical or unique visual features. The authors characterize two types of directional signals in the area that they have previously reported: classic head direction cells anchored to the global allocentric reference frame and multi-direction cells (MDCs), which have a rotationally preserved directional field anchored to local compartments. The primary finding of this work is that MDCs seem sensitive to local environmental geometry rather than visual context. They also show that MDC tuning persists in the absence of hippocampal place field repetition, further dissociating the RSC local directional signal from the broader allocentric representation of space. A novel observation is that RSC non-directional spatial signals are anchored to the local environment, which could and should be explored further. While the data is solid and the analyses are mostly appropriate, the primary findings are incremental, and more interesting novel claims are not explored in detail or not explicitly tested.

Strengths:

The environmental manipulations clearly demonstrate that tuning is not modulated by complex visual information.

The finding that RSC two-dimensional spatial responses are stable and anchored to environmental features is novel and can be further explored in future work.

Weaknesses:

The observation that BDCs and MDCs are insensitive to visual context builds upon the author's previous work (and replicates aspects of Zhang et al., 2022) but leaves many open questions that are not addressed with the current set of experiments. Specifically, what exactly are MDCs anchoring to? The primary theory is that they anchor to environmental geometry, but there are no explicit experimental manipulations to test this theory. It is important to note that 2- and 4-compartment environments share many features, including the same cardinal axes, making any differences/similarities in these two conditions difficult to interpret.

The main finding presented with respect to BDC/MDs tuning is that they are not sensitive to visual context as manipulated by distinct visual patterns on the wall and floor in multicompartment environments. One could argue that the individual rooms are, in actuality, quite similar in low-level visual features - each possesses a large white background square visual feature on a single wall with a fixed relationship to the door(s). How can the authors rule out that i) BDC/MDC responses are modulated by these low-level features rather than geometry and/or ii) that the rats are not paying attention to any visual features at all? There is no task requiring them to indicate which room they are in. Furthermore, the doorways themselves are prominent visual features that are present in each context. It would be interesting to see if MDC/BDC tuning persisted in a square room where the number of doorways was manipulated to rule out this possibility.

A strong possibility is that the rotational symmetry of both MDCs and non-directional spatial neurons is related to i) door-related firing, 2) stereotyped movement, and 3) stereotyped directional sampling. In Supplemental Figure 8, the authors begin to address this by comparing a 'population ratemap' to a 'population speed map.' I do not think this is sufficient and is difficult to interpret. Instead, the authors should assess whether MDC and BDCs fire more at doorways and what the overlap is with the speed-modulated cells they report. Moreover, they should assess whether the spatial speed profile itself is rotationally symmetric within each session. It would also be useful to look at the confluence of the variables simultaneously using some form of regression analysis. The authors could generate a directional predictor that captures the main response property of these cells and see if it accounts for greater variability in spiking than speed or x,y position. Finally, rotationally symmetric directional sampling biases could arise from the doors being present on the same two walls in each room. The authors should assess whether MDC tuning is still present if directional sampling is randomly downsampled to match directional observations in each compartment.

Recent work has demonstrated that neurons with egocentric corner or boundary tuning are observed in RSC. The authors do not address whether egocentric tuning contributes to MDC signals. An explicit analysis of the relationship and potential overlap of MDC and egocentric populations is warranted.

Many of the MDCs presented in the main figures are not especially compelling. This includes alterations to MDC tuning in Figure 2, which is a key datapoint. The authors should show significantly more (if not all) examples of MDCs in each environment. It would similarly be useful to see all/more examples of non-directional spatially tuned neurons with rotationally symmetric firing patterns.

"One might hypothesize that specific environmental cues, such as door orientation or landmark positioning, drive these tuning shifts. However, our results argue against this interpretation. In four-room environments, each room had multiple entry points, yet MDCs never exhibited multidirectional activity within a single room."

I do not understand the logic here. Can the authors unpack this? Also, it is clear that some of the example cells have more than one peak in individual compartments. How is this quantified?

