Reviewer #1 (Public review):

[Editor's note: this version has been assessed by the Reviewing Editor with further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

Summary:

Urination requires precise coordination between the bladder and external urethral sphincter (EUS), while the neural substrates controlling this coordination remain poorly understood. In this study, Li et al. identify estrogen receptor 1-expressing neurons (ESR1+) in Barrington's nucleus as key regulators that faithfully initiate or suspend urination. Results from peripheral nerve lesions suggest that BarEsr1 neurons play independent roles in controlling bladder contraction and relaxation of the EUS. Finally, the authors performed region-specific retrograde tracing, claiming that distinct populations of BarEsr1 neurons target specific spinal nuclei involved in regulating the bladder and EUS, respectively.

Strength:

Overall, the work is done with high quality. The authors integrate several cutting-edge technologies and sophisticated, thorough analyses, including opto-tagged single unit recordings, combined optogenetics and urodynamics, particularly those following distinct peripheral nerve lesions.

Comments on revised version:

During the revision, the authors have adequately addressed my concerns and made the suggested changes accordingly. I have no additional comments.

Reviewer #2 (Public review):

Summary:

The authors have performed a rigorous study to assess the role of ESR1+ neurons in the PMC to control coordination of bladder and sphincter muscles during urination. This is an extension of previous work defining the role of these brainstem neurons, and convincingly adds to the understanding of their role as master regulators of urination. This is a thorough, well-done study that clarifies how the Pontine micturition center coordinates different muscle groups for efficient urination, but there are some questions and considerations that remain.

Strengths:

These data are thorough and convincing in showing that ESR1+ PMC neurons exert coordinated control over both the bladder and sphincter activity, which is essential for efficient urination. The anatomical distinctions in pelvic versus pudendal control is clear, and it's an advance to understand how this coordination occurs. This work offers a clearer picture of how micturition is driven.

Weaknesses:

The dynamics of how this population of ESR1+ neurons is engaged in natural urination events remains unclear. Not all ESR1+neurons are always engaged, and it is not measured whether this is simply variation in population activity, or if more neurons are engaged during more intense starting bladder pressures, for instance. In particular, the response dynamics of single and doubly-projecting neurons are not defined. Additionally, the model for how these neurons coordinate with CRH+ neuron activity in the PMC is not addressed, although these cell types seem to be engaged at the same time. Lastly, it would be interesting to know how sensory input can likely modulate the activity of these neurons, but this is perhaps a future direction.

Reviewer #3 (Public review):

Summary:

The paper by Li et al explored the role of Estrogen receptor 1 (Esr1) expressing neurons in the pontine micturition center (PMC), a brainstem region also known as Barrington's nucleus (Hou wt al 2016, Keller et al 2018). First the author conducted bulk Ca2+ imaging/unit recording from PMCESR1 to investigate the correlations of PMCESR1 neural activity to voiding behavior in conscious mice and bladder pressure/external urethral muscle activity in urethane anesthetized mice. Next the authors conducted optogenetics inactivation/activation of PMCESR1 to confirm the contribution to the voiding behavior also conducted peripheral nerve transection together with optogenetics activation to confirm the independent control of bladder pressure and urethral sphincter muscle.

Comments on revised version:

No concerns. All my major questions were addressed.

Author response:

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

We would like to express our deep appreciation to the editor and reviewers for their constructive comments and suggestions, which have significantly improved the quality of our manuscript. In response, we have carefully revised the manuscript, addressed all comments, and performed additional experiments and analyses to strengthen our findings.

(1) We repeated retrograde tracing using CTB-647 to verify precise targeting of SPN and DGC neurons, as shown in the new Figure 7.

(2) We performed dual retrograde tracing combined with fiber photometry or optogenetic activation to investigate the role of PMC dual-projecting neurons in the control of urination, as shown in Figure supplements 11 and 12.

(3) We conducted new experiments activating PMC^ESR1+ neurons after PDNx to assess their role in urination, as shown in new Figure 6.

(4) We added a more detailed analysis of the dynamics of neural responses in PMC^ESR1+ neurons in Figure supplements 3F-3G.

(5) We analyzed peak Ca²⁺ signals in the PMC during and after the onset of EMG bursting, as shown in Figure supplement 4F.

(6) We added a comparison of spontaneous and light-induced spikes in PMC^ESR1+ neurons, as shown in Figure supplements 3B–3C.

(7) We expanded the Discussion to address how PMC^ESR1+ neurons coordinate bladder contraction and sphincter relaxation to control both the initiation and suspension of urination.

We hope these revisions meet the reviewers' expectations and contribute to the improvement of our manuscript.

Reviewer #1 (Public review):

Summary:

Urination requires precise coordination between the bladder and external urethral sphincter (EUS), while the neural substrates controlling this coordination remain poorly understood. In this study, Li et al. identify estrogen receptor 1-expressing neurons (ESR1+) in Barrington's nucleus as key regulators that faithfully initiate or suspend urination. Results from peripheral nerve lesions suggest that BarEsr1 neurons play independent roles in controlling bladder contraction and relaxation of the EUS. Finally, the authors performed region-specific retrograde tracing, claiming that distinct populations of BarEsr1 neurons target specific spinal nuclei involved in regulating the bladder and EUS, respectively.

Strengths:

Overall, the work is of high quality. The authors integrate several cutting-edge technologies and sophisticated, thorough analyses, including opto-tagged single unit recordings, combined optogenetics, and urodynamics, particularly those following distinct peripheral nerve lesions.

We are grateful for your insightful and constructive comments, which affirmed the importance and technical depth of our work. Thank you for dedicating your expertise and time to reviewing our manuscript. Guided by your suggestions, we have revised the paper as detailed below.

Weaknesses:

(1) My major concern is the novelty of this study. Keller et al. 2018 have shown that BarEsr1 neurons are active during urination and play an essential role in relaxing the external urethral sphincter (EUS). Minimally, substantial content that merely confirms previous findings (e.g. Figures 1A-E; Figures 3A-E) should be move to the supplementary datasets.

Thank you for this valuable and constructive comment. We fully agree that the novelty of our study relative to Keller et al., 2018 must be made explicit. Keller et al. established that PMC^ESR1+ neurons are active during socially evoked urine-marking behavior (voluntary urination) and demonstrated their essential role in relaxing the EUS. Their study mainly focused on behavioral context and EUS relaxation. In contrast, our work addresses a distinct, mechanistic question: how these same neurons participate in reflexive, physiological urination and coordinate both bladder detrusor contraction and EUS relaxation.

Novel aspects of the present study:

(1) Temporal dynamics of PMC^ESR1+ neurons during reflexive micturition.

Using opto-tagging and single-unit recordings, we reveal the precise firing pattern of PMC^ESR1+ neurons during reflexive voiding. Simultaneous fiber photometry, cystometry, and EUS-EMG recordings demonstrate that population-level activity of PMC^ESR1+ neurons precedes and tightly correlates with both bladder contraction and EUS relaxation a coordination not previously demonstrated.

(2) Causal role in reflexive urination.

Manual closed-loop optogenetic inhibition at the onset of reflexive voiding acutely terminates EUS bursting and bladder contraction, immediately halting urine release.

(3) Dual control of bladder and EUS.

Optogenetic activation combined with selective pelvic or pudendal nerve transection shows that PMC^ESR1+ neurons drive both bladder contraction and EUS relaxation, revealing a coordinating role beyond EUS relaxation alone.

(4) Anatomical substrate for coordinated control of bladder contraction and EUS relaxation in reflexive urination.

Retrograde tracing identifies three spinal-projecting sub-populations: SPN-only, DGC-only, and dual-targeting neurons, providing a circuit-level explanation for the simultaneous control of bladder and EUS.

Following your suggestion, panels that merely replicate Keller et al. (former Figures 1A–1E and Figures 3A–3E) have been moved to new Figure Supplements 1 and 7, respectively, so that the main figures now emphasize the new mechanistic findings.

(2) I also have concerns regarding the results showing that the inactivation of BarEsr1 neurons led to the cessation of EUS muscle firing (Figures 2G and S5C). As shown in the cartoon illustration of Figure 8, spinal projections of BarEsr1 neurons contact interneurons (presumably inhibitory) that innervate motor neurons, which in turn excite the EUS. I would therefore expect that the inactivation of BarEsr1 should shift the EUS firing pattern from phasic (as relaxation) to tonic (removal of relaxation), rather than stopping their firing entirely. Could the authors comment on this and provide potential reasons or mechanisms for this finding?

Thank you for this crucial comment. We apologize that the representative EUS-EMG traces in Figures 2G and S5C were too small to be clearly seen and that the corresponding results description was not sufficiently accurate. We have now replaced these EMG traces with enlarged versions (revised Figures 2G and S5C) and revised the corresponding Results section (lines 184, 197, 340-341). Based on the enlarged traces, we found that acute photoinhibition of PMC^ESR1+ neurons at the onset of phasic EUS-EMG bursting shifted the EUS firing pattern from large-amplitude phasic bursts to low-amplitude tonic firing. This suggests that ongoing activity of PMC^ESR1+ neurons is required to maintain phasic EUS bursting. A similar shift from phasic to tonic EUS-EMG activity during optogenetic silencing of PMC^ESR1+ neurons was reported by Keller et al., 2018 (Figure supplement 8C), confirming the reproducibility of the phenotype. We propose that the potential mechanism of this low-amplitude tonic activity may be mediated in part by a spinal reflex pathway (the guarding reflex) for preventing urination, whereby the loss of PMC^ESR1+ neurons-mediated supraspinal facilitation reduces inhibition of spinal interneurons, leading to enhanced baseline excitability of EUS motor neurons in response to bladder afferent input during bladder distension (William C. de Groat et al., Comprehensive Physiology. 2015, PMID: 25589273).

(3) Current evidence is insufficient to support the claim that the majority of BarEsr1 neurons innervate the SPN but not DGC. The current spinal images are uninformative, as the fluorescence reflects the distribution of Esr1- or Crh-expressing neurons in the spinal cord, along with descending BarEsr1 or BarCrh axons. Given the close anatomical proximity of these two nuclei, a more thorough histological analysis is required to demonstrate that the spinal injections were accurately confined to either the SPN or the DGC.

Thank you for raising this important concern. To rigorously verify that our spinal injections were confined to either the SPN or the DGC, we performed new retrograde-tracing experiments in ESR1-Cre and CRH-Cre mice. We injected a mixture of AAV-Retro-DIO-mCherry or AAV-Retro-DIO-EGFP with the retrograde tracer CTB-647 specifically into the SPN or DGC (Methods, lines 465-466). Only animals in which CTB-647 fluorescence was strictly limited to the target nucleus, without detectable spread to the adjacent region, were included in the analysis (new Figures 7A and 7E). These results confirm our original observation that PMC^ESR1+ neurons comprise three distinct spinal-projection subpopulations: one (19.0%) targeting the SPN, one (52.2%) innervating the DGC, and a third (28.8%) projecting to both regions (Results, lines 304–306; new Figures 7F–7H). In addition, the majority of PMC^CRH+ neurons project to the SPN but not the DGC (new Figures 7B–7D; Results, lines 297–301). We have assembled new Figure 7 using the newly acquired spinal images and the validated data.

Reviewer #1 (Recommendations for the authors):

From the abstract: "Anatomically, PMCESR1+ cells possess two subpopulations projecting to either the pelvic or pudendal nerve". I don't think these neurons directly project to either nerve.

Thank you for this precise comment. We apologize for incorrectly stating that PMC^ESR1+ cells project directly to the pelvic or pudendal nerves. In the revised Abstract (lines 32–36) we have rephrased the sentence to clarify the actual anatomy: “Anatomically, PMC^ESR1+ neurons consist of three distinct spinal-projection-based subpopulations: one targeting the sacral parasympathetic nucleus (SPN), one innervating the dorsal gray commissure (DGC), and a third that projects to both regions, thereby enforcing the coordination of bladder contraction and sphincter relaxation in a rigid temporal sequence.”. We trust this revision now accurately reflects the anatomical findings.

Reviewer #2 (Public review):

Summary:

The authors have performed a rigorous study to assess the role of ESR1+ neurons in the PMC to control the coordination of bladder and sphincter muscles during urination. This is an important extension of previous work defining the role of these brainstem neurons, and convincingly adds to the understanding of their role as master regulators of urination. This is a thorough, well-done study that clarifies how the Pontine micturition center coordinates different muscle groups for efficient urination, but there are some questions and considerations that remain.

Strengths:

These data are thorough and convincing in showing that ESR1+PMC neurons exert coordinated control over both the bladder and sphincter activity, which is essential for efficient urination. The anatomical distinctions in pelvic versus pudendal control are clear, and it's an advance to understand how this coordination occurs. This work offers a clearer picture of how micturition is driven.

We sincerely thank you for highlighting the rigor of our study and for recognizing the advance in understanding how PMC^ESR1+ neurons exert coordinated, anatomically segregated control over bladder and sphincter. We also appreciate the constructive suggestions that helped us further improve clarity, which we address point-by-point below.

Weaknesses:

The dynamics of how this population of ESR1+ neurons is engaged in natural urination events remains unclear. Not all ESR1+ neurons are always engaged, and it is not measured whether this is simply variation in population activity, or if more neurons are engaged during more intense starting bladder pressures, for instance. In particular, the response dynamics of single and doubly-projecting neurons are not defined. Additionally, the model for how these neurons coordinate with CRH+ neuron activity in the PMC is not addressed, although these cell types seem to be engaged at the same time. Lastly, it would be interesting to know how sensory input can likely modulate the activity of these neurons, but this is perhaps a future direction.

Thank you for this insightful comment. First, we agree that not all ESR1+ neurons are consistently engaged during urination (Figure 1B). Because bladder pressure was not measured during the opto-tagging experiments, we cannot determine whether this reflects trial-to-trial variability in population activity or pressure-dependent recruitment of additional neurons. We speculate that stronger starting bladder pressures may recruit a larger subset of ESR1+ neurons, analogous to graded, pressure-dependent recruitment observed in peripheral sensory neurons (Bruns et al., J Neural Eng. 2011, PMID: 21878706; Marshall et al., Nature. 2020, PMID: 33057202).

Second, using fiber photometry recording and optogenetic activation, we examined the dynamics of dual-projecting neurons in the PMC that were retrogradely labeled from the SPN and DGC. Their activity correlated with bladder contraction and sphincter relaxation, and optogenetic activation sequentially induced these events to trigger urination (see Recommendation #8). Although retrograde labeling captured only a subset of dual-projecting neurons, the results indicate that they coordinate bladder and sphincter activity.

Third, previous studies suggest that PMC^CRH+ cells are associated with bladder contraction and likely serve as an integration center for context-dependent micturition behavior (Hou et al., Cell. 2016, PMID: 27662084; Ito et al., Elife. 2020, PMID: 32347794). We therefore propose that PMC^CRH+ cells establish the baseline conditions and contextual readiness for voiding, whereas PMC^ESR1+ cells act as the executive command to reliably initiate and execute the event.

Finally, we agree that sensory inputs likely modulate PMC^ESR1+ neuron activity. Although this falls beyond the scope of the present study, it represents an important avenue for future investigation.

Reviewer #2 (Recommendations for the authors):

(1) In the introduction, the authors write that Keller 2018 only showed this ESR1 population to induce EUS relaxation, but those results also do show bladder contraction with photostimulation of this population. While the authors' work extends this finding in important ways, this should be acknowledged (line 60).