Reviewer #3 (Public review):

Summary:

The authors examine firing of dysgranular retrosplenial cortex (dRSC) neurons in relation to head orientation and location for rats exploring open-field environments. One environment utilized was a square arena with high walls that is split into two rectangular spaces connected by a doorway. Another environment is a square arena split into quadrants connected by doors near the center. For each, the different sub-spaces of the environments are either identical in terms of visual and tactile cues or different. For head direction neurons, the authors present one population where each neuron maintains a single tuning direction for the two or four sub-compartments of the two environments. A second population exhibits what is termed multi-directional firing, wherein neurons exhibit (overall) two or four head direction peaks in firing. For such neurons, firing in each of the sub-compartments is associated with only a single preferred direction, but the directions across compartments are shown to be at 180-degree (two-compartment environment) or 90-degree offsets. The offsets evidence tuning to the "same" orientation for the sub-compartments that are, in the global reference frame, oriented at 180 or 90 degree offsets. The results are similar whether or not the sub-compartments have the same or different tactile and visual cues. Thus, the first population is said to be global in its head direction tuning, while the second relates to each local environment in a way that is systematic across sub-compartments. Spatially-specific activity of another population of non-direction-tuned RSC neurons is examined, and comparisons of sub-compartment spatial firing maps suggest that spatial tuning in RSC also repeats across compartments when the firing maps for the compartments are rotated to match each other (as in physical space). Finally, a population of hippocampal "place" cells exhibited different location mapping across sub-compartments. The findings are interpreted to indicate that RSC can simultaneously map orientation in both local and global reference frames, possibly forming a mechanism whereby the sub-compartments' shared geometry (given by the boundary shapes and the door locations) can be related to each other and to the global space they share.

Strengths:

This paper addresses an interesting problem and expands how the field will think about directional tuning.

Weaknesses:

It is not clear that the experimental design allows for a clear interpretation of the data. Rates for preferred turning are low, as are ratemap correlations for spatially-tuned neurons.

(1) It is concerning that the neurons with head direction tuning have fairly low peak firing rates (mean close to 5 Hz), where prior studies examining head direction tuning in dRSC found head direction-tuned neurons with peak rates more than an order of magnitude higher (100 Hz or more). Under circumstances where neurons are tuned well to variables other than head direction (for example, angular velocity of movement), weak head direction tuning may be observed if those other variables are not sampled equally across head directions. The manuscript contains no rigorous control for this possibility. One place to start to address this issue would be to map out variables such as angular velocity by head orientation, and to test whether such relationships also carry 90 and 180 degree offsets.

(2) There is some question as to whether dRSC neurons (spatial or directional) following the sub-compartment "geometry" is appropriate in terms of interpreting the data. In the condition with sub-compartments carrying different tactile and visual cues, it seems that such cues pertain only to the floor of the environments. The distal visual space of the boundaries appears to be identical. One is left to wonder whether distinguishing environments according to boundary wall visual cues would lead to different results. The CA1 data does not help to rule this possibility out. A second reason to doubt the "shared geometry" interpretation is that there is no condition where sub-compartment geometry is varied. It is also the case that the sub-compartment doorways may stand as the only salient distal visual cue linking the environments. Local sensory cues and geometry seem not so disentangled in this study, but this is a major claim in the abstract.

(3) There is some concern with the interpretation that the spatial tuning of some dRSC neurons repeats in rotated form across sub-compartments. The firing rate map correlations are very low on average (~0.2), and far lower than the population of CA1 having repeating fields across the same vs different visual/tactile cue conditions. The authors should define the chance level of ratemap correlation by shuffling neuron identities. Apologies if this is indeed the current approach, but it seems not to be (I was left a bit lost by the description in the methods). For any population of hippocampal place cells, the cross-neuron correlations of firing rate maps are typically not zero, and correlations at 0.2 would normally be evidence for remapping.

(4) A somewhat picky point here that is not meant to claim that multi-compartment studies are not useful - the introduction states that real-world environments typically consist of multi-compartment rooms. This is certainly not true for rodents and is only sometimes true in humans.

(5) The discussion lacks a consideration of how such dRSC output might impact the target structures of dRSC.

(6) The discussion speaks to the idea that multi-directional neurons may aid in transitioning between contexts (sub-compartments). But it is notable that none of the multidirectional neurons have multi-directional tuning in all sub-compartments, but such firing was seen in the 2017 Nature Neuroscience study by Jacob/Jeffery. The discussion should address this difference and perhaps posit a means by which the firing of global and local head direction neurons can be related to each other to yield navigation that depends on both scales.

(7) The authors should provide the size of the smoothing function for spatial firing rate maps.

(8) The authors should devise a measure to define directional tuning in 4 directions (with 90-degree offsets).

(9) Figures 2D and 2H - The offsets in preferred tuning across sub-compartments are rather variable.

Wow, I didn’t really think about Cole as the dispatchable alternative to renewables but in retrospect that’s literally what China has been doing for the last five years. Duh.

Reviewer #1 (Public review):

Summary:

This manuscript presents a tunable Bessel-beam two-photon fluorescence microscopy (tBessel-TPFM) platform that enables high-speed volumetric imaging with stable axial focus. The work is technically strong and broadly significant, as it substantially improves the flexibility and practicality of Bessel-beam-based two-photon microscopy. The demonstrations are generally strong and bridge a wide range of neuroimaging applications, namely vascular dynamics, neurovascular coupling, optogenetic perturbation, and microglial responses. These convincingly show that the approach enables biological measurements that are difficult or impractical with existing methods.

The evidence supporting the technical and biological claims is generally strong. The optical design is carefully motivated, clearly described, and validated through a combination of simulations and experimental characterization. The biological applications are diverse and well chosen to highlight the strengths of the proposed method, and the data are of high quality, with appropriate controls and comparative measurements where relevant.

Strengths:

(1) The optical innovation addresses a well-recognized limitation of existing Bessel-TPFM implementations, namely axial focus drift during tuning, and does so using a relatively simple, light-efficient, and cost-effective design.

(2) The manuscript provides convincing experimental evidence for this being a versatile platform to map flow dynamics across diverse vessel sizes and orientations in both healthy and pathological states.

(3) Biological demonstrations are comprehensive and span multiple domains such as hemodynamics, neurovascular coupling, and neuroimmune responses.

(4) Quantitative analyses of blood flow across vessel sizes and orientations, including kilohertz line scanning, are particularly compelling and clearly beyond the reach of standard Gaussian TPFM.

(5) Particular advantages are that higher blood slow speeds become measurable up to 23mm/sec (20x more than conventional frame scanning), and that simultaneous (Bessel-)imaging and (Gaussian-)perturbation are possible because of the stable axial focus.

Weaknesses:

(1) At present, the paper does not properly position the new Bessel-beam method against previous work, and fails to compare it to alternative fast volumetric imaging methods without Bessel beams.

(2) The cost-effectiveness of the proposed method is not well described or supported by evidence; it would be useful to include more detail or remove this claim.

(3) Some biological conclusions, e.g., regarding novel features of microglial dynamics (i.e., the observed two-wave responses and coordinated extension-retraction), are based on relatively limited sample size and would benefit from clearer discussion of variability across animals and fields of view.

(4) The use of neural network-based denoising for microglial imaging is reasonable but introduces potential concerns about trustworthiness; additional clarification of validation or failure modes would strengthen confidence in these results.

To conclude, most of the authors' claims are well supported by the data. The central conclusion, namely that tBessel-TPFM provides tunable volumetric imaging enabling experiments not feasible with existing two-photon approaches, is justified. Some biological interpretations would benefit from a more cautious framing, but they do not undermine the main technical and methodological contributions of the study. This is a strong and technically rigorous manuscript that makes a substantial methodological advance with clear relevance to neuroscience and intravital imaging. Minor clarifications and a slightly more measured discussion of certain biological findings are recommended.

Reviewer #2 (Public review):

Summary:

The authors describe a tunable Bessel beam two-photon microscope (tBessel-TPFM) designed to overcome a common limitation of Bessel-based volumetric imaging: axial shifts of the effective focus during Bessel beam parameter tuning. Their optical design allows independent control of axial beam length and resolution while keeping the axial center fixed. This is extensively validated through simulations and experiments.

Strengths:

A major strength of the work is the breadth of validation combined with the level of technical detail provided. The authors carefully characterize the optical performance of the system and clearly explain the design choices and underlying derivations, which will make it easier for others to understand and implement. The authors demonstrate the utility of the method across several in vivo applications, including neurovascular imaging, blood flow measurements, optogenetic stimulation, and microglial dynamics.

Weaknesses:

In the in vivo demonstrations, the authors employ different Bessel beam configurations across experiments, but the beam parameters are not dynamically tuned during live imaging. A video example showing continuous or interactive tuning of the Bessel beam within a single in vivo imaging sequence would further highlight the practical advantages of this platform and strengthen the case for its potential applications. In addition, while excitation powers are reported, the manuscript does not place these values in the broader context of known photodamage thresholds for two-photon microscopy, which would be helpful to the readers. Denoising/image restoration are applied in one of the in vivo examples, but it is unclear why this step was used specifically for this dataset and whether it was necessary to achieve adequate SNR or primarily included as an additional demonstration.

Reviewer #1 (Public review):

Summary:

The study investigates the Drosophila non-visual light receptor rhodopsin7 with regard to its role in light information processing and resulting consequences for behavioral patterns and circadian clock function. Using behavioral, in situ staining, and receptor activation assays together with different fly mutants, the authors show that rhodopsin7 is an important determinant of activity under and response to darkness, which likely signals via a pathway distinct from other, visual Drosophila rhodopsins. Based on phylogenetic analysis, the authors further discuss a potentially conserved functional role of non-visual photoreceptors like rhodopsin7 and the mammalian melanopsin light information processing and circadian clock modulation.