Thank you for this important correction. We have now revised the Introduction to explicitly acknowledge that stimulation of neurons expressing estrogen receptor 1 (ESR1) in the PMC (PMC^ESR1+) contributes to sphincter relaxation and increased bladder pressure (Introduction, lines 60-62), as originally reported by Keller et al., 2018.

(2) I think a more detailed analysis of the dynamics of neural responses in the PMC ESR1 neurons would be valuable. For example: are the same cells always engaged before micturition, or do different populations activate on different trials? Can the authors comment on the half of the opto-tagged ESR1 population that is not firing during urination? Do they ever fire? A cell-by-cell analysis of which neurons are engaged over multiple trials would be very valuable to understand the dynamics of population activity. Figure 1H shows cumulative sessions, but what do single sessions look like?

Thank you for these valuable comments. In response, we have performed refined single-trial analyses of neuronal activity, as detailed in the point-by-point replies below.

For example: are the same cells always engaged before micturition, or do different populations activate on different trials?

Among 11 PMC^ESR1+ units that showed urination-related excitation, 8 units exhibited a consistent firing increase in every voiding trial, whereas the remaining 3 increased their discharge in >78 % of trials (Figure 1B; new Figure supplement 3F). Thus, the same PMC^ESR1+ cells are recruited repeatedly, rather than distinct populations being activated on different trials. We have added this clarification to Results (lines 106–108).

Can the authors comment on the half of the opto-tagged ESR1 population that is not firing during urination? Do they ever fire? A cell-by-cell analysis of which neurons are engaged over multiple trials would be very valuable to understand the dynamics of population activity.

Approximately half of the opto-tagged PMC^ESR1+ cells showed no increase in firing rate during urination, yet exhibited spontaneous spikes at other times (new Figure supplement 3G), confirming their electrical competence. Because the PMC also participates in defecation, uterine activity, and other pelvic functions (Rouzade-Dominguez et al., Eur J Neurosci. 2003, PMID: 14686905; Schellino et al., Frontiers in Neuroanatomy. 2020, PMID: 33013330; Quaghebeur et al., Auton Neurosci. 2021, PMID: 34391125), these ESR1+ neurons may serve functions other than urination. We have now added this cell-by-cell analysis and discussion to the manuscript (Results, lines 108-112).

Figure 1 H shows cumulative sessions, but what do single sessions look like?

As shown in new Figure supplements 3F–3G, single-session raster plots reveal that PMC^ESR1+ neurons display consistent firing patterns across individual trials. Neurons whose firing rate increased during urination did so in most trials (Figure supplement 3F), whereas neurons unrelated to voiding remained silent or showed no discernible rate change during voiding across trials (Figure supplement 3G). These single-session observations are consistent with the cumulative population analysis shown in Figure 1H (new Figure 1B).

(3) Supplemental Figure 4: It seems clear from this figure that NVCs are only occurring when the sphincter fails to engage. Can the authors quantify how often this is the case?

Thank you for this important point. We have now quantified the occurrence of non-voiding contractions (NVCs) across all 229 bladder contraction events from 3 mice shown in Supplemental Figure 4. NVCs were observed exclusively when the external urethral sphincter failed to relax, accounting for 62/229 events (27.1 %), whereas coordinated voiding contractions (VCs) occurred in the remaining 167 events (72.9 %). These new data are presented in Figure supplement 4C.

(4) Continuing from the above point: the authors say that the insufficient top-down drive or strength of activity from PMC ESR1 neurons is why NVCs occur. In looking closely, it also seems there is a small hump and subsequent increase in the calcium signal when the EUS bursting begins (particularly clear in Supplementary Figure 4). Could this instead mean that the bursting/urethral activity itself is feeding back onto the PMC to continue/enhance its activity, and it is instead the lack of sphincter bursting that results in the NVC? Could the authors analyze the signal during and after bursting starts? This model is consistent with one of the classic reflexes defined by Barrington, in which urethral fluid flow/activation enhances bladder contraction. The Figure 4 transection experiments do not fully answer this, as the authors are driving activity in the PMC at this time, but they could test this using PDN transection with fiber photometry recording.

Thank you for this important point. We fully agree that EUS bursting may provide excitatory feedback to the PMC that sustains or even amplifies its activity, and that the absence of such feedback could underlie NVCs. To test this possibility, we re-analyzed the fiber-photometry traces aligned to the onset and offset of each EUS bursting (new Figure supplement 4). A small but consistent hump in the Ca²⁺ signal appeared before bursting onset and the Ca²⁺ signal continued to rise throughout the bursting (Figure supplement 4B, yellow arrow). The amplitude at bursting offset was significantly higher than both the NVC peak and the level recorded at bursting onset. These observations support the interpretation that urethral fluid flow/activation supplies excitatory feedback that reinforces PMC activity and bladder contraction, consistent with Barrington’s classic reflex. We have incorporated these new analyses into the revised manuscript (lines 145–155 and Figure supplement 4F).

We agree that the positive-feedback loop described by Barrington’s classic urethra-to-bladder reflex is an intriguing mechanism. However, the PDN-transection experiment in Figure 4 was designed to determine if bladder contractions triggered by PMC^ESR1+ cells can proceed in the absence of sphincter bursting, not to evaluate this reflex. Incorporating simultaneous fiber-photometry recording into the PDN-transection experiment would therefore go beyond the scope of the present study. In future work we are keen to combine PDN transection with fiber photometry to further determine whether the urethra-to-bladder reflex contributes to the sustained PMC activity observed in our paradigm.

(5) In Figure 4, is the timing of sphincter engagement different with ChR2 stimulation from what normally occurs? It appears that the bursting happens immediately upon activation whereas bladder contraction is a bit delayed.

Thank you for this important observation. We have carefully re-examined the EMG traces from all animals shown in Figure 4. We confirm that the onset of sphincter bursting activity during ChR2 stimulation is indeed more rapid than during natural reflex voiding; nevertheless, the onset of phasic sphincter bursting during ChR2 stimulation remained delayed relative to the intravesical pressure rise (see Figure 8B).

The immediate sphincter discharge visible in some trials was tonic EUS discharge or rare irregular bursting, not the typical EUS bursting. This tonic pattern corresponds to the spinal guarding reflex that suppresses urine leakage (Fowler et al., Nature Reviews Neuroscience. 2008, PMID: 18490916; Keller et al., Nature Neuroscience. 2018, PMID: 30104734). These segments were identified by their amplitude and spectral content and excluded from burst-onset analysis. Our analysis protocol therefore distinguishes tonic guarding activity from true phasic bursting, ensuring that only the latter was used to determine burst timing.

(6) The explanation on line 299 about how spinal reflexes are impinging on this circuit is confusing. I agree that the bladder contraction stopping later than the EUS signal likely has something to do with spinal reflexes, but it seems this could instead be feedback from the urethral fluid flow, which continues bladder contractions (urethra-destrusor facilitative reflex). Could the authors clarify their thoughts here?

Thank you for highlighting this ambiguity. We agree that the delayed cessation of bladder contraction could equally reflect either (1) the urethra-to-bladder facilitative reflex driven by ongoing urethral fluid flow or (2) spinal reflexes that we described. In the revised manuscript (Results, lines 343–349), we have re-worded the paragraph to make this dual possibility explicit, thereby avoiding an overly strong emphasis on spinal mechanisms alone.

(7) A note on phrasing: the authors frequently say PMCESR1 cells drive sphincter relaxation, but then show an effect on sphincter bursting. Experienced readers might realize that relaxation and bursting are connected, but this might be confusing for readers and should be clarified in the text.

Thank you for highlighting the potential ambiguity. We agree that the sentence “PMC^ESR1 cells drive sphincter relaxation” can seem paradoxical when our data show increased EUS bursting. In adult mice, the EUS does not remain continuously relaxed during voiding; instead, it generates rhythmic bursting composed of high-frequency spike clusters (active periods) alternating with low tonic activity (silent periods), resulting in rhythmic contractions and relaxations of EUS. This phasic activity acts as a pump that facilitates urine flow through the narrow rodent urethra (Kadekawa et al., Am J Physiol Regul Integr Comp Physiol, 2016, PMID: 26818058). The EUS bursting activity we recorded is consistent with the results reported in previous studies (Keller et al., Nat Neurosci, 2018, PMID:30104734; Ito et al., Elife, 2020, PMID:32347794).

Consequently, when PMC^ESR1 neurons initiate bursting, they simultaneously generate the relaxation phases that separate the spikes. To make this explicit we have replaced the phrase “PMC^ESR1+ cells drive sphincter relaxation” with “PMC^ESR1 neurons trigger EUS bursting, which generates rhythmic sphincter contractions and relaxations.” (Results, page 7, lines 219-221). We have applied similar clarifications throughout the revised manuscript (Results, lines 125-129). We hope this revision eliminates any apparent contradiction.

(8) The question remains as to which neurons (dual projecting, single projecting, or all?) are active in natural urination. This is possible to do through dual injection of retrograde virus in SPN and DGC that could coordinately turn on Gcamp, but this challenging experiment is perhaps beyond the scope of this paper. Even still, the authors could discuss their model for whether the dual- and single-projecting neurons are all engaged at once in a natural urination event. Do the authors have any data that could provide insight as to when these sub-populations are active? Results from the opto-tagging in Figure 1 (and comment #2 about single neuron firing properties) might provide a foundation for hypotheses or insights.

Thank you for this valuable suggestion. We have now performed the experiment you proposed: dual injection of retrograde virus (AAV-Retro-Cre and AAV-Retro-DIO-GCaMP6s) in SPN and DGC were used to selectively label PMC dual-projecting neurons, and a 200-µm optic fiber was implanted above the PMC to record their Ca²⁺ dynamics during natural urination (Figure supplement 11A and Methods, lines 470–474, 652-655). Dual-projecting neurons exhibited robust activation throughout the entire voiding phase that was tightly correlated with intravesical pressure rise and EUS bursting (Figure supplements 11A–11H). However, technical limits of current retrograde tools preclude selective isolation of single-projecting (SPN-only or DGC-only) subsets for independent fiber-photometry recordings and injection restricted to one target unavoidably labels both single- and dual-projecting cells. We now state this technical limitation explicitly (Discussion, lines 426-430).

Accordingly, in the revised Discussion (lines 389-406), we integrate fiber-photometry Ca²⁺ signals with single-unit data from opto-tagged recordings to propose several testable, non-mutually-exclusive models for how dual- and single-projecting PMC^ESR1+ neurons are engaged during natural urination: “Based on population dynamics obtained by fiber photometry (Figures 1D-1H, Figure supplements 1A-1F, and Figure supplements 11A-11H) and single-neuron firing properties recorded via optrode (Figures 1A-1C), we propose several mechanistic models for the engagement of dual- and single-projecting PMC^ESR1+ neurons during natural micturition. One possibility is that all three populations (dual-projecting, SPN-projecting and DGC-projecting neurons) are co-activated, with the dual-projecting subset acting as a “bridging amplifier” that sustains rising bladder pressure while coordinating EUS relaxation. Alternatively, SPN-projecting neurons may be recruited first to initiate bladder contraction, followed by DGC-projecting neurons that evoke EUS bursting and facilitate urine entry into the urethra; once flow begins, the urethro-detrusor facilitative reflex could recruit dual-projecting neurons to further enhance voiding efficiency. In addition, contextual or state-dependent urination—such as scent-marking behavior characterized by multiple voiding events with smaller volumes than reflexive urination—may predominantly rely on sequential and cooperative activation of single-projecting neurons. Other recruitment sequences remain conceivable. Future studies combining diverse urination-related behavioral paradigms with simultaneous recordings from projection-specifically labeled PMC neurons will be required to validate and refine these models.”

Reviewer #3 (Public review):

Summary:

The paper by Li et al explored the role of Estrogen receptor 1 (Esr1) expressing neurons in the pontine micturition center (PMC), a brainstem region also known as Barrington's nucleus (Hou et al 2016, Keller et al 2018). First, the author conducted bulk Ca2+ imaging/unit recording from PMCESR1 to investigate the correlations of PMCESR1 neural activity to voiding behavior in conscious mice and bladder pressure/external urethral muscle activity in urethane anesthetized mice. Next, the authors conducted optogenetics inactivation/activation of PMCESR1 to confirm the contribution to the voiding behavior also conducted peripheral nerve transection together with optogenetics activation to confirm the independent control of bladder pressure and urethral sphincter muscle.

We sincerely thank you for providing a thoughtful summary and insightful comments on our study.

Weaknesses:

(1) The study demonstrates that pelvic nerve transection reduces urinary volume triggered by PMC ESR1+ cell photoactivation in freely moving mice. Could the role of pudendal nerve transection also be examined in awake mice to provide a more comprehensive understanding of neural involvement?

Thank you for this valuable suggestion. We conducted an additional experiment to determine the contribution of the pudendal nerve to PMC^ESR1+ neuron-driven voiding in awake mice. Bilateral pudendal nerve transection (PDNx) reduced the optogenetically evoked urine volume compared with sham-operated controls, yet photoactivation of PMC^ESR1+ neurons still reliably induced urination after PDNx (new Figure 6). Thus, bilateral integrity of the pudendal nerve is required for efficient PMC^ESR1+ neuron-driven voiding, most likely by transmitting the signals that entrain rhythmic EUS bursting. These data and experimental details have been incorporated into Figure 6, Results (lines 272–276), and Methods (lines 542–545).

(2) While the paper primarily focuses on PMCESR1+ cells in bladder-sphincter coordination, the analysis of PMCESR1+-DGC/SPN neural circuits - given their distinct anatomical projections in the sacral spinal cord - feels underexplored. How do these circuits influence bladder and sphincter function when activated or inhibited? Also, do you have any tracing data to confirm whether bladder-sphincter innervation comes from distinct spinal nuclei?

Thank you for this critical comment. To determine how PMC^ESR1+ neurons that target distinct sacral nuclei influence bladder–sphincter coordination, we first focused on the dual-projecting subset in a new experiment (Figures supplement 11 and Methods, lines 470–477, 652-655, 669-673). Dual retrograde virus injections into SPN and DGC selectively labelled PMC dual-projecting neurons, a subset of which are ESR1+. Fiber-photometry recordings showed that these cells were active during bladder contraction and sphincter relaxation (Figure supplements 11E-11H), whereas optogenetic activation reliably initiated urination: bladder pressure rose immediately and was followed by rhythmic EUS bursting (Figure supplements 11I-11N and 12B; Results, lines 309-313, 332-335). Thus, the dual-projecting sub-population is sufficient to coordinate bladder contraction with sphincter relaxation. Current retrograde tools do not allow selective isolation of single-projecting (SPN-only or DGC-only) subsets; injecting only one target unavoidably labels both single- and dual-projecting cells. Consequently, we cannot yet compare the functional impact of pure SPN-only versus DGC-only PMC populations. This limitation is now stated explicitly in the revised Discussion (lines 426–430).

In our 2025 paper (Yan et al., Commun Biol, 2025, PMID: 40259086), we used PRV-based retrograde tracing to show that SPN and DGC constitute two separate spinal nuclei controlling the bladder and the EUS, respectively. Classic studies have reached the same conclusion (Yao et al., Nat Neurosci, 2018, PMID: 30361547; Karnup & De Groat, IBRO Reports, 2020, PMID: 32775758; Karnup, Auton Neurosci, 2021, PMID: 34391124). These citations and a concise summary have been added to the Results (lines 289–294).

(3) Although the paper successfully identifies the physiological role of PMCESR1+ cells in bladder-sphincter coordination, the study falls short in examining the electrophysiological properties of PMC ESR1+-DGC/SPN cells. A deeper investigation here would strengthen the findings.

Thank you for this thoughtful suggestion. While a detailed electrophysiological characterization of PMC^{ESR1+-DGC/SPN} neurons would provide complementary information, the primary goal of the present study was to define the in vivo functional dynamics and behavioral role of these neurons during natural urination. As you suggested, further electrophysiological analysis of PMC^{ESR1+-DGC/SPN} neurons will be an important direction for our future work.

(4) The parameters for photoactivation (blue light pulses delivered at 25 Hz for 15 ms, every 30 s) and photoinhibition (pulses at 50 Hz for 20 ms) vary. What drove the selection of these specific parameters? Moreover, for photoactivation experiments, the change in pressure (ΔP = P5 sec - P0 sec) is calculated differently from photoinhibition (Δpressure = Ppeak - Pmin). Can you clarify the reasoning behind these differing approaches?

Thank you for this opportunity to clarify our experimental design. The photoactivation protocol (25 Hz, 15 ms pulses) was chosen because PMC^ESR1+ neurons faithfully follow this frequency without depolarisation block and it reliably triggers voiding (Keller et al., Nat Neurosci, 2018, PMID:30104734). For photoinhibition we originally stated “50 Hz, 20 ms pulses”, but this was an error. Consistent with the same study (Keller et al., Nat Neurosci, 2018, PMID:30104734), we used continuous light (constant illumination) to maintain sustained suppression. The Methods section has been corrected (lines 659-661, 690-691).

The ΔP formula was tailored to the temporal profile of each manipulation. For activation, ΔP (P_{5 sec} - P_{0 sec}) captures the rapid pressure rise after light onset; the same window was used in (Hou et al., Cell. 2016, PMID: 27662084). For inhibition, because saline infusion produces rhythmic reflex voiding, we delivered light at the onset of EUS bursting (i.e. when pressure was already at ~peak). Inhibition abruptly stops the bladder contraction, so the bladder cannot return to its pre-void baseline. The Δpressure (P_peak – P_min) was therefore used to quantify the extent to which the ongoing pressure wave was aborted by photoinhibition. P_min is the lowest value reached before the next infusion-driven upswing, making the metric insensitive to the slow baseline drift produced by continuous infusion. These clarifications have been added to the Methods (Methods, lines 676-677, 679-680, 692-693).

(5) The discussion could further emphasize how PMCESR1+ cells coordinate bladder contraction and sphincter relaxation to control urination, highlighting their central role in the initiation and suspension of this process.

Thank you for this valuable comment. We have revised the Discussion to emphasize that PMC^ESR1+ neurons coordinate urination by sequentially driving bladder contraction followed by sphincter relaxation through their dual projections to the SPN and DGC. We also emphasized that this coordination is essential for the initiation and effective execution of voiding (Discussion, lines 369-388). In addition, in the revised Discussion (Discussion, lines 389-406), we integrate fiber-photometry Ca²⁺ signals with single-unit data from opto-tagged recordings to propose several testable, non-mutually-exclusive models for how PMC^ESR1+ cells are engaged during natural urination.

(6) In Figure 8, The authors analyze the temporal sequence of bladder pressure and EUS bursting during natural voiding and PMC activation-induced voiding. It would be acceptable to consider the existence of a lower spinal reflex circuit, however, the interpretation of the data contains speculation. Bladder pressure measurement is hard to say reflecting efferent pelvic nerve activity in real time. (As a biological system, bladder contraction is mediated by smooth muscle, and does not reflect real-time efferent pelvic nerve activity. As an experimental set-up, bladder pressure measurement has some delays to reflect bladder pressure because of tubing, but EUS bursting has no delay.) Especially for the inactivation experiment, these factors would contribute to the interpretation of data. This reviewer recommends a rewrite of the section considering these limitations. Most of the section is suitable for the results.

We agree with the reviewer that bladder pressure, mediated by smooth muscle contraction, provides an indirect measure of efferent pelvic nerve activity and is subject to both physiological and experimental delays. Regarding potential delay from the tubing system, pressure propagates in fluid at approximately 1000 m/s (Kela & Pekka, Proceedings of World Academy of Science Engineering & Technology, 2009, DOI: 10.5281/zenodo.1080526). Given that the total tubing length in our setup is 0.5-1 meter, this gives an estimated transmission delay of only 0.5-1 ms. However, this delay is negligible compared with the observed time difference (~700 ms) between the cessation of EUS bursting and the termination of bladder contraction. Theoretically, pressure transmission is not expected to introduce a temporal delay. However, we cannot exclude the possibility that the pressure measurement itself may impose such a delay, because bladder pressure does not necessarily reflect efferent pelvic nerve activity in real time. Future studies using simultaneous recordings of bladder pressure and pelvic nerve discharges will help clarify whether a true temporal delay exists. Nevertheless, we agree that additional physiological or peripheral factors may also contribute to this difference in timing. As suggested by the reviewer, we have revised the discussion to consider the potential influence of other factors, such as urethra-detrusor facilitative reflex (Results, lines 343-349).

Reviewer #3 (Recommendations for the authors):

(1) In opto-tag experiments, a comparison of average AP waveform during behavior and during light stimulation should be included as criteria. It should be mostly the same waveform.

Thank you for bringing this to our attention. We have now added this comparison as an inclusion criterion in the revised manuscript. Figure supplement 3B shows representative examples of the average waveforms, and Figure supplement 3C displays the distribution of correlation coefficients between spontaneous and light-evoked spikes for all recorded PMC^ESR1+ units, all of which exhibited r > 0.8.

(2) Optical fiber implantation seems to be done in two different methods. In Figure 1 and Figure 2, the fiber tip is positioned just above PMC but in Figure 3 it seems to be angled. The information should be included in the Methods section.

Thank you for this important comment. We have now clarified in the Methods that for Figures 1 and 2, the optical fibers were implanted vertically above the PMC, whereas for Figure 3, the left optical fiber was implanted at a 33° lateral angle targeting the PMC (Methods, lines 499-503).

(3) In the closed-loop inhibition experiments of Figure 2, the parameters to start closed-loop photo-inactivation were not described in the method. If it is a manual closed loop, it should be described clearly.

Thank you for raising this important point. We apologize for omitting these details in the original Methods. We have now added a complete description of the manual closed-loop photo-inhibition protocol, including the triggering criteria and operator-controlled timing, in the revised Methods section (lines 602–605).

(4) In Figure 7A/E the authors provide a spinal cord image to show the injection site, but the image is misleading. The figure only shows AAV-infected CRH/ESR1 neurons in the spinal cord section. It does not indicate the AAV injection site or the terminal distribution.

Thank you for your important comment. We apologize for providing a spinal cord image that did not accurately depict the injection site. To rigorously verify that our spinal injections were confined to SPN or DGC, we performed new retrograde-tracing experiments in ESR1-Cre and CRH-Cre mice. A mixture of AAV-Retro-DIO-mCherry or AAV-Retro-DIO-EGFP with the retrograde tracer CTB-647 was injected specifically into SPN or DGC. Only animals in which CTB-647 fluorescence was strictly limited to the target nucleus, without spread to the adjacent region, were included (new Figures 7A and 7E). These data confirmed our original observations and have been pooled in Figure 7. The manuscript and figure have been updated accordingly (Results, lines 297-301, 304-306; Methods, lines 465–466).

eLife Assessment

This manuscript applies a theoretical analysis to two published datasets on yeast and bacterial evolution to compare different ways of quantifying fitness. It makes an important advance by clarifying how discrepancies can arise by using different approaches and provides recommendations for best practices. Overall, this is an impressive and highly beneficial study that is based on convincing evidence and has the potential of setting standards in this rapidly growing field.

Reviewer #1 (Public review):

The authors point out that the fitness estimates obtained from different experimental assays (monoculture, pairwise competition or bulk competition) are not generally equivalent, not even with regard to the fitness ranking of different genotypes. Using a computational model based on experimentally measured growth phenotypes for knockout strains in yeast, as well as data from Lenski's Long Term Evolution Experiment (LTEE), they derive a set of best practice rules aimed at extracting the optimal amount of information from such experiments.

The study is very complete on a technical level, and the conceptual weaknesses raised in the first round of reviews have been fully addressed in the revision.

Reviewer #2 (Public review):

Summary:

The manuscript "Quantifying microbial fitness in high-throughput experiments" provides a comprehensive analysis of the various approaches to quantifying fitness in microbial evolution, focusing on three primary factors: encoding of relative abundance, time scale of measurement, and the choice of reference subpopulation. The authors systematically explore how these choices impact fitness statistics and provide recommendations aimed at standardizing practices in the field. This manuscript aims to highlight the impact of differing fitness definitions and the methodologies utilized for analysis and how that can significantly alter interpretations of mutant fitness, affecting evolutionary predictions and the overall understanding of genetic interactions in the experiments.

Strengths:

The choices for quantifying fitness in evolution experiments are critical and highly relevant given the increasing prevalence of high-throughput experiments in evolutionary biology. The authors methodically categorize fitness statistics and their implications, providing clarity on a complex subject. This structured approach aids in understanding the nuances of fitness measurement. The manuscript effectively highlights how different choices in fitness measurement can influence fitness rankings and the understanding of epistasis, which is important for modeling evolutionary dynamics.

Comments on revisions:

The authors have comprehensively addressed all previous comments and suggestions. In particular, the addition of the new methods section: 'A guide to calculate pairwise relative fitness under the logit encoding from bulk competition data' - significantly improves the clarity of the implementation and helps in the overall interpretation of the framework.

Reviewer #3 (Public review):

Summary:

The authors present analyses of different fitness measures derived from empirical data from yeast knock-out mutants and the long-term evolution experiment (LTEE) with Escherichia coli to explore discrepancies and identify preferred methods to estimate relative fitness in high-throughput experiments. Their work has three components. They first discuss the different "encodings" of relative abundance data and conclude that logit-transformations are preferred, because they transform nonlinear abundance trajectories into linear trajectories with greater predictive power. Next, they compare per-generation with per-growth cycle relative fitness estimates inferred from simulations of pairwise competitions based on published growth traits for the yeast strains and on published pairwise competition measurements for the LTEE data. Both data sets show quantitative and qualitative (i.e. rank order) discrepancies of estimates across different time scales, which are highlighted by considering possible underlying causes (i.e. trade-offs between growth traits) and consequences (i.e. epistasis among mutations affecting different growth traits). Finally, the authors compare simulated pairwise and bulk (i.e. where many mutants compete during a growth cycle in a single environment) competition assays based on the yeast knock-out mutants and demonstrate an optimal ratio of collective mutants to wild-type strains that minimizes both sampling error and overestimation of fitness estimates when compared with pairwise competitions.

Strengths:

The study deals with a highly relevant topic. Fitness is central to general evolutionary theory, but also poorly defined and implies different traits for different organisms and conditions. For microbes, which are often used in evolution experiments, high-throughput experiments may yield different measures to quantify abundance over time, from individual growth traits to bulk competition experiments. Hence, it is relevant to consider discrepancies among those measures and identify preferred measures with respect to predicting population dynamic and evolutionary processes. The present study contributes to this aim by (i) making readers aware of differences among commonly used fitness estimates, (ii) showing that simulated (yeast) and calculated (E. coli) competitive fitness may differ across time scales, and (iii) showing that bulk competitions may yield relative fitness estimates that are systematically higher than pairwise competitions. The study is rather thorough on the theory side, with extensive derivations and analyses of various fitness measures using their resource competition model in the Supplementary Information. The study ends with a few practical recommendations for preferred methods to infer relative fitness estimates, that may be useful for experimentalists and stimulate further investigations.

Weaknesses:

The study has a few limitations. Perhaps the most apparent limitation is the lack of a clear answer to the question which fitness measure is best "in the light of first principles". The authors show clear discrepancies between fitness estimates across different time scales or using different reference genotypes in bulk competition and provide useful recommendations based on practical considerations (e.g. using pairwise competitions as "golden standard"), but it remains unclear whether these measures provide the greatest value for the questions researchers may want to answer with them (e.g. predict shifts in genotype frequencies). -- The authors have convinced me in their response that their recommendations were fundamentally related to the resource competition model, and the changes in introduction and discussion help to appreciate the choice of fitness measure in relation to the research question.

A second limitation is that the authors analyse fitness differences arising solely from resource competition, whereas microbes often interact via other mechanisms, e.g. the production of anticompetitor toxins, cross-feeding of metabolites or lack of growth to enhance their persistence in stress conditions. Without simulations of these processes, understanding discrepancies among fitness measures is necessarily limited. In addition, the analysis of trade-offs between growth traits causing these discrepancies during resource competition seems confounded by biases in measurement error or parameter estimation, at least for growth rate and lag time (Fig. 2B), where the replicate estimates for the wildtype show a similar negative correlation. -- The motivation to use a resource competition model for fitness inference is generally well motivated now. I accept their argument that resource competitive differences are most important for microbial strains with small genetic differences (e.g. from mutant libraries or from the same evolution experiment). However, it is relevant to note that this ignores situations that are rather common, where the wild-type strain produces an anticompetitor toxin or causes growth inhibition through metabolite products that lower the pH (and derived strains will likely contain resistant mutations).

Third, the study does not validate relative fitness predictions from growth traits (as is done for the yeast mutants) with measured relative fitness estimates using competition assays, while such data are available, e.g. for the LTEE. This would strengthen their inferences about preferred fitness measures. -- In their response, the authors explain that their aim was different, i.e. the provide "proof of principle" that the choices of fitness measure can produce discrepancies even when they follow the same growth model.

Fourth, the analysis of epistasis between mutations affecting different growth traits (shown in Fig. 3) based on the LTEE data could be better introduced and analysed more comprehensively. Now, the examples given in panels C-F seem rather idiosyncratic and readers may wonder how general these consequences of using fitness estimates based on different time scales are. -- The authors have made extensive improvements to address how different growth parameters, especially lag and growth rate, differently affect apparent epistasis based on measures at different time scale (per generation vs per cycle). These provide a more comprehensive analysis of down-stream consequences for epistasis detection.

Finally, the study is generally less accessible to experimentalists due to the extensive and principled treatment of specific population dynamic models and fitness inferences. This may distract from the overarching aim to identify fitness measures that are most accurate and useful for predictions of population dynamic and evolutionary processes. In this light, the motivation for the initial discussion of the importance of how to best encode relative abundance (Fig. 1) is unclear. Also, the conclusion, that logit encoding is preferred, because it linearizes logistic growth dynamics and "improves the quality of predictions", is not further motivated. Experimentalists using non-linear models to infer fitness from growth curves or competition assays may miss the relevance of this discussion. -- Thanks for this explanation (indeed, I confused "logistic dynamics" with "logistic growth model"); the additional explanations and text reductions have improved accessibility for experimentalists.

Comments on revisions:

I appreciate the thorough and effective response to all recommendations and have no further comments.

Author response:

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

We thank both editors and the three reviewers for their constructive criticism of our work. As a result of these comments, we have made several significant revisions to the paper that we believe strengthen and clarify our major results:

(1) Following suggestions from Reviewers #1 and #3, we have have improved our introduction to the different fitness concepts (lines 105–148) and streamlined the discussion of the logit encoding (lines 175–190). In particular, we have moved the most technical points to the SI (Sec. S3).

(2) Based on criticisms of our usage of the population dynamics model from Reviewers #1 and #3, we significantly revised our explanation of the motivation and interpretation of this model (lines 284–310 and 323–336) and our discussion of the generalizability of these results (lines 678–728), including the possible effects of interactions besides resource competition.

(3) Following a request from Reviewer #3, we have expanded our analysis of epistasis to systematically test all possible double mutants between qualitative types of trait perturbations in the model. We have added a new main text figure (Fig. 3), new SI figures (Figs. S9–S15), a new subsection in the Results (lines 344–395), and corresponding new sections in the Methods (lines 864–892) and SI (Sec. S8).