Strengths:

The manuscript follows a very clear structure with all investigations logically building onto each other. Background information and methodology are provided in appropriate detail so that readers can fully understand why and how experiments were conducted. It is further praiseworthy that the authors provide the details that allow also non-experts in the field to fully understand their approaches. Experimental work was conducted in a highly standardized manner, and also considered potential "side-aspects" like the consequences of temperature cycles and changed photoperiods. The detailed and clear description of the obtained results makes them very convincing, with (almost) all observable patterns being addressed.

By highlighting the evolutionary old phylogenetic position of rhodopsin7 and its conservation across numerous clades, the authors provide strong reasoning for the relevance of their work, also pointing out the similarities to the mammalian melanopsin. The postulated hypothesis regarding protein structure and functioning, as well as the role in light information processing and behavioral and circadian clock modulation are well based on the authors' observations, and speculative aspects are correctly pointed out.

Weaknesses:

Where the manuscript still has potential for improvement is the discussion, which in its current form does seem slightly self-contained and does not fully integrate the findings of previous studies on Drosophila rhodopsin7. As the introduction specifically points out that previous findings have been contradictory, this seems like a missed opportunity. Further details on this are provided in the recommendations below.

Similarly, the manuscript currently lacks a discussion of the possible relevance of rhodopsin7 (and other non-visual light receptors in other organisms) in the context of a species' environment and lifestyle, i.e., what is the relevance/benefit of having rhodopsin7 in the fly's everyday life? While this clearly involves speculation, when done carefully, it can elevate the paper's relevance from a primarily academic to a societal one.

An additional point concerns the title and abstract, which postulate rhodopsin7 roles in contrast vision as well as motion and brightness perception. Contrast remains poorly defined in the text, leaving it ambiguous whether it refers to bright/dark contrasts, e.g., along edges, or the temporal contrast that results from dark pulses (startle response). While the latter seems to apply here, the former is likely more intuitive. Thus, this aspect should be rephrased (also in the title) or properly clarified early on. Regarding motion detection, this is backed up by the optomotor response results, but the findings stand somewhat isolated from the other results, lacking a clear connection aside from general visual processing. Lastly, brightness perception is mentioned in the abstract, but never again, possibly due to inconsistent phrasing throughout the manuscript.

Reviewer #2 (Public review):

Summary:

This is a very interesting paper bringing new and important information about the poorly understood rhodopsin 7 photoreceptive molecule. The very ancient origin of the gene is revealed in addition to data supporting a signaling pathway that is different from the one known for the canonical rhodopsins. Precise expression data, particularly in the optic lobe of the fly, as well as clear behavioral phenotypes in responses to light changes, make this study a strong contribution to the understanding of the still-debated function of rhodopsin 7.

Specific comments

(1) Title and abstract: Contribution of Rh7 to circadian clock regulation

(a) It is not that clear to me what rhodopsin does in terms of circadian regulation (even though its function might be circadianly regulated). The clear role in the light/dark distribution of activity might not be circadian per se, but mostly light/dark-driven, and there is no evidence here for a role in the entrainment of the clock.

(b) The authors should cite Lazopulo, which nicely shows that Rh7 has an important role in peripheral neurons to allow flies to escape from blue light (see below).

(2) Figure 2 C

The finding showing that Galphaz but not Galphaq can trigger signaling from light-excited Rh7 is a very intriguing finding to better understand Rh7 function. Since Galphaz is related to Gi/o, it would be interesting to test those, for example, by expressing RNAi with Rh7-gal4 and testing the Light-dark or light-off response behavior.

(3) Figures 3-4

The change in the locomotor activity distribution between light and dark in LD conditions provides a nice assay for Rh7 function. Since Lazopulo et al. (2019) have shown that wild-type but not Rh7 mutants do escape from blue light, it would be important to compare and discuss these LD behavior data with the Lazopulo results. Precisely, is this nighttime preference linked to blue light?

The expression data are really nice and show that Rh7 is mostly a non-retinal photoreceptor. However, the paper would be strongly reinforced by correlating this with the LD behavior. The LD phenotype should be tested in flies with Rh7 expression rescued under Rh7gal4 control (as done for the startle response). This is important to show whether the expression pattern is likely responsible for the described Rh7 function in LD. If L5 and or M11 drivers are available, they should be used to rescue Rh7? Since expression in some clock neurons is shown, the rescue experiment should also be done with a clock neuron driver.

In the same line, can the LD phenotype (or startle response phenotype of Figure 4) be restored by expressing Rh7 under ppk control, as shown for the blue light avoidance phenotype by Lazopulo et al?