(4) Following concerns from Reviewers #2 and #3 about the limited empirical data, we have expanded our analysis of the LTEE data (new main text Fig. 4, revised text on lines 416–439, and revised SI Figs. S16–S18) and have analyzed two new benchmarking datasets for bulk fitness to test our predictions (new main text Fig. 6, new Results subsection on lines 561–590, and new SI Figs. S24 and S25).

(5) Following the criticism of Reviewer #3 about the lack of a clear recommendation on fitness quantification that provides the greatest value for a given scientific question, we have better explained what we think the scientific consequences of fitness are as a motivation for our analysis (lines 82–88, 319–322, and 615–630) and replaced the final flowchart figure with a step-by-step guide in the Methods to implement our recommendations in practice (lines 964–982).

Reviewer #1 (Public review):

The authors point out that the fitness estimates obtained from different experimental assays (monoculture, pairwise competition, or bulk competition) are not generally equivalent, not even with regard to the fitness ranking of different genotypes. Using a computational model based on experimentally measured growth phenotypes for knockout strains in yeast, as well as data from Lenski’s Long Term Evolution Experiment (LTEE), they derive a set of best practice rules aimed at extracting the optimal amount of information from such experiments.

The study is very complete on a technical level and I have no suggestions for further analyses. However, I feel the readability and the conceptual focus of the manuscript could be significantly improved by rearranging the material with regard to the contents of the main text vs. the Methods and the Supplement. Detailed recommendations:

(1) Regarding readability, the large number of references to material in the Methods and Supplement fragment the main text and make it difficult to follow.

We understand the challenges these references pose to the flow of the main text; we have attempted to keep those references to a minimum, while ensuring that technical details of the work are fully documented and referenced for completeness.

(2) Conceptually, it seems to me that the current presentation obscures the reasons why we should care about fitness in the first place. In the first paragraph of Results, the authors define fitness “as any number that is sufficient to predict the genotype’s relative abundance x(t) over a short-time horizon”. To me, this seems like an extremely narrow and not very interesting definition. Instead, I view fitness as an intrinsic property of a genotype that allows us to predict its performance under a range of conditions, including in particular conditions that are different from the experimental setup that was used to obtain the fitness estimates. The latter viewpoint is well expressed in Supplementary Section S1, where the authors discuss the notion of fitness potential. I would recommend to move at least part of this discussion to the main text.

We appreciate the reviewer’s viewpoint and have moved that conceptual discussion from the SI to the beginning of the Results section to give readers a broader perspective on fitness (lines 105–148). We use “potential” in analogy with potential energy in physics and have clarified this on lines 126–135.

What we call fitness potential, like the other notions of fitness we discuss in this paper (relative and absolute fitness), is still specific to an environmental condition. Fitness as a property intrinsic to a genotype and independent of any environment, as the reviewer mentions, is an interesting concept but beyond the scope of this paper, which is focused on analyzing fitness measurements that are inevitably environment-specific and we have clarified this on lines 142–148. While it is true that this definition of fitness is narrow, it is what can be empirically measured directly, and thus we believe it is crucial to understand how to best interpret that data.

By comparison, the arguments in favor of the logit encoding that currently opens the Results session are rather straightforward and could be shortened significantly.

We agree and have condensed this section (lines 175–192).

(3) Similarly, the modeling strategy used in this work is quite subtle and needs to be explained more fully in the main text. The authors use growth traits (lag time, growth rate, and yield) extracted from monoculture experiments on a yeast knockout collection and feed them into a specific mathematical model to simulate pairwise and bulk competition scenarios. Since a key claim of the work is that monoculture experiments are generally poor predictors of competitive fitness, the basis for this conclusion and the assumptions on which it is based need to be described clearly in the main text. In the current version of the manuscript, this information has been largely relegated to the Methods section.

We agree that our motivation for the population dynamics model and growth curve data was not clearly explained. We have significantly revised this section of the Results in the main text (lines 284–310).

In particular, we recognize the potential for misunderstanding this material we do not intend the relative fitness values calculated from this model to be interpreted as predictions of the true relative fitness between yeast deletion strains. Rather, we use the population dynamics model for our proof of principle: that the most basic features of microbial population dynamics in laboratory experiments, as captured by this model (resource competition, lag phase, growth phase, saturation), are sufficient to create discrepancies between common fitness statistics used in these experiments (different encodings, time scales, choices of reference subpopulations). We have added a statement to highlight existing work on monoculture predictors for competition outcomes [32, 34, 36, 37] on lines 453–459.

Reviewer #1 (Recommendations for the authors):

In the discussion of the LTEE in Section S8, the authors write on page 8 that “we couldn’t fit the fitted values a,b in ref. 29 so we were unable to check it”. I don’t understand this sentence - is the claim that the fit in ref. 29 was incorrect?

We have clarified this point in the SI (now Sec. S9). Our point was not that the fit in Wiser et al. 2013 is incorrect, but merely that we could not find the exact values of the fitted parameters they obtained documented in their paper, so we could not compare our own fitted parameters directly to theirs.

Also, at the end of the section, the authors refer to theory work on the long-term fitness trend in the LTEE. Here, two early references arguing for a logarithmic increase in fitness could be mentioned as well:

International Journal of Modern Physics B 12,:361-391 (1998) Evolution and Extinction Dynamics in Rugged Fitness Landscapes Paolo Sibani, Michael Brandt, and Preben Alstrøm

J. Stat. Mech. (2008) P04014 Evolution in random fitness landscapes: the infinite sites model Su-Chan Park and Joachim Krug

We thank the reviewer for providing these two references and have added them to the list of previous works on long-term fitness trends at the end of the section (now Sec. S9).

Reviewer #2 (Public review):

Summary:

The manuscript “Quantifying microbial fitness in high-throughput experiments” provides a comprehensive analysis of the various approaches to quantifying fitness in microbial evolution, focusing on three primary factors: encoding of relative abundance, time scale of measurement, and the choice of reference subpopulation. The authors systematically explore how these choices impact fitness statistics and provide recommendations aimed at standardizing practices in the field. This manuscript aims to highlight the impact of differing fitness definitions and the methodologies utilized for analysis and how that can significantly alter interpretations of mutant fitness, affecting evolutionary predictions and the overall understanding of genetic interactions in the experiments. Although this manuscript focuses on a critical issue in the quantification of fitness in high throughput experiments, it heavily relies on only one experimental dataset (Warringer et al 2003) and one organism i.e, Yeast (Saccharomyces cerevisiae) grown in a defined medium, the environmental influence is not completely captured. While the theoretical framework is strong, more experimental examples with more organisms (i.e., more datasets) in their analysis and comparison would enhance the manuscript, especially its conclusion.

We have expanded our analysis of competition data from the Long-Term Evolution Experiment in E. coli (lines 416– 439), including adding a main text figure (Fig. 4) along with the three SI figures (Figs. S16–S18). We have also added two completely different data sets that directly test our predicted discrepancies in fitness estimates from bulk competition experiments. From this data we have added a new main text figure (Fig. 6), two new SI figures (Figs. S24 and S25), and a new section at the end of the Results (lines 563–590).

We wish to clarify, though, that the aim of this study is to develop theory on fitness quantification choices and minimal examples to demonstrate the potential for discrepancies between these choices. While we appreciate the reviewer’s interest in understanding how discrepancies in fitness statistics vary across organisms and environments, that is an empirical question beyond the scope of this paper.

Strengths:

The choices for quantifying fitness in evolution experiments are critical and highly relevant given the increasing prevalence of high-throughput experiments in evolutionary biology. The authors methodically categorize fitness statistics and their implications, providing clarity on a complex subject. This structured approach aids in understanding the nuances of fitness measurement. The manuscript effectively highlights how different choices in fitness measurement can influence fitness rankings and the understanding of epistasis, which is important for modeling evolutionary dynamics.

Weaknesses:

The theoretical framework is robust, but the manuscript could benefit from more empirical examples to illustrate how different fitness quantification methods lead to varied conclusions in experiments.

Please see our response to the previous comment on this point.

The discussion on the choice of reference subpopulation could be expanded with the influence of the environment or the condition. Different types of reference groups might yield different implications for fitness calculations, and further elaboration would enhance this section.

While we agree that studying how environmental conditions affect fitness is an important and interesting problem, it goes beyond the scope of this paper, which focuses on the basic theory of quantifying microbial fitness from highthroughput experiments. Applications of this theory to empirical questions about environmental variation would be best served by their own studies. We have added a statement clarifying this goal (lines 144–148).

We are unsure how the choice of reference subpopulation is related to this issue. In our view, if the goal of a mutant fitness measurement is to predict how that mutant would behave when arising spontaneously and competing against its immediate ancestor, the gold-standard reference subpopulation must always be the mutant’s immmediate ancestor, or another mutant that is known to be phenotypically equivalent to the ancestor (e.g., neutral mutants in the case of a large mutant library). Other choices of reference subpopulations would not provide directly meaningful information in this regard.

The authors overgeneralize some findings; for instance, the implications of fitness measurement choices could vary significantly across different microbes or experimental conditions. A more detailed discussion would strengthen the conclusion.

We certainly agree that the consequences of fitness quantification choices could vary significantly across organisms and environments; our goal for this paper is to demonstrate what discrepancies are possible in principle and in particular how they depend on basic features of microbial population dynamics (e.g., variation in yield). We have added two separate paragraphs in the Discussion section to address the generalizability of our results in the context of pairwise (lines 678–710) and bulk fitness measurements (lines 711–728).

Overall, this manuscript is a significant contribution to the field of evolutionary biology, addressing a critical issue in the quantification of fitness but lacks more experimental support to make it a wider claim. By systematically exploring the factors that influence fitness measurements, the authors provide valuable insights that can guide future research - the framework is computationally thorough but needs a more detailed explanation of concepts instead of generalizing.

We have improved our explanation of several of the important concepts. In particular, we have significantly revised our explanation of the population dynamics model (lines 284–310) to emphasize its role as a null model to demonstrate how fundamental aspects of microbial growth are sufficient to cause discrepancies between fitness statistics. We have also revised two paragraphs on the generalizability of our results in the Discussion section (lines 678–728).

Further work is needed, particularly to incorporate empirical examples and expand certain discussions to include environmental variation and their impact, which would improve clarity and applicability.

We have added a sentence at the beginning of the Results section to acknowledge the environmental dependence of fitness (lines 142–148). We believe further discussion of that issue is beyond the scope of this paper, as it would require a significant amount of additional data and/or environmental modeling.

Reviewer #2 (Recommendations for the authors):

In addition to the comments from the previous sections, other specific comments:

(1) Figure 5 needs to be populated with additional parameter details. For example, include brief descriptions of each parameter involved in the encoding, time scale, and reference choices. This will help users understand the implications of each choice. Adding these details will make the flow diagram more comprehensive, aiding researchers in implementing these steps more clearly.

Following this comment and another comment about this figure from Reviewer #3, we decided to replace this figure with a new Methods section with step-by-step instructions (lines 964–982).

(2) Duplication in Line 620: “Nevertheless, the fact that we see the fact that we see...” This redundancy needs to be corrected.

We thank the reviewer for pointing this out; we have rewritten this paragraph.

(3) More experimental data comparisons and their assessment concerning various microbial systems and multiple environmental conditions are recommended to support the claim.

Please see our responses to the related public comments.

Reviewer #3 (Public review):

Summary:

The authors present analyses of different fitness measures derived from empirical data from yeast knockout mutants and the long-term evolution experiment (LTEE) with Escherichia coli to explore discrepancies and identify preferred methods to estimate relative fitness in high-throughput experiments. Their work has three components. They first discuss the different “encodings” of relative abundance data and conclude that logit transformations are preferred because they transform nonlinear abundance trajectories into linear trajectories with greater predictive power. Next, they compare per-generation with per-growth cycle relative fitness estimates inferred from simulations of pairwise competitions based on published growth traits for the yeast strains and on published pairwise competition measurements for the LTEE data. Both data sets show quantitative and qualitative (i.e. rank order) discrepancies of estimates across different time scales, which are highlighted by considering possible underlying causes (i.e. trade-offs between growth traits) and consequences (i.e. epistasis among mutations affecting different growth traits). Finally, the authors compare simulated pairwise and bulk (i.e. where many mutants compete during a growth cycle in a single environment) competition assays based on the yeast knock-out mutants and demonstrate an optimal ratio of collective mutants to wild-type strains that minimizes both sampling error and overestimation of fitness estimates when compared with pairwise competitions.

Strengths:

The study deals with a highly relevant topic. Fitness is central to general evolutionary theory, but also poorly defined and implies different traits for different organisms and conditions. For microbes, which are often used in evolution experiments, high-throughput experiments may yield different measures to quantify abundance over time, from individual growth traits to bulk competition experiments. Hence, it is relevant to consider discrepancies among those measures and identify preferred measures with respect to predicting population dynamics and evolutionary processes. The present study contributes to this aim by (i) making readers aware of differences among commonly used fitness estimates, (ii) showing that simulated (yeast) and calculated (E. coli) competitive fitness may differ across time scales, and (iii) showing that bulk competitions may yield relative fitness estimates that are systematically higher than pairwise competitions. The study is rather thorough on the theory side, with extensive derivations and analyses of various fitness measures using their resource competition model in the Supplementary Information. The study ends with a few practical recommendations for preferred methods to infer relative fitness estimates, that may be useful for experimentalists and stimulate further investigations.

Weaknesses:

The study has several limitations. Perhaps the most apparent limitation is the lack of a clear answer to the question of which fitness measure is best “in the light of first principles”. The authors show clear discrepancies between fitness estimates across different time scales or using different reference genotypes in bulk competition and provide useful recommendations based on practical considerations (e.g. using pairwise competitions as the “golden standard”), but it remains unclear whether these measures provide the greatest value for the questions researchers may want to answer with them (e.g. predict shifts in genotype frequencies).

We agree on the importance of considering the scientific questions researchers want to answer in determining the best way to quantify fitness. We have revised both the Introduction (lines 82–88) and the Discussion (lines 615–630) to more clearly explain possible downstream questions researchers may wish to answer with fitness data, and thus why discrepancies in that data based on analysis choices may be important.

We believe that the text does provide a specific recommendation (second subsection of the Discussion, lines 635– 658) for how to quantify relative fitness: using the logit encoding (rather than other encodings), measuring fitness per-cycle (rather than per-generation), and using the wild-type or a phenotypically-equivalent proxy as reference subpopulation to calculate pairwise fitness in a bulk competition (rather than using the mutant library as a whole). This recommendation is based on first principles: the logit encoding is based on the principle of the logistic equation as the null model of relative abundance dynamics (lines 635–637), the choice of the per-cycle timescale is based on the principle that in non-steady state environments the time scale for measuring selection should not depend on the wild-type growth (lines 640–645), and the choice of reference population is based on the principle that a mutant’s fitness should serve as a predictor of its dynamics when arising de novo at low frequency and competing against its wild-type (lines 648–653).

A second limitation is that the authors analyse fitness differences arising solely from resource competition, whereas microbes often interact via other mechanisms, e.g. the production of anticompetitor toxins, cross-feeding of metabolites, or lack of growth to enhance their persistence in stress conditions. Without simulations of these processes, understanding discrepancies among fitness measures is necessarily limited.

We agree that other interactions are important in many microbial ecosystems and could affect measurements of fitness. We discuss the possibility of these other interactions and their potential consequences for fitness on lines 697– 710.

We focus on resource competition in this paper, however, for two reasons. One is that we are using it as a null model: resource competition is always present, and thus it provides an important baseline for discrepancies in fitness statistics in the absence of any other assumptions. Indeed, our results are that this minimal assumption alone is sufficient to produce a wide range of significant discrepancies, which provides the proof of principle that choices of fitness quantification matter. We have clarified this in a revised explanation of the population dynamics model on lines 294–304.

The second reason is that fitness measurements of the type discussed in this paper are typically performed on mutants that have only small genetic differences with their ancestor (e.g., a point mutation or gene deletion). While more complex interactions between such similar genotypes are not impossible, we expect them to be rare, in which case resource competition is the only interaction. Explicit modeling of other interactions is an important question for future work, but would require more detailed models and data of those phenomena, and thus would go beyond the scope of the present study. We have added a sentence to explain our emphasis on resource competition on lines 298–301 and 690–697.

In addition, the analysis of trade-offs between growth traits causing these discrepancies during resource competition seems confounded by biases in measurement error or parameter estimation, at least for growth rate and lag time (Figure 2B), where the replicate estimates for the wildtype show a similar negative correlation.

The tradeoff between growth traits was only an incidental observation and is not necessary for the fitness statistic discrepancies we analyze in this paper; the only important pattern in the growth traits is the existence of mutants with reduced yields (so as to reduce the wild-type log fold-change in a competition) as well as variation in one other trait under selection (lag time or growth rate in this model). We have clarified this mechanism on lines 328–336, which is demonstrated by Fig. S7. Since these tradeoffs are not relevant to the results and we agree that their significance may be unreliable due to the noisiness of the data, we have removed mention of them.

Third, the study does not validate relative fitness predictions from growth traits (as is done for the yeast mutants) with measured relative fitness estimates using competition assays, while such data are available, e.g. for the LTEE. This would strengthen their inferences about preferred fitness measures.

The goal of our modeling with the yeast growth trait data is not to test the ability to predict competition experiments from monoculture data; that has been the focus of previous studies [32, 34, 36, 37]. Rather, we use the population dynamics model for a proof of principle: that the most basic features of microbial population dynamics in laboratory experiments, as captured by this model (resource competition, lag phase, growth phase, saturation), are sufficient to create discrepancies between common fitness statistics used in these experiments (different encodings, time scales, choices of reference subpopulations). The yeast growth curve data merely provides realistic parameters for this model, to ensure we are studying a biologically relevant regime of the dynamics. To avoid this misconception, we have revised our explanation of this model and the data on lines 284–310.

Fourth, the analysis of epistasis between mutations affecting different growth traits (shown in Figure 3) based on the LTEE data could be better introduced and analysed more comprehensively. Now, the examples given in panels C-F seem rather idiosyncratic and readers may wonder how general these consequences of using fitness estimates based on different time scales are.

We agree that this analysis was incomplete and missed an opportunity to emphasize this important consequence of fitness quantification. We have thus expanded this analysis into a systematic test of all possible double mutants between qualitative types of trait perturbations in the model. We have added a new main text figure (Fig. 3), new SI figures (Figs. S9–S15), a new subsection in the Results (lines 346–395), and corresponding new sections in the Methods (lines 864–892) and SI (Sec. S8).

Finally, the study is generally less accessible to experimentalists due to the extensive and principled treatment of specific population dynamic models and fitness inferences. This may distract from the overarching aim to identify fitness measures that are most accurate and useful for predictions of population dynamics and evolutionary processes.

We appreciate this concern as we do hope to make the paper as broadly accessible as possible, especially to experimentalists who measure microbial fitness. To this end, we have reduced the technical discussion of encodings in the first section of the Results (lines 164–187); revised explanations of the population dynamics model (lines 284–310), importance of growth trait variation (lines 328–336), and epistasis (lines 346–395) to better emphasize the conceptual intuition of these parts; and added a step-by-step guide for our recommended best practices of quantifying fitness in bulk competition experiments (lines 964–982).

In this light, the motivation for the initial discussion of the importance of how to best encode relative abundance (Figure 1) is unclear. Also, the conclusion, that logit encoding is preferred, because it linearizes logistic growth dynamics and “improves the quality of predictions”, is not further motivated. Experimentalists using non-linear models to infer fitness from growth curves or competition assays may miss the relevance of this discussion.

The motivation for the discussion of encodings is that it is one of the choices made differently by researchers, mainly using either the logit (more common in experimental evolution and population genetics studies) or log encoding (more common in TnSeq analyses). As such we believe it is important to explain where this choice comes from (a transformation of relative abundance data to make it approximately linear in time, and thus amenable to characterization by a single slope parameter) and why we believe the logit encoding is more logical in most cases. We have streamlined and revised this subsection to make it clearer (lines 164–187).

Our argument for favoring the logit encoding in most cases is based on the logistic model being a null model for relative abundance dynamics (Sec. S3). In light of the reviewer’s comments, we have realized this may be confusing because there are two common usages of logistic dynamics that are biologically distinct. What we mean by logistic model is the dynamics of relative abundance x of a mutant in competition with other genotypes:

Here s turns out to be the relative fitness under the logit encoding. On the other hand, researchers also use a logistic ODE to describe the dynamics of absolute abundance N of a single strain in monoculture (e.g., as in a growth curve):

We believe the reviewer’s last point refers to Eq. (2), whereas our argument about the logit encoding is based on Eq. (1). We have added a note to clarify this distinction for the reader (lines 192–196).

Reviewer #3 (Recommendations for the authors):

In addition to my general comments in the public review, I have several more specific recommendations:

(1) Line 183-189: unclear why logit-based relative fitness is preferred. Abundance data are not typically binomial.

We agree this claim about abundance data was incorrect and have removed it. We have revised the section to focus on motivating the logit encoding from logistic dynamics of relative abundance as a null model for most systems (main text lines 175–187 and Sec. S3).

(2) Line 205: it may be mentioned that s(logit) is the same as the “selection rate constant” often used in microbial studies.

We have added a sentence clarifying the equivalence of the logit-encoded relative fitness to the selection coefficient in population genetics (lines 188–190).

(3) Line 368: why do mutations that increase biomass yield also increase WT LFC? Is this, because they grow slower and hence allow the WT more time to grow?

Mutants with higher yield allow the wild-type to achieve higher log fold-change because those mutants consume fewer resources per cell, which frees up more resources for the wild-type to consume and increase its overall growth. It’s not about growth rate or time, as this would occur even for mutants whose growth rates are identical to the wild-type’s. We have revised our explanation of how variation in growth traits differentially affects fitness statistics (lines 323–340) and epistasis (lines 361–378).

(4) Line 382-386: you may want to cite Ram et al. (2019, 10.1073/pnas.1902217116), who also did such analyses for experimental data from E. coli.

We have cited this work as Ref. [34].

(5) Line 415: perhaps use “bulk relative fitness” instead of “total relative fitness”, to contrast with “pairwise relative fitness”.

We acknowledge the language in this section can be subtle. However, “bulk” is not a sufficient identifier for the concept of total relative fitness as bulk competition experiments (with many genotypes competing simultaneously) can be used to measure either total relative fitness or pairwise relative fitness. (In pairwise competition experiments with only two genotypes, these two types of fitness are identical.) As such we adhere to our original language but have added words to clarify which type of experiment (bulk or pairwise) we are talking about in a given context (e.g., on lines 495–504).

(6) Line 451-453: why does a population in bulk competition consume resources more slowly than in pairwise competitions?

Mutant libraries used in bulk competition experiments usually include a large number of deleterious mutants, which grow more slowly than the wild-type. Thus these populations typically consume resources more slowly than a population in a pairwise competition would, where a large part of the population is the wild-type.

(7) Line 565: I don’t understand how one can compare relative fitness to other timescales.

Relative fitness, as we’ve defined it, has units of rate, since it describes the rate of change of relative abundance (or an encoding of it) over some time scale (e.g., a batch growth cycle or a generation). Therefore it can be compared to other times scales of the system, such the rate of new mutations arising or the rate of genetic drift fluctuations, as long as they are measured in the same units. This comparison is important to population genetics analyses, such as determining whether the population is in the strong selection-weak mutation limit or the clonal interference regime.

(8) Line 620 repeats text.

Thank you, we have revised this paragraph and removed the typo.

(9) Figure 1C+D: the link between the scenarios on the left and the graphs on the right may be better explained. For example, it may help to make explicit that the 4 scenarios in panel C show the same relative fitness per cycle and that mutant and wildtype have the same growth rate, but different growth periods in both scenarios in panel D. It is also unclear whether the grey dot links to the upper scenario in D.

We have clarified this issue in the caption and changed the colors to avoid this confusion.

(10) Figure 2E: it is unclear why “mutants with equal fitness are assigned the lowest rank”.

This was a technical comment about how to handle ties in our analysis of mutant rankings, but it is moot since no exact ties actually occur in our simulations. We have removed this remark to avoid confusion.

(11) Figure 2F: the axis labels are confusing, as for the WT estimates no LFC mutant exists. It would also help to make explicit in the legend against which WT replicate/reference strain each strain has competed.

We agree the inclusion of wild-type replicates in this plot was confusing and unnecessary, so we have removed them. The mutants compete against a wild-type with traits defined by their median values across all wild-type replicates; this is noted in Fig. 2A and the Methods section on our analysis of this data (lines 809–813).

(12) Figure 5: I am not sure this is needed, as its information is rather limited.

We agree and have removed this figure.

eLife Assessment

This is a valuable study presenting solid data indicating that the bacterial GTPases EngA and ObgE enable single-step reconstitution of functional 50S ribosomal subunits under near-physiological conditions. The study elegantly bridges the gap between the non-physiological aspects of the previous two-step reconstitution method and the extract-dependent iSAT system to enable assembly of highly functional ribosomes under translation-compatible conditions. The reported findings represent progress towards achieving a bottom-up reconstruction of the translation machinery from synthetic parts.

Reviewer #1 (Public review):

Summary:

This study presents evidence that addition of the two GTPases EngA and ObgE to reactions comprised of rRNAs and total ribosomal proteins purified from native bacterial ribosomes can bypass the requirements for non-physiological temperature shifts and Mg+2 ion concentrations for in vitro reconstitution of functional E. coli ribosomes.

Strengths:

This advance allows ribosome reconstitution in a fully reconstituted protein synthesis system containing individually purified recombinant translation factors, with the reconstituted ribosomes substituting for native purified ribosomes to support protein synthesis. This represents a significant development in the long-term effort to produce synthetic cells.

Weaknesses:

- The authors carried out additional experiments indicating that ~60% of the reconstituted ribosomes are functional and that a significant proportion are capable of synthesizing GFP from the correct initiation codon to the correct stop codon, and also of producing an enzymatically active protein at appreciable levels. Their SDS-PAGE and MS analyses of N-terminally tagged GFP are also quite useful but did not assess the frequency of initiation at the wrong start codon, termination at the incorrect stop codon, or the frequency of frameshifting during elongation. This would require examining additional reporters designed to examine dependence on a Shine-Dalgarno sequence or the impact of an in-frame stop codon to assess the fidelity of initiation and termination events, respectively, and one with a programmed frameshift site to assess the elongation fidelity of their reconstituted ribosomes.

- Reconstitution studies in the past have succeeded by using all recombinant, individually purified RPs that, if successful here, would have eliminated the possibility that one or more unknown ribosome assembly factors that co-purify with native ribosomes was added to their reconstitution reactions.

Reviewer #2 (Public review):

This study has developed a single-step method to assemble active bacterial ribosomes under near-physiological conditions by using the GTPase factors EngA and ObgE. These factors eliminate the need for the traditional, harsh manipulations of temperature and magnesium levels. This integration is an important step toward the bottom-up construction of synthetic cells.

Comments on revisions:

The authors have addressed my concerns in the previous round of review.

Author response:

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

eLife Assessment

This is a useful study presenting solid data indicating that the bacterial GTPases EngA and ObgE enable single-step reconstitution of functional 50S ribosomal subunits under near-physiological conditions. The study elegantly bridges the gap between the non-physiological aspects of the previous two-step reconstitution method and the extract-dependent iSAT system to enable ribosome assembly under translation-compatible conditions; however, it is limited by reliance on rRNA and proteins extracted from native ribosomes and does not achieve a true bottom-up reconstruction from all synthetic components. The evidence is incomplete in not characterizing the spectrum of reporter polypeptides produced and not comparing their rate and yield of synthesis from reconstituted ribosomes to that obtained with pure native ribosomes; and the impact of the study is limited by not including reporters to examine the fidelity of initiation, elongation or termination achieved with the reconstituted ribosomes.

As described below, based on the comments from the public reviewers, we have summarized at the end of the Discussion how this study contributes toward true bottom-up reconstruction from fully synthetic components, as well as the aspects that will require further development. In addition, we have newly provided data characterizing the reporter polypeptides from multiple perspectives, demonstrating that the assembled ribosomes do not exhibit issues such as reduced fidelity (Fig. 6, 7, Supplementary Data 2, 3). We believe that these data adequately address the limitations that were pointed out in the eLife Assessment.

Public Reviews:

Reviewer #1 (Public review):

This study presents evidence that the addition of the two GTPases EngA and ObgE to reactions comprised of rRNAs and total ribosomal proteins purified from native bacterial ribosomes can bypass the requirements for non-physiological temperature shifts and Mg+2 ion concentrations for in vitro reconstitution of functional E. coli ribosomes.

Strengths:

This advance allows ribosome reconstitution in a fully reconstituted protein synthesis system containing individually purified recombinant translation factors, with the reconstituted ribosomes substituting for native purified ribosomes to support protein synthesis. This work potentially represents an important development in the long-term effort to produce synthetic cells.

Weaknesses:

While much of the evidence is solid, the analysis is incomplete in certain respects that detract from the scientific quality and significance of the findings:

(1) The authors do not describe how the native ribosomal proteins (RPs) were purified, and it is unclear whether all subassemblies of RPs have been disrupted in the purification procedure. If not, additional chaperones might be required beyond the two GTPases described here for functional ribosome assembly from individual RPs.

Native ribosomal proteins (RPs) were prepared from native ribosomes, according to the well-established protocol described by Dr. Knud H. Nierhaus [Nierhaus, K. H. Reconstitution of ribosomes in Ribosomes and protein synthesis: A Practical Approach (Spedding G. eds.) 161-189, IRL Press at Oxford University Press, New York (1990)]. In this method, ribosome proteins are subjected to dialysis in 6 M urea buffer, a strong denaturing condition that may completely disrupt ribosomal structure and dissociate all ribosomal protein subassemblies. To make this point clear, we described the detailed ribosomal protein (RP) preparation procedure in the manuscript, rather than merely referring to the book.

In addition, we would like to clarify one point related to this comment. The focus of the present study is to show that the presence of two factors is required for single-step ribosome reconstitution under translation-compatible, cell-free conditions. We do not intend to claim that these two factors are absolutely sufficient for ribosome reconstitution. Hence, we have revised the manuscript to more explicitly state what this work does and does not conclude.

(2) Reconstitution studies in the past have succeeded by using all recombinant, individually purified RPs, which would clearly address the issue in the preceding comment and also eliminate the possibility that an unknown ribosome assembly factor that co-purifies with native ribosomes has been added to the reconstitution reactions along with the RPs.

As noted in the response to the Comment (1), the focus of the present study is the requirement of the two factors for functional ribosome assembly. Therefore, we consider that it is not necessary to completely exclude the possibility that unknown ribosome assembly factors are present in the RP preparation. Nevertheless, we agree that it is important to clarify what factors, if any, are co-present in the RP fraction. To address this, we performed proteomic analysis of the TP70 preparation (Supplementary Data 3) and stated the possibility of other factors’ inclusion.