Finally, the Rh7 "darkfly" rescued flies should be tested in LD.

Reviewer #3 (Public review):

Summary:

While our knowledge regarding visual opsins is largely very good, a lot more uncertainty exists around the role of non-visual opsins. Using the power of the Drosophila melanogaster model system, Kirsh et al. investigate the role of the non-visual opsin Rhodopsin7 (Rh7). Expression analysis, based on Rh7-Gal4>UAS-GFP and HRC in situ staining, reveals strong expression in the optic lobes and somewhat weaker, but nevertheless extensive expression in the brain. An investigation of motor activity reveals that loss of function leads to an altered day and night rhythm, specifically decreasing activity during the dark phase. These flies were also less sensitive, but still responsive to a light-induced startle response and showed deficiencies in the optomotor response. To further investigate how Rh7 may modulate these responses, inspired by the Dark line of flies (which were kept in the dark for ~1400 generations) and which has accumulated C-terminal related losses, the authors conducted rescues with an intact and a C-terminal-deficient Rh7 and were able to pinpoint that region as an important driver of related behavioral shifts. These findings are particularly intriguing as Rh7 represents an ancient opsin with phylogenetic and mechanistic parallels to mammalian melanopsin.

Strengths:

The paper is well-written and contains high-quality data with appropriate sample sizes, and the conclusions are well supported.

Weaknesses:

No weaknesses were identified by this reviewer, but the following recommendations are made:

(1) The authors should clarify exactly what tissues were taken for the comparative qPCR. This is particularly interesting in terms of the retina. Since Rh7 appears not to be expressed within the photoreceptor cells of the retina, this raises the important question as to which cells it is expressed in. To address this important question, it would also be helpful to include an expression analysis of the retina itself (by extending the RH7-GFP expression patterns and/or adding HCR in situ of the ommatidia array). The cell types of the retina are very well classified, and some evidence already exists for Rh7 expression in support cells (e.g., Charlton-Perkins et al., (2017); PMID: 28562601). This study has a unique opportunity to investigate this further by adding these critical data for a more complete picture of Rh7.

(2) Mammalian opsins should be included in the phylogenetic analysis illustrated in Figure 2A and indicate their position on the tree. This will allow readers to better put the authors' statements regarding the intermediate position of Rh7 into perspective. In addition, note that the distinction between red and deep red is easy to miss regarding the Rh7 cluster. Perhaps the authors could use a more distinct colour scheme, for example, orange and deep red.

(3) More details should be provided on the optomotor response experiments. Specifically, specifications of the frequencies used for the optomotor response are needed. Results show a relatively large level of variation, which may be due to different angular perspectives that flies may have had while viewing the stimulus. If possible, provide videos as examples, as they will make it clearer to viewers how much flies could move around in the setup (from the methods, it seems they could move within the 2.2 of the 3 cm diameter of the arena, which would lead to substantial differences in the visual angle of the viewed grating.

This section is a fun and clear way to show that “going viral” isn’t just an internet phenomenon. The examples of books, chain letters, and sourdough starters nicely illustrate how ideas and practices spread through effort, incentives, and social networks long before digital platforms existed. I especially like the chain letter example because it clearly shows how emotional pressure and fear helped drive sharing, which feels very similar to modern online virality. Overall, this makes the concept of cultural evolution and memes much more concrete and easy to understand.

This section does a great job showing how recommendation algorithms can unintentionally amplify polarization and even contribute to radicalization. The examples (Facebook reactions, Twitter quote-tweet dynamics, and the flat earth → QAnon pipeline) clearly illustrate how engagement-based systems can reward negativity and extreme content. I also appreciate the nuance at the end that polarization itself isn’t always bad, which keeps the discussion balanced rather than alarmist. Overall, this is a clear, well-supported explanation of why algorithmic design choices have serious social consequences beyond individual user intent.

When designers default to a narrow idea of the average user, their work can unintentionally exclude people whose experiences differ. That exclusion isn’t limited to disability, it shows how design choices quietly decide who gets to participate easily and who doesn’t.

It would be interesting to contrast how effective it is to continually survey disabled people compared to getting a disabled designer. I can see it being beneficial to have a person who intimately knows the issues disabled people face, but at the same time there may be many disabilities and experiences from intersecting disabilities.

Ability only matters in relation to the environment: tetrachromats may see more, but because the world isn’t built for that perception, it has little value. What we call a disability often reflects society norms, not an objective lack of ability.