We also agree that additional, as-yet-unidentified components, including factors involved in rRNA modification, could plausibly further improve assembly efficiency. We also consider that such studies may contribute to extending the system to the use of in vitro-transcribed rRNA and fully recombinant ribosomal proteins, which could be essentially a next step of this study. We noted the possibility of as-yet-unidentified components and the future perspectives in the Discussion.

(3) They never compared the efficiency of the reconstituted ribosomes to native ribosomes added to the "PURE" in vitro protein synthesis system, making it unclear what proportion of the reconstituted ribosomes are functional, and how protein yield per mRNA molecule compares to that given by the PURE system programmed with purified native ribosomes.

According to this suggestion, we measured the sfGFP synthesis rate from the increase in fluorescence over time under conditions where the template mRNA is in excess, and compared this rate directly between reconstituted and native ribosomes. We consider that this comparison provides insight into what fraction of ribosomes reconstituted in our system are functionally active (Fig. 6).

As noted in the provisional responses, quantifying protein yield per mRNA molecule is substantially more challenging. The translation system is complex, and the apparent yield per mRNA can vary depending on factors such as differences in polysome formation efficiency. In addition, the PURE system is a coupled transcription–translation setup that starts from DNA templates, which further complicates rigorous normalization on a per-mRNA basis. Because the main focus of this study is to determine how many functionally active ribosomes can be reconstituted under translation-compatible conditions, we addressed this comment by just carrying out the experiment comparing sfGFP synthesis rate.

(4) They also have not examined the synthesized GFP protein by SDS-PAGE to determine what proportion is full-length.

We have added an affinity tag to the sfGFP reporter, and then, purified the synthesized products from the reaction mixture and analyzed it by SDS–PAGE (Fig. 7a).

(5) The previous development of the PURE system included examinations of the synthesis of multiple proteins, one of which was an enzyme whose specific activity could be compared to that of the native enzyme. This would be a significant improvement to the current study. They could also have programmed the translation reactions containing reconstituted ribosomes with (i) total native mRNA and compared the products in SDS-PAGE to those obtained with the control PURE system containing native ribosomes; (ii) with specifc reporter mRNAs designed to examine dependence on a Shine-Dalgarno sequence and the impact of an in-frame stop codon in prematurely terminating translation to assess the fidelity of initiation and termination events; and (iii) an mRNA with a programmed frameshift site to assess elongation fidelity displayed by their reconstituted ribosomes.

Following the recommendation, we selected DHFR as an enzymatically active protein and used it as a reporter, confirming that it exhibited enzymatic activity comparable to that observed when synthesized by native ribosomes (Fig. 7c). In addition, MS analysis of the purified sfGFP used for SDS-PAGE analysis showed that nearly all peptide fragments were detected, covering almost the entire sequence from the initiator amino acid to the amino acid immediately preceding the stop codon (Fig. 7b, Supplementary Data 2. These results suggest that protein synthesis by the newly assembled ribosomes proceeds smoothly from initiation to termination, with no apparent problem in fidelity, and therefore indicate that functional ribosomes were successfully reconstituted.

Reviewer #2 (Public review):

This study presents a significant advance in the field of in vitro ribosome assembly by demonstrating that the bacterial GTPases EngA and ObgE enable single-step reconstitution of functional 50S ribosomal subunits under near-physiological conditions-specifically at 37 {degree sign}C and with total Mg²⁺ concentrations below 10 mM.

This achievement directly addresses a long-standing limitation of the traditional two-step in vitro assembly protocol (Nierhaus & Dohme, PNAS 1974), which requires non-physiological temperatures (44-50 {degree sign}C), and high Mg²⁺ concentrations (~20 mM). Inspired by the integrated Synthesis, Assembly, and Translation (iSAT) platform (Jewett et al., Mol Syst Biol 2013), leveraging E. coli S150 crude extract, which supplies essential assembly factors, the authors hypothesize that specific ribosome biogenesis factors-particularly GTPases present in such extracts-may be responsible for enabling assembly under mild conditions. Through systematic screening, they identify EngA and ObgE as the minimal pair sufficient to replace the need for temperature and Mg²⁺ shifts when using phenol-extracted (i.e., mature, modified) rRNA and purified TP70 proteins.

However, several important concerns remain:

(1) Dependence on Native rRNA Limits Generalizability

The current system relies on rRNA extracted from native ribosomes via phenol, which retains natural post-transcriptional modifications. As the authors note (lines 302-304), attempts to assemble active 50S subunits using in vitro transcribed rRNA, even in the presence of EngA and ObgE, failed. This contrasts with iSAT, where in vitro transcribed rRNA can yield functional (though reduced-activity, ~20% of native) ribosomes, presumably due to the presence of rRNA modification enzymes and additional chaperones in the S150 extract. Thus, while this study successfully isolates two key GTPase factors that mimic part of iSAT's functionality, it does not fully recapitulate iSAT's capacity for de novo assembly from unmodified RNA. The manuscript should clarify that the in vitro assembly demonstrated here is contingent on using native rRNA and does not yet achieve true bottom-up reconstruction from synthetic parts. Moreover, given iSAT's success with transcribed rRNA, could a similar systematic omission approach (e.g., adding individual factors) help identify the additional components required to support unmodified rRNA folding?

We fully recognize the reviewer’s point that our current system has not yet achieved a true bottom-up reconstruction. Although we intended to state this clearly in the manuscript, the fact that this concern remains indicates that our description was not sufficiently explicit. We therefore added the paragraph to ensure that this limitation is clearly communicated to readers.

(2) Imprecise Use of "Physiological Mg²⁺ Concentration"

The abstract states that assembly occurs at "physiological Mg²⁺ concentration" (<10 mM). However, while this total Mg²⁺ level aligns with optimized in vitro translation buffers (e.g., in PURE or iSAT systems), it exceeds estimates of free cytosolic [Mg²⁺] in E. coli (~1-2 mM). The authors should clarify that they refer to total Mg²⁺ concentrations compatible with cell-free protein synthesis, not necessarily intracellular free ion levels, to avoid misleading readers about true physiological relevance.

We agree that this is a very reasonable point and revised the manuscript to clarify that we are referring to the total Mg²⁺ concentration compatible with cell-free protein synthesis, rather than the intracellular free Mg²⁺ level under physiological conditions. We also changed the term “physiological” to “near-physiological” to avoid the misunderstanding.

In summary, this work elegantly bridges the gap between the two-step method and the extract-dependent iSAT system by identifying two defined GTPases that capture a core functionality of cellular extracts: enabling ribosome assembly under translation-compatible conditions. However, the reliance on native rRNA underscores that additional factors - likely present in iSAT's S150 extract - are still needed for full de novo reconstitution from unmodified transcripts. Future work combining the precision of this defined system with the completeness of iSAT may ultimately realize truly autonomous synthetic ribosome biogenesis.

Recommendations for the authors:

Reviewing Editor Comments:

Recommendations for improvement:

(1) Assess the length distribution of GFP polypeptides being produced using SDS-PAGE.

SDS-PAGE was performed according to the comment 4 of the Reviewer #1 (Fig. 7b). Please refer to our response addressing the comment.

(2) Compare the rate and yield of GFP synthesized per mRNA using their reconstituted ribosomes to that obtained with pure native ribosomes.

The efficiency of the reconstituted ribosomes was compared to native ribosomes according to the comment 3 of the Reviewer #1 (Fig. 6). Please refer to our response addressing the comment.

(3) Expand the panel of reporter mRNAs being examined to compare the fidelity of initiation, elongation or termination achieved with reconstituted ribosomes to that obtained using native ribosomes.

DHFR synthesis was addressed and also MS analysis of synthesized sfGFP was performed according to the comment 5 of the Reviewer #1 (Fig. 7b, c). Please refer to our response addressing the comment.

(4) Revise the manuscript to clarify that the in vitro assembly demonstrated here is contingent on using native rRNA and thus does not achieve a true bottom-up reconstruction from synthetic parts.

We added to the Discussion a paragraph summarizing the findings of this study, limitations, and future perspectives according to the comment 1 and 2 of the Reviewer #1 and the comment 1 of the Reviewer #2. Please refer to our responses addressing these comments.

(5) Revise the manuscript to clarify that they are referring to total Mg2+ concentrations compatible with cell-free protein synthesis, not necessarily intracellular free ion levels, to avoid misleading readers about the physiological relevance of the reconstitution.

We revised the manuscript to clarify this point according to the comment 2 of the Reviewer #2. Please refer to our response addressing the comment.

(6) Revise the text to fully describe how the native ribosomal proteins (RPs) were purified and indicate whether all subassemblies of RPs were disrupted in the purification procedure.

We revised the Methods section to clarify how the native RPs were purified and that all subassemblies of RPs were disrupted according to the comment 1 of the Reviewer #1.

(7) Revise the text to indicate that achieving ribosome reconstitutions using all recombinant, individually purified RPs is required to achieve a true bottom-up reconstruction from all synthetic components.

As with our response to the comment 4, we have added the point at the end of the Discussion as a future perspective toward true bottom-up reconstruction from all synthetic components.

(8) Consider conducting a similar systematic omission approach (e.g., adding individual factors) to help identify the additional components required to support unmodified rRNA folding.

As with our response to the comment 4 and 7, we have added the point at the end of the Discussion as a future perspective toward identification of additional essential factors for true bottom-up reconstruction.

Reviewer #1 (Recommendations for the authors):

(1) Assessing the spectrum of GFP polypeptides being produced by SDS-PAGE and comparing the rate and yield of GFP produced to that obtained with pure native ribosomes would seem to be essential additional measurements needed to bolster the evidence supporting the main conclusions of the work.

SDS-PAGE and MS analysis of the synthesized sfGFP were performed (Fig. 7a, b). Comparison of the assembled ribosomes and native ones were also performed (Fig. 6).

(2) Examining translation of other reporter mRNAs designed to compare the fidelity of initiation, elongation or termination achieved with reconstituted ribosomes to that produced by native ribosomes in the PURE system would be required to elevate the scientific quality of the work and its significance to the field.

DHFR synthesis and its activity measurement were performed (Fig. 7c). Also, MS analysis of the purified sfGFP showed that nearly all peptide fragments were detected, covering almost the entire sequence from the initiator amino acid to the amino acid immediately preceding the stop codon (Fig. 7b). We consider that these findings indicate that there is no apparent problem with fidelity.

eLife Assessment

This is an important study that develops multiple human iPSC-based models to study the consequences of DNMT3A mutations in Tatton-Brown-Rahman Syndrome. Convincing evidence shows dysregulation of GABAergic interneuron development and function, and the authors identify some of the key signaling mechanisms underlying these changes. This study will be of interest for understanding the functions of DNMT3A in brain development and the causes of neurological dysfunction in Tatton-Brown-Rahman Syndrome.

Reviewer #1 (Public review):

Summary:

This is an important study that describes the consequences of the DNMT3A mutation in human neuronal development for the first time. The selective impact of DNMT3A function on GABAergic interneurons is interesting and an important feature of future therapeutics. The claims made in that manuscript are supported by strong evidence for the most part. And the data are of high quality in general and presented well.

Strengths:

The strengths of the work include: Characterization of multiple DNMT3A loss-of-function alleles, including two misense variants, R882H, P904L, and a deletion allele. The missense mutation lines both include an ideal control with the same genetic background. The CRISPRi-mediated DNMT3A knockdown has also been included. The study identifies the mTOR-PI3K pathway as a factor of overgrowth issues found in the mutant organoid. In bulk mRNA sequencing and whole-genome bisulfite sequencing, identify hypomethylated genomic regions associated with gene expression repression. Again, this is more pronounced in the ventral organoid compared to the dorsal organoid. In addition, the extensive electrophysiological characterizations with a high-density microelectrode array support the more mature status of mutant interneurons.

Weaknesses:

Although a strong study overall, some weaknesses are noted. These include:

(1) The lack of validation data for the generated iPSCs and hESCs, such as the chromosomal contents, ploidy, and pluripotency states.

(2) Other weaknesses relate to data interpretation and insufficient discussion of related matters, as detailed in the recommendations to the authors.

(3) Also, some errors are noted and detailed in the recommendation section.

Reviewer #2 (Public review):

Summary:

Chapman, Determan et al. investigate how pathogenic mutations in DNMT3A, which cause Tatton-Brown-Rahman Syndrome (TBRS), disrupt human cortical developmental processes using a comprehensive panel of human pluripotent stem cell models spanning DNMT3A loss-of-function severity. The authors aim to identify the cellular and molecular mechanisms underlying TBRS-associated brain overgrowth and intellectual disability, and to test whether mechanistic convergence exists between TBRS and other overgrowth-intellectual disability disorders (OGIDs) caused by mutations in EZH2 (Weaver syndrome) or PIK3CA pathway components. Their central conclusion is that GABAergic interneuron development is selectively vulnerable to DNMT3A mutation, where reduced DNA methylation causes premature de-repression of neuronal and synaptic genes, driving precocious neuronal maturation and hyperactivity sufficient to disrupt neuronal network synchrony. This report adds to a growing literature supporting the vulnerability of GABAergic interneurons in NDDs and further provides a mechanistic view of this vulnerability, potentially convergent across OGIDs. The mechanistic claims around H3K27me3 compensation and mTOR-based therapeutic convergence, while promising, rest on more preliminary evidence and would benefit from the distinction between correlation and mechanism being made more explicit in the text. Overall, this is a compelling study with a rigorous experimental design and novel findings with a potential impact on a better understanding of the OGID pathophysiology.

Strengths:

(1) A major strength of this work is the breadth and rigor of the disease modeling approach. Four independent TBRS model systems are used in tandem: a patient-derived iPSC line with isogenic CRISPR-corrected control (R882H), a knock-in hESC model (P904L) with its wild-type isogenic, patient deletion iPSC lines (Del1/2), and CRISPRi knockdown models (G1/G2), collectively spanning a range of DNMT3A loss-of-function that correlates with phenotypic severity. This allelic series design substantially strengthens causal inference beyond what any single isogenic pair could provide.

(2) The multi-omic integration across matched developmental stages provides a strong mechanistic foundation for the cellular phenotyping and provides significantly enhanced novelty. RNA-seq, whole-genome bisulfite sequencing, and H3K27me3 CUT&Tag are combined in the same cell types, and timepoints show that DNMT3A loss reduces CG methylation at neuronal and synaptic gene loci, leading to premature transcriptional activation.

(3) The selective vulnerability of ventral (GABAergic) versus dorsal (glutamatergic) progenitors is one of the study's most important findings. This lineage specificity is consistently observed across all model systems and in both 2D and organoid formats, where ventral NPCs show increased proliferation, premature neuronal gene expression, and increased neurogenesis, while dorsal NPCs are largely unaffected at the transcriptomic and cellular level despite exhibiting comparable DNA methylation changes. This adds to a body of emerging work showing GABAergic interneuron vulnerability in NDDs where ubiquitously expressed genes such as chromatin modifiers are perturbed, and provides additional molecular insights into potential mechanisms of "resilience" of dorsal populations.

(4) The functional characterization follows a logical progression from single-neuron electrophysiology (demonstrating GABAergic hyperactivity with increased action potential amplitude and firing rate) to network-level analysis using high-density multi-electrode arrays. The HD-MEA experimental design - pairing TBRS or control GABAergic neurons with a constant background of control iGlut neurons - cleanly isolates GABAergic dysfunction as the driver of network hypersynchrony.

Weaknesses:

(1) The concomitant induction of proliferation and differentiation in TBRS V-NPCs is conceptually striking, since these are generally considered antagonistic developmental programs. The authors partially address this tension by noting that DNMT3A LOF alone is insufficient to initiate neuronal differentiation, i.e., V-NPCs upregulate neuronal and synaptic genes while retaining progenitor identity, implying that transcriptomic priming and commitment to differentiation are decoupled. However, the relationship between the proliferative phenotype and the epigenetic priming phenotype remains mechanistically unresolved. The manuscript documents mTOR pathway upregulation at the protein level and identifies shared DEGs that include proliferative regulators, but it does not establish whether mTOR-driven proliferation and mCG-loss-driven neuronal gene de-repression/enhanced differentiation are causally linked or represent two independent consequences of DNMT3A LOF.

(2) Relatedly, the rapamycin rescue experiment is a valuable proof-of-concept for the PIK3/AKT/mTOR convergence but is limited to a single dose in a single model (882) with a single readout (Ki67+ proliferation). Given the prominence of mTOR pathway convergence in the manuscript as a potential shared therapeutic avenue across OGIDs, the data supporting this claim are somewhat preliminary. It remains unknown whether mTOR inhibition rescues downstream phenotypes (neurogenesis, gene expression, neuronal maturation) or whether less severe TBRS models respond similarly. This might also help tackle the first comment above. e.g., if mTOR inhibition rescued proliferation but not the transcriptomic priming, that would support two independent mechanisms.

(3) The claim that H3K27me3 compensates for mCG loss is an important mechanistic point, but the current data do not distinguish between active compensation, in which EZH2 is recruited in response to methylation loss, and functional redundancy, in which H3K27me3 is independently established and becomes the dominant repressive mark once DNA methylation is reduced. The EZH2 knockdown/inhibition experiments show that H3K27me3 is sufficient to maintain repression at hypo-DMR sites, but they do not establish that H3K27me3 gain is itself a response to methylation loss. Because H3K27me3 profiling was performed only in the severe 882 model, it is also unclear whether H3K27me3 gain scales with DNMT3A LOF severity, as a compensatory model would predict. Finally, the EZH2 overexpression rescue is performed in V-NPCs, whereas the compensation model is developed primarily in D-NPCs, making it difficult to assess whether the same mechanism operates in the lineage where it was originally inferred.

(4) The narrative framing of dorsal neuron development as unaffected by DNMT3A LOF is somewhat at odds with the data presented. The 882 D-NPCs show substantial DNA methylation changes, and TBRS D-INs exhibit what the authors describe as "substantive transcriptomic differences" involving persistent expression of pluripotency and progenitor genes, which seems to be a distinct but potentially significant phenotype. The impact of DNMT3A loss between ventral and dorsal lineages might be more accurately framed as divergent in nature rather than specific to a certain population.

(5) SST stainings are not entirely convincing. They appear mostly nuclear, and some instances localized to rosettes in organoids, whereas the protein is largely confined to processes and is expected to be found outside progenitor-rich zones like rosettes.

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors investigated TBRS etiology by using new human pluripotent stem cell models, modeling varying levels of TBRS-associated loss of DNMT3A function. They identified increased lineage-specific proliferation of precursors in TBRS ventral MGE-like progenitors, which they propose was related to increased signaling through the PIK3/AKT/mTOR pathway. Furthermore, they show that reduced DNA methylation during MGE-like progenitor differentiation into GABAergic interneurons can cause a premature expression of neuronal and synaptic genes, triggering precocious neuronal maturation. In conclusion, they propose that TBRS-derived GABAergic neurons exhibit hyperactivity that can alters the development and structure of neuronal networks.

Strengths:

Overall, the data presented is convincing, from an early developmental point of view, given that the iPSC-derived 2D cultures or organoids used do not get to reach a mature state. Nonetheless, the data clearly show the effects that deleterious mutations in TBRS can cause during the period of neurogenesis, which was missing in the field.

Weaknesses:

(1) Li et al., 2022 (referred to in the manuscript) seems to already show the interplay between H3K27me3 and Dnmt3a discussed in this study i.e., that in the absence of DNA methylation, there is an expansion of polycomb-like repression. These data should be better acknowledged in the paragraph 'Repressive H3K27me3 compensates for severe loss of DNA methylation' (page 9), given it supports the data presented in this manuscript and suggests this as a common mechanism in the interplay between these two repressive marks, as it is well established in the literature.

(2) The authors should acknowledge that the omics data come from a mixed population of cells.

(3) The authors are encouraged to further discuss whether the overgrowth observed in ventral GABAergic cultures or organoids compares to the overgrowth observed in diseased patients. One expects MRIs to have been performed in patients and that these could be harnessed to discern if overgrowth occurs in the cortex or ventral regions of the brain.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

This is an important study that describes the consequences of the DNMT3A mutation in human neuronal development for the first time. The selective impact of DNMT3A function on GABAergic interneurons is interesting and an important feature of future therapeutics. The claims made in that manuscript are supported by strong evidence for the most part. And the data are of high quality in general and presented well.

Strengths:

The strengths of the work include: Characterization of multiple DNMT3A loss-of-function alleles, including two misense variants, R882H, P904L, and a deletion allele. The missense mutation lines both include an ideal control with the same genetic background. The CRISPRi-mediated DNMT3A knockdown has also been included. The study identifies the mTOR-PI3K pathway as a factor of overgrowth issues found in the mutant organoid. In bulk mRNA sequencing and whole-genome bisulfite sequencing, identify hypomethylated genomic regions associated with gene expression repression. Again, this is more pronounced in the ventral organoid compared to the dorsal organoid. In addition, the extensive electrophysiological characterizations with a high-density microelectrode array support the more mature status of mutant interneurons.

Weaknesses:

Although a strong study overall, some weaknesses are noted. These include:

(1) The lack of validation data for the generated iPSCs and hESCs, such as the chromosomal contents, ploidy, and pluripotency states.

We thank the reviewer for their constructive feedback. We previously validated our 882 models with whole genome sequencing and teratoma formation upon mouse fat pad injection, while the parental human embryonic stem cell line (WA01 hESCs) used for P904L variant knock-in was validated by our Genome Engineering Stem Cell (GESC) core upon derivation of that variant knock-in model. We have now added both karyotyping and pluripotency staining (SOX2/OCT4) for all other hPSC lines as (new) Supplementary Figure S17 and included further description in our Methods section under “hPSC Model Generation and Culture”.

New Data: Supplemental Figure S17 (SOX2/OCT4 staining in hPSCs and karyotyping of all lines used)

Text edits: Additional language confirming hPSC line validation will be added to the Methods section under “hPSC Model Generation and Culture” on page 18.

(2) Other weaknesses relate to data interpretation and insufficient discussion of related matters, as detailed in the recommendations to the authors.

We thank the reviewer for their insightful suggestions and have detailed our responses in the “recommendations to the authors” section.

(3) Also, some errors are noted and detailed in the recommendation section.

We thank the reviewer for catching these errors and have since corrected them, with detailed responses below.

Reviewer #2 (Public review):

Summary:

Chapman, Determan et al. investigate how pathogenic mutations in DNMT3A, which cause Tatton-Brown-Rahman Syndrome (TBRS), disrupt human cortical developmental processes using a comprehensive panel of human pluripotent stem cell models spanning DNMT3A loss-of-function severity. The authors aim to identify the cellular and molecular mechanisms underlying TBRS-associated brain overgrowth and intellectual disability, and to test whether mechanistic convergence exists between TBRS and other overgrowth-intellectual disability disorders (OGIDs) caused by mutations in EZH2 (Weaver syndrome) or PIK3CA pathway components. Their central conclusion is that GABAergic interneuron development is selectively vulnerable to DNMT3A mutation, where reduced DNA methylation causes premature de-repression of neuronal and synaptic genes, driving precocious neuronal maturation and hyperactivity sufficient to disrupt neuronal network synchrony. This report adds to a growing literature supporting the vulnerability of GABAergic interneurons in NDDs and further provides a mechanistic view of this vulnerability, potentially convergent across OGIDs. The mechanistic claims around H3K27me3 compensation and mTOR-based therapeutic convergence, while promising, rest on more preliminary evidence and would benefit from the distinction between correlation and mechanism being made more explicit in the text. Overall, this is a compelling study with a rigorous experimental design and novel findings with a potential impact on a better understanding of the OGID pathophysiology.

Strengths:

(1) A major strength of this work is the breadth and rigor of the disease modeling approach. Four independent TBRS model systems are used in tandem: a patient-derived iPSC line with isogenic CRISPR-corrected control (R882H), a knock-in hESC model (P904L) with its wild-type isogenic, patient deletion iPSC lines (Del1/2), and CRISPRi knockdown models (G1/G2), collectively spanning a range of DNMT3A loss-of-function that correlates with phenotypic severity. This allelic series design substantially strengthens causal inference beyond what any single isogenic pair could provide.

(2) The multi-omic integration across matched developmental stages provides a strong mechanistic foundation for the cellular phenotyping and provides significantly enhanced novelty. RNA-seq, whole-genome bisulfite sequencing, and H3K27me3 CUT&Tag are combined in the same cell types, and timepoints show that DNMT3A loss reduces CG methylation at neuronal and synaptic gene loci, leading to premature transcriptional activation.

(3) The selective vulnerability of ventral (GABAergic) versus dorsal (glutamatergic) progenitors is one of the study's most important findings. This lineage specificity is consistently observed across all model systems and in both 2D and organoid formats, where ventral NPCs show increased proliferation, premature neuronal gene expression, and increased neurogenesis, while dorsal NPCs are largely unaffected at the transcriptomic and cellular level despite exhibiting comparable DNA methylation changes. This adds to a body of emerging work showing GABAergic interneuron vulnerability in NDDs where ubiquitously expressed genes such as chromatin modifiers are perturbed, and provides additional molecular insights into potential mechanisms of "resilience" of dorsal populations.

(4) The functional characterization follows a logical progression from single-neuron electrophysiology (demonstrating GABAergic hyperactivity with increased action potential amplitude and firing rate) to network-level analysis using high-density multi-electrode arrays. The HD-MEA experimental design - pairing TBRS or control GABAergic neurons with a constant background of control iGlut neurons - cleanly isolates GABAergic dysfunction as the driver of network hypersynchrony.

Weaknesses:

(1) The concomitant induction of proliferation and differentiation in TBRS V-NPCs is conceptually striking, since these are generally considered antagonistic developmental programs. The authors partially address this tension by noting that DNMT3A LOF alone is insufficient to initiate neuronal differentiation, i.e., V-NPCs upregulate neuronal and synaptic genes while retaining progenitor identity, implying that transcriptomic priming and commitment to differentiation are decoupled. However, the relationship between the proliferative phenotype and the epigenetic priming phenotype remains mechanistically unresolved. The manuscript documents mTOR pathway upregulation at the protein level and identifies shared DEGs that include proliferative regulators, but it does not establish whether mTOR-driven proliferation and mCG-loss-driven neuronal gene de-repression/enhanced differentiation are causally linked or represent two independent consequences of DNMT3A LOF.

We thank the reviewer for their comment and agree that this phenotype, whereby progenitors exhibited both increased proliferation and hallmarks of gene expression associated with neuronal differentiation is striking and interesting, given that these are typically antagonistic paradigms during normal development.

We documented that these phenotypes involve upregulated expression of both neuronal/synaptic and proliferative genes in V-NPCs (Figure 2d), with concomitant loss of repressive DNA methylation at regulatory elements associated with these genes (Figure 2f, Supplemental Data 5). In this work, DNMT3A mutation had a more prominent role in de-repressing neuronal and synaptic gene expression to promote hallmarks of neuron differentiation, while playing a relatively less central role in direct regulation of proliferation genes, as seen from the relative prominence of neuronal/synaptic- versus proliferation-related GO terms in our Supplemental Data 5 table.

To examine the mechanisms underlying increased V-NPC proliferation in our TBRS models, we assessed a potential relationship with the PIK3/AKT/mTOR pathway, as this is implicated in increased proliferation resulting from DNMT3A-associated mutation in myeloid leukemia (Dai et al., 2017, PMID: 28461508). In our work, DNMT3A mutation increased the expression and/or phosphorylation of mTOR signaling pathway targets specifically in V-NPCs (Figure 1q-r, Supplemental Figure S3a-d). However, while TBRS mutation directly affected repressive DNA methylation at a suite of cell proliferation-related genes, these did not include the PIK3/AKT/mTOR pathway genes themselves, suggesting an indirect relationship between altered DNA methylation and increased mTOR signaling.

Text Edits: We will incorporate further discussion of how DNMT3A-mediated gene repression and levels of PIK3/AKT/mTOR pathway signaling may be interacting, providing a framework for future studies to identify how these related OGID gene mutations may converge mechanistically.

(2) Relatedly, the rapamycin rescue experiment is a valuable proof-of-concept for the PIK3/AKT/mTOR convergence but is limited to a single dose in a single model (882) with a single readout (Ki67+ proliferation). Given the prominence of mTOR pathway convergence in the manuscript as a potential shared therapeutic avenue across OGIDs, the data supporting this claim are somewhat preliminary. It remains unknown whether mTOR inhibition rescues downstream phenotypes (neurogenesis, gene expression, neuronal maturation) or whether less severe TBRS models respond similarly. This might also help tackle the first comment above. e.g., if mTOR inhibition rescued proliferation but not the transcriptomic priming, that would support two independent mechanisms.

We thank the reviewer for their comment. We explored both the overall levels and phosphorylation of proteins involved in PIK3/AKT/mTOR signaling in the 882, 904, Del1, Del2, and KO V-NPC models (Figure 1q-r, Supplementary Figure S3a-d), finding specific increases of all proteins. We showed that rapamycin addition reversed the increased proportion of KI67+ proliferating cell nuclei resulting from 882 mutation in V-NPCs in main Figure 1s, while demonstrating that rapamycin also reduced the proportion of KI67+ nuclei observed in both less severe 904 and Del1 V-NPC models (Supplementary Figure S3e-f).

We agree that understanding whether rapamycin treatment can rescue TBRS neuronal phenotypes would be very interesting, as previous work on Tuberous Sclerosis Complex has utilized rapamycin and other mTOR inhibitors to effectively reverse TSC-related alterations of neuronal morphology and neuronal hyperexcitability (Buttermore et al., 2025, PMID: 40792287). Future studies examining convergent mechanisms and therapeutics for OGIDs should examine how similarly targeting this and related pathways rescues altered neuronal morphology, maturation, and function, as we have demonstrated that TBRS mutation has subsequent consequences for V-IN differentiation, maturation, and function. This point has been detailed in the discussion section on pages 15-16.

(3) The claim that H3K27me3 compensates for mCG loss is an important mechanistic point, but the current data do not distinguish between active compensation, in which EZH2 is recruited in response to methylation loss, and functional redundancy, in which H3K27me3 is independently established and becomes the dominant repressive mark once DNA methylation is reduced. The EZH2 knockdown/inhibition experiments show that H3K27me3 is sufficient to maintain repression at hypo-DMR sites, but they do not establish that H3K27me3 gain is itself a response to methylation loss. Because H3K27me3 profiling was performed only in the severe 882 model, it is also unclear whether H3K27me3 gain scales with DNMT3A LOF severity, as a compensatory model would predict. Finally, the EZH2 overexpression rescue is performed in V-NPCs, whereas the compensation model is developed primarily in D-NPCs, making it difficult to assess whether the same mechanism operates in the lineage where it was originally inferred.