This section explains disability as something created by social expectations, not just by a person’s body or mind. A disability happens when environments, designs, or systems assume everyone has certain abilities, like walking, seeing, hearing, or focusing, and fail to accommodate people who don’t. What counts as a disability can change across societies and situations, and disabilities can be visible or invisible, permanent or temporary. Overall, the reading emphasizes that disability is closely tied to design choices and social norms, and many difficulties could be reduced if society made more inclusive accommodations.

I think that this better encapsulates the meaning of a disability rather than the traditional connotation. In this way, every disability is situational and the responsibility to mitigate is on the society, rather on the individual.

This text describes disability as not being about the person’s body, but rather about society’s design. It illustrates this by demonstrating how disability is not about one’s body, but rather about how society’s design of objects and space affects people. For example, stairs, picture books, grocery shelves, and airplane seats are all objects and space designed by society and are meant to assume that “normal” people are able to do certain things. If someone cannot do these things, then they are disabled in this particular space.

In a few of the paragraphs above, the authors detailed how traffic counters would break and remain in disrepair for a year or more.

Devices like the pneumatic tube traffic counter are not fundamentally complex, and are relatively cheap to maintain and replace. Think about the cost of a few thousand dollars at most and comparing that to the cost of some world famous national parks potentially being harmed due to some negligence.

I'm actually quite curious about how these formulas are derived from regression analysis, so did brief search and learned that its the correlating easy-to-track metrics (visitor counts) with historical field audits. It’s a pragmatic but imperfect attempt to bridge the "data-reality gap" in a park with no front door.

This part makes it clear that data isn’t just a neutral “record” — it can become a political tool. Even choosing not to publicize a dataset is a form of power, because it shapes what the public thinks is happening and what feels “true” or “important.” The dataset doesn’t change, but the story around it does.

Universal Design shifts responsibility from individuals to systems, which feels fair on the surface, but fairness is not always the same as justice. In social media, treating everyone the same may still disadvantage people who start from very different positions. True justice may require platforms to offer different tools, protections, or visibility to different users based on their needs. So the goal should not only be “fairness,” but a more thoughtful form of equity that actively reduces harm rather than assuming equal access works for everyone.

Universal Design shifts responsibility from individuals to systems, which feels fair on the surface, but fairness is not always the same as justice. In social media, treating everyone the same may still disadvantage people who start from very different positions. True justice may require platforms to offer different tools, protections, or visibility to different users based on their needs. So the goal should not only be “fairness,” but a more thoughtful form of equity that actively reduces harm rather than assuming equal access works for everyone.

Universal Design shifts responsibility from individuals to systems, which feels fair on the surface, but fairness is not always the same as justice. In social media, treating everyone the same may still disadvantage people who start from very different positions. True justice may require platforms to offer different tools, protections, or visibility to different users based on their needs. So the goal should not only be “fairness,” but a more thoughtful form of equity that actively reduces harm rather than assuming equal access works for everyone.

This quote really made me stop and think. The idea that essays are scary because you have to be “certain” makes a lot of sense to me and describes why writing can feel stressful. I like how Stielstra reminds us that essays don’t have to be about having all the answers, but about exploring ideas.

The irony example stood out to me. Memorizing definitions without really understanding them feels like how we often study just to pass exams. I’ve also had moments where something only made sense after I experienced it myself. This shows that repeating information isn’t the same as truly learning it.

I understand her augment about having to much structure but the five paragraph model can be really helpful with students who are just starting to write.

The author's humor is displayed through a full circle moment of how her school didn't teach to learn, but to recite and remember.

This is an important point to note, that quantitative data can reveal patterns, but not lived experience or subjective reality.

The "Husk" Diagnostic Fatigue is usually a signal to sleep. But "Extraction Fatigue" is different—it is a signal to Disconnect. If you sleep while still connected to the "IV drip" of the feed, you will wake up tired because your subconscious is still being mined.

The "Emergency Brake" is a tactical interrupt. You must distinguish between Physical Exhaustion (requires sleep) and Systemic Depletion (requires silence).

Sakal vs. Tsalah The English word "success" in the Old Testament often conflates two very different Hebrew concepts. There is Tsalah (to push forward, to rush, to break through by force), and there is Sakal (שָׂכַל).

Sakal implies a deliberate, intelligent structuring of reality. It is the wisdom mentioned in Proverbs 14:8: "The wisdom of the prudent is to understand his way." When David "behaved himself wisely" (Sakal) in 1 Samuel 18, he wasn't just brave; he was observant. He understood the political and spiritual physics of Saul's court. The "Sakal Protocol" is the refusal to rely on brute force (Tsalah) when tactical insight is required. It is the difference between kicking down a door and unlocking it.