We thank the reviewer for the opportunity to clarify our findings and experimental reasoning. A previous study using a conditional Dnmt3a knockout mouse model (Li et al., 2022, PMID: 35604009) demonstrated increased expression of multiple PRC2 components following the loss of Dnmt3a. This study demonstrated that sites which lost DNA methylation gained H3K27me3 in postnatal neurons upon Dnmt3a loss. Therefore, we hypothesize that the gain of H3K27me3 likely occurs in response to loss of DNMT3A methylation.

While we did not perform CUT&Tag for H3K27me3 in our less severe models, we did validate gene expression changes following EZH2 knockdown and inhibition in both the R882H (Figure 4g-h) and P904L (Supplementary Figure S8b) models, finding that gene expression was unchanged in the model with the less severe DNMT3A mutation (P904L). Based upon these findings, we hypothesized that compensatory H3K27me3 may occur only upon severe DNMT3A loss, as seen in the dominant-negative R882H model. Furthermore, as H3K27me3 compensation was more prominent in D-NPCs, we hypothesized that this might be sufficient to prevent de-repression and aberrant neuronal gene repression upon loss of DNMT3A-mediated repression in D-NPCs. However, since TBRS mutation caused the most prominent de-repression of neuronal gene expression in V-NPCs, we also tested whether EZH2 overexpression could reverse this, finding that it partially suppressed this dysregulated neuronal gene expression. To better clarify this logic and the findings, we will make text edits to this results section.

Text edits: We will clarify the reasoning for performing the EZH2 overexpression experiments in V-NPCs and reference Li et al., 2022 in both the results (pg. 9-10) and discussion.

(4) The narrative framing of dorsal neuron development as unaffected by DNMT3A LOF is somewhat at odds with the data presented. The 882 D-NPCs show substantial DNA methylation changes, and TBRS D-INs exhibit what the authors describe as "substantive transcriptomic differences" involving persistent expression of pluripotency and progenitor genes, which seems to be a distinct but potentially significant phenotype. The impact of DNMT3A loss between ventral and dorsal lineages might be more accurately framed as divergent in nature rather than specific to a certain population.

We thank the reviewer for their comment. While TBRS mutations appear to have a significantly stronger effect on V-NPCs and subsequently V-INs, both transcriptomic and methylation alterations do also occur upon TBRS mutation in D-NPCs and D-INs, as noted in Supplemental Figure S4d, S11, and Supplemental Data 2. However, we observed substantially greater molecular alterations in V-NPCs/V-INs, a lack of overt cellular phenotypes in D-NPCs where assayed, and a lack of functional consequences in matured D-INs, suggesting a more significant requirement for DNMT3A in regulating the differentiation and subsequent maturation of cortical inhibitory interneurons during embryonic and early pre-natal development, the developmental periods that we can readily model in hPSC-derived neurons.

It should also be noted that these hPSC differentiation models do not recapitulate post-natal deposition of non-CpG (mCA) DNA methylation, a mechanism disrupted postnatally by TBRS-associated mutations in our prior work in murine models (Harrison Gabel; e.g. Beard et al., 2023, PMID: 37952155). Therefore, we hypothesize that if we could sufficiently mature D-INs to a state that modeled postnatal development and recapitulated this non-CpG methylation, we might be able to detect cellular and functional phenotypes in later stage D-INs. To avoid misinterpretation, we will alter the language in the results section to confirm that there are both transcriptomic and methylation changes in our D-NPCs/D-INs, but that these are not accompanied by cellular phenotypes or neuronal dysfunction.

Text edits: We will better clarify that there are transcriptomic and methylation changes in D-NPCs/D-INs, but that these changes are minimal compared to those in V-NPCs/V-INs, as supported by the lack of cellular and functional phenotypes seen in D-NPCs/D-INs.

(5) SST stainings are not entirely convincing. They appear mostly nuclear, and some instances localized to rosettes in organoids, whereas the protein is largely confined to processes and is expected to be found outside progenitor-rich zones like rosettes.

We agree that the perinuclear SST staining detected in these young ventral telencephalic-patterned organoids at day 30 differs somewhat from the more process-localized and cytosolic signal seen in later stage organoids in other studies. This may be related to the use of different commercial SST antibodies across studies but also likely reflects SST immunoreactivity in newborn neurons near the onset of SST expression. For example, immature SST-immunoreactive neurons in the early postnatal rat cortex exhibit predominant SST staining in perinuclear cytoplasm and short processes (e.g. Fig. 3 in Lee et al, PMID: 9664223) while acquiring more cytosolic and process-localized staining as postnatal neuron maturation occurs. Evaluation of immunopositivity for other markers of neurogenesis (ASCL1) and immature neurons (TUJ1) is also congruent with these findings for SST, with TBRS-associated mutations increasing in the fraction of cells in V-NPCs/V-ORGs that express these three markers.

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors investigated TBRS etiology by using new human pluripotent stem cell models, modeling varying levels of TBRS-associated loss of DNMT3A function. They identified increased lineage-specific proliferation of precursors in TBRS ventral MGE-like progenitors, which they propose was related to increased signaling through the PIK3/AKT/mTOR pathway. Furthermore, they show that reduced DNA methylation during MGE-like progenitor differentiation into GABAergic interneurons can cause a premature expression of neuronal and synaptic genes, triggering precocious neuronal maturation. In conclusion, they propose that TBRS-derived GABAergic neurons exhibit hyperactivity that can alters the development and structure of neuronal networks.

Strengths:

Overall, the data presented is convincing, from an early developmental point of view, given that the iPSC-derived 2D cultures or organoids used do not get to reach a mature state. Nonetheless, the data clearly show the effects that deleterious mutations in TBRS can cause during the period of neurogenesis, which was missing in the field.

Weaknesses:

(1) Li et al., 2022 (referred to in the manuscript) seems to already show the interplay between H3K27me3 and Dnmt3a discussed in this study i.e., that in the absence of DNA methylation, there is an expansion of polycomb-like repression. These data should be better acknowledged in the paragraph 'Repressive H3K27me3 compensates for severe loss of DNA methylation' (page 9), given it supports the data presented in this manuscript and suggests this as a common mechanism in the interplay between these two repressive marks, as it is well established in the literature.

We thank the reviewer for this suggestion and will incorporate this reference into both the results and the discussion when discussing the respective roles of DNMT3A and PCR2-mediated repression.

Text edits: We will add Li et al., 2022 to both the results section (pg. 9-10) and our discussion section.

(2) The authors should acknowledge that the omics data come from a mixed population of cells.

We thank the reviewer for their comment. We have validated that the established 2-D differentiation methods we used in this study generate cell populations with >85-90% enrichment for the desired progenitor and neuronal cell type, based upon marker expression, but acknowledge that these are bulk -omics data obtained from cells that may represent a mixed population and have now detailed this in the methods section under “Sequencing”.

Text edits: we will add language acknowledging that our omics data (bulk) was generated from mixed populations of cells.

(3) The authors are encouraged to further discuss whether the overgrowth observed in ventral GABAergic cultures or organoids compares to the overgrowth observed in diseased patients. One expects MRIs to have been performed in patients and that these could be harnessed to discern if overgrowth occurs in the cortex or ventral regions of the brain.

We thank the reviewer for their suggestion and do note that at least one published study documents increased cortical thickness in the MRIs of TBRS patients (Jiménez de la Peña et al., 2024, PMID: 37795572); however, to our knowledge studies have not examined regional or cell type-selective overgrowth of cortical tissue in TBRS patients. Future clinical studies examining the nature of the neuronal progenitor overgrowth and resulting consequences for patient brain imaging would be of interest to better understand TBRS-associated etiology of brain overgrowth and its manifestations.

eLife Assessment

This is a useful study investigating the role of peristalsis in the elongation of the gut, using the chick ceca as a model. The work employs optogenetics together with embryological approaches to establish links between peristaltic muscle contractions and downstream cell behaviors that lead to tube elongation. However, the work is somewhat incomplete, limited in mechanistic insights that would extend beyond prior work in the literature, which has already suggested a role for smooth muscle contractility in avian gut elongation.

Reviewer #1 (Public review):

Kawamura et al. investigated the role of circumferential smooth muscle contractions in chick gut tube elongation, addressing the hypothesis that "peristaltic activity generated by the gut promotes its own elongation during embryogenesis". Although not acknowledged in the current manuscript, this interesting premise was, in fact, previously demonstrated.

Indeed, the experiments in the present manuscript closely parallel a previous study (Khalipina et al, 2019: "Smooth muscle contractility causes the gut to grow anisotropically") that also cultured chick gut tissue and performed time-lapse analyses to quantify peristalsis. Both studies showed that inhibiting peristalsis with Ca-channel blockers induces a switch from elongational to radial growth in the gut.

However, one of the main strengths of the current study is the innovative use of optogenetic manipulation to rescue gut lengthening in drug-inhibited gut tissue by re-stimulating peristaltic contractions. In addition, the authors use aphidicolin to show that peristalsis-mediated gut elongation is independent of cell division. They also track individual smooth muscle cells and show that they divide circumferentially, but become redistributed along the length of the gut tube with peristalsis.

While these data are solidly quantitative, they do not provide mechanistic insight into how peristaltic contractions cause smooth muscle cells to be redistributed.

The evidence presented in this manuscript supports the main conclusion that peristalsis plays a critical role in embryonic gut elongation, but this conclusion itself is not novel. In addition to corroborating previous work, this manuscript provides some useful additions to our existing knowledge of the role of mechanical forces in embryonic gut morphogenesis and illustrates the utility of a previously published optogenetic manipulation technique.

Reviewer #2 (Public review):

Summary:

This study uses the chicken caecum ex vivo culture to show that embryonic peristaltic activity is a key mechanical factor for gut elongation. It is shown that pharmacological inhibition arrests intestinal growth, while optogenetic restoration rescues longitudinal elongation. The authors propose a two-step mechanism in which circular smooth muscle cells proliferate circumferentially, but peristalsis pushes them toward longitudinal rearrangement, which explains the anisotropic growth of the gut.

Strengths:

The experiments combine loss-of-function (peristalsis inhibition) with gain-of-function (optogenetic rescue) experiments and quantifiable readouts in an embryonic gut culture model. The work is clearly presented with nice microscopy videos and offers a potentially valuable conceptual framework linking tissue-scale mechanics to smooth muscle cell behaviors during development.

Weaknesses:

Some results appear conceptually inconsistent with the claim of peristalsis-essential rearrangement (e.g., longitudinal separation of daughter cells even without peristalsis), and the mechanistic link would benefit from clearer quantification and reconciliation. The study largely overlooks contributions from other gut layers and the ECM (and aphidicolin affects all proliferating cells), limiting interpretation of how smooth muscle rearrangement translates into whole-wall elongation.

Reviewer #3 (Public review):

Summary:

The authors noted a steep increase in the rate of growth with the onset of more frequent peristaltic-like movements and hypothesized that peristaltic activity rearranges the orientation of cell growth from circumferential to longitudinal. This study sought to alter peristalsis and then (1) carefully examine the growth of the chick cecum relative to the frequency of peristaltic-like movements and (2) examine the orientation of cells relative to the circumferential and longitudinal axes to determine whether peristalsis is required for cecum lengthening. To alter peristaltic-like movements, contraction was inhibited through treatment with nifedipine (a calcium channel blocker that acts to relax smooth muscle) or Ani9 (inhibits Ca-activated chloride channels), and contractions were induced through activation of a blue light-activatable channel rhodopsin 2 (introduced through electroporation).

Strengths:

(1) Use of multiple methods to alter peristalsis in initial studies.

(2) Live imaging.

(3) Careful measurements.

(4) Nicely presented figures.

Weaknesses:

(1) Only Nifedipine inhibition was examined for cell positional changes.

(2) Ki67 was not carefully analysed, and apoptosis was not shown at all.

(3) The results shown are suggestive of a role for peristalsis in the lengthening of the cecum. Demonstration that increased peristalsis could further increase lengthening would be helpful.

(4) The novelty of this work is incremental for the field in that the reagents used and the model of smooth muscle driving gut lengthening in mouse and chick small intestines have both previously been published. This manuscript does suggest that the role of smooth muscle in longitudinal growth may extend to other tubular organs (chick cecum).

Author response:

We sincerely appreciate the efforts of the Senior and Reviewing Editors, as well as the three reviewers, for their careful evaluation of our manuscript and their insightful comments. Previous studies have suggested that smooth muscle activity contributes to gut elongation; however, these studies do not directly demonstrate that peristaltic movements per se drive elongation. For example, studies in mouse have primarily focused on residual stress of smooth muscle (Yang et al., 2021), rather than the dynamic spatiotemporal nature of peristalsis. In chickens, inhibition of peristalsis by nifedipine has been interpreted as evidence for a role of peristalsis in gut elongation (Khalipina et al., 2019). However, because nifedipine broadly affects calcium-dependent cellular processes, these experiments cannot distinguish whether the observed effects arise specifically from loss of peristalsis or from other cellular perturbations. In our current study, we aimed to challenge this limitation by combining pharmacological inhibition with optogenetic reactivation. This approach allows us to selectively restore peristaltic movements under conditions in which endogenous peristalsis are suppressed. Based on these experiments, we provide evidence supporting a causal contribution of peristalsis to the anisotropic gut growth. We agree with the reviewers that the positioning of our study relative to previous work should be clarified. In a revised manuscript, we will more clearly distinguish between static mechanical tension and endogenous peristaltic movements, and better define the conceptual advance of our study. In addition to macroscopic growth analysis, we identified cellular dynamics associated with elongation, including circumferentially oriented cell division and peristalsis-dependent longitudinal cell rearrangement. We agree that the mechanistic link between peristalsis and downstream cellular behaviors remains incompletely understood. In the revised manuscript, we will clarify this limitation and outline future directions, including experiments to test the role of mechanical cues (e.g., mechanical perturbation and pharmacological manipulation of mechanotransduction pathways).

Public Reviews:

Reviewer #1 (Public review):

The mechanism by which peristalsis and the cell rearrangement are mediated

We appreciate this important point. As suggested, the possibility that mechanical aspects of peristalsis contribute to the gut elongation is highly plausible. To address this, we plan to perform additional experiments aimed at isolating the mechanical component of peristalsis. Furthermore, we will investigate the involvement of mechanotransduction pathways, including Piezo-mediated pathway, using pharmacological approaches. We will revise the manuscript to better discuss these possibilities and clarify the current limitations of our study.

The novelty and positioning of our study

We appreciate this comment and have addressed this point in the General response above. In the revised manuscript, we will more clearly position our study relative to the previous studies.

Reviewer #2 (Public review):

Longitudinal separation of daughter cells even without peristalsis

We appreciate this insightful and important comment. As noted, daughter cells can exhibit longitudinal separation even under nifedipine treatment, whereas the divergence index (DI) shows a clear increase only in the control (with peristalsis) condition. We interpret this as follows; immediately after cell division, two daughter cells occupy nearly identical positions along the longitudinal axis, and stochastic fluctuations may cause them to separate each other. Such local separation does not necessarily reflect population-level cell rearrangement. In contrast, DI captures collective dispersion of a cell population, which reflects organized tissue-level rearrangement associated with elongation. We will revise the manuscript to clarify this distinction between local cell behavior and population-level dynamics, and to better explain how DI reflects elongation-related processes.

Contributions from other gut layers and ECMs

We agree that contributions from other tissue layers and extracellular matrix (ECM) components might be important. To address this, we plan additional experiments including targeted ablation of specific tissue layers and pharmacological manipulation of ECM remodeling (e.g., using MMP modulators). We will also expand the Discussion to better acknowledge these factors.

Reviewer #3 (Public