The "Kingdom" logic fundamentally re-wires the concept of rank through the Greek principle of Sōma (σῶμα)—the Body. In 1 Corinthians 12:26, the spiritual mechanics are clear: "If one part suffers, every part suffers with it; if one part is honoured, every part rejoices with it." In the Empire, rank is a zero-sum game (Comparison). In the Kingdom, rank is replaced by Function (Collaboration). The Hebrew concept of Anavah (Humility) is not about thinking less of oneself, but about "occupying your designated space." When you occupy your specific "vector" in the Body, the threat of "rank loss" evaporates because your value is intrinsic to your function, not your height on a ladder. The Kingdom system replaces Social Dominance with Covenantal Security.

This stood out to me because I have seen this first hand. This show how kairos on social media can be risky. Being one of the first people to post about something can bring attention to it, but it can also spread misinformation quickly if the information is not correct.

But also, how do we get this message across to the lay person, because as much as we learn this unless we communicate it well politics will not change. Politics are often optics and semantics, so we must learn how to use this data to speak in a manner that is heard. It took 10 years for the TEA stuff to begin to be corrected and that was blatant misuse of data.

Hawaii and California are richer states so they have more money but less students in special education. The cylce keeps turning...

But lets do away with the department of education! (sracasm...)

But also how often do we see leaders who lose sight of the children? I have seen many a leader climbing the ladder at the expense of marginalized communities. I have also seen good leaders but data is a double edged sword.

Where was New Spain? What happened in Mexico in the 1500s that would affect California in the 1700s? (34 -36) [Mexico is Mexico City before a time period (~1500s - look up to confirm)] - Cortez conquered central Mexico, continued on - he founded New Spain - vast colony, split into multiple provinces - 1521 - 1769 - 1521: conquering what would become New Spain - 1603: When San Diego and Monterrey got their name - 1769: Franciscan mission in San Diego - 1821: Mexico becomes independent country - 1848: California becomes a part of the US - people tend to think Cali history starts with the missions (1769) but it goes back so much further - (time period prior has relatively little documentation, not many records)

Why did it take so long for California to be called California? - (separate) Many recollections of the indigenous comes from the Spaniards [consider bias], due to lack of written records - lots of contact not documented (including some trade info

Discussed in class: Maidu Tribe- stands for "man" some people relocated to Oklahoma along with other tribes from area. A lot allowed to return, because they were not faring well. - never were more than a few thousand people - they nearly disappeared but they rebounded, there are now thousands again.

Discussed in class: - largest language groups in Cali - primary labor force for missions - location discussed - diet lots of fish - trade and craft skills (fish nets, storage baskets) - activities (leisure) - gambling, sports

Comparison to Tongva - different languages, activities, religion - but still trade

Generally: - no evidence of them trying to change minds; no wars, battles on the basis of religion. no territory disputes. - no evidence of them trying to conquer others - they did fight, skirmish, but it was temporary. (ex for reason: someone feels wronged)

look into more what aspects were taken on? What became a part of cultural practice?

Denial vs. Regulation The "Spa Day" fallacy is a misapplication of mindfulness that can lead to Dissociation. When the brain’s Anterior Cingulate Cortex (ACC) detects a conflict (the "burning room"), the healthy response is to mobilise the Sympathetic Nervous System for a solution.

The "PsyOp" of passive peace encourages a "freeze" response under the guise of relaxation. This causes the Dorsal Vagal branch of the Vagus Nerve to shut down system functions rather than engaging the Ventral Vagal state of "Social Engagement and Safety." True Shalom requires Neural Integration—where the brain acknowledges the chaos but maintains the Executive Function needed to act, rather than simply suppressing the stress signal until the system collapses from within.

A recent example of disinformation online comes from the Ottawa Senators fanbase on X. In January, goaltender Linus Ullmark stepped away from the team, fans speculated about troubles in the locker room with teammates without verified sources. The Senators later put out a post clarifying the situation, demonstrating how misinformation can rise from uncertainty rather than deception.

In this case, the original burner account created false rumours about Ullmark, and without an official statement, created a reason for why he was gone. Ullmark eventually came out and said his leave was due to mental health, including panic attacks and anxiety issues, not issues with the team and other players.

Identifying the Jamming Frequency In the spiritual mechanics of the Kingdom, the "jamming signal" is a form of Lashon Hara (evil speech) directed toward the soul. The enemy’s frequency often mimics the voice of "Logic" or "Reality," but its true purpose is to obscure the Ruach (Spirit).

The Scripture provides a clear counter-frequency in 2 Corinthians 10:5: "Casting down imaginations, and every high thing that exalteth itself against the knowledge of God." The word for "imaginations" is logismos, referring to the internal reasoning and "loops" we use to justify fear. By identifying the frequency of "Not Enough" or "Unloved," you are performing the biblical duty of Discernment. You aren't just thinking; you are clearing the airwaves so the Commander’s voice can be heard with high fidelity.

The Attentional Switching Cost This describes a failure of the Default Mode Network (DMN). When you transition from a high-stress task to a screen, you aren't resting; you are engaging in "Attention Switching." Each switch incurs a metabolic cost in the Prefrontal Cortex, depleting your stores of glucose and oxygen.

Instead of the Vagus Nerve signalling a "Rest and Digest" state to lower your heart rate, the "blue light" and rapid-fire stimuli of the feed keep your Amygdala on high alert. You are essentially trying to recharge a battery while the "flashlight" is still turned on. You end the day "tired" but "wired" because your nervous system never received the signal that the "hunt" was over.

The FieldGuide Application: "The Dark Room Protocol" Action: For the next 60 seconds, place your palms over your eyes to create total darkness.

The Goal: Physically cut the visual input to the Superior Colliculus. Force the brain to acknowledge that the "feed" has stopped so the actual recovery can begin.

The Physics of Sakal In the Kingdom, we differentiate between Asah (doing/labour) and Sakal (שָׂכַל), which is the Hebrew term for tactical wisdom or "success through insight."

In Nehemiah 4, the wall was not finished because the builders ran faster; it was finished because they "had a mind to work" and refused to let the mockery of Sanballat break their focus. The Treadmill is the spiritual equivalent of the Wilderness Wandering—40 years of movement with zero geographical progress. The Kingdom operates on the Vector of Faith, which requires a stationary moment to hear the "Still Small Voice" before the next stone is laid.

The FieldGuide Application: "The Static Reset" Action: Stand perfectly still for 60 seconds. Do not fidget.

The Goal: Recognise that your value to the King is not your RPM (Revolutions Per Minute), but your availability to be placed where He needs a stone.

In the Kingdom, the "Muzzle" is the antithesis of the Prophetic Vector. The Greek term Parrhesia (παρρησία)—often translated as "boldness" or "plainness of speech"—literally means "all-speech" or "telling it all." It is the refusal to leave parts of the truth behind for the sake of the audience.

When the Muzzle locks, we lose our Ruach (Breath/Spirit) alignment. Just as a physical muzzle restricts the intake of oxygen, spiritual domestication restricts the flow of the Ruach. In 2 Timothy 1:7, we are reminded that we haven't been given a spirit of Deilia (cowardice/timidity), but of Dunamis (Power). The "Golden Muzzle" replaces Dunamis—which is the raw, unpredictable kinetic energy of the Kingdom—with the static "Influence" of the Empire.

SECTION 1B - KEY QUOTE. Shows R/S integration supports client autonomy within ethical bounds. Directly addresses ACA A.4.b concern.

This statement makes a really important point about how we interpret data. When we look at large sets of information, it’s common to find patterns that seem meaningful, but they often only exist by random chance.

This qoute inspires me because I agree with this due to the fact that systems to achieve your goals are often overlooked because most people only look at their goals instead of building systems that would help them achieve their goals .

Thinking of culture as something that “reproduces” is kind of unsettling but also makes a lot of sense online. It explains why simple, catchy, or extreme ideas often survive better than nuanced ones. Platforms don’t need to push bad ideas intentionally—if something spreads easily, it will spread anyway.

The Meta-Layer Infrastructure SIG board is being sunsetted but the SIG itself will remain as an interface for certain kinds of communications.

We are sunseting the board but it did review and refine the initial draft.

After reading this section I felt like my understanding of disability, it is not just limited to those who are disability due to illness, but also those who cannot do something that we assume everyone can do. This made me realise that some design of infrastructures nowadays are improving for those who are disabled, but there are more to be changed

Supports the idea that metacognition might be the most powerful skill to build as we learn to navigate AI

captures my thoughts exactly!

but education is always political...

In 1970, Britain was facing many serious problems. The economy was weak, with slow growth, rising inflation, and increasing unemployment. There were many strikes by trade unions, which disrupted daily life and caused conflict with the government. Politically, the Conservative government under Edward Heath tried to control unions and improve the economy, but with limited success. Britain was also losing confidence as an international power, and many people felt the country was in decline.

Slightly unrelated, but making me think of how society can be conditioned into consuming through media

In this sentence we see that malfunctions don't only occur when AI is trying to generate and image but it also goes for text generator and researchers have found some errors in it. I had an English teacher tell me her students that were academically dishonest had gotten caught because the AI generator would make up false articles that didn't exist which gave them away in their assignments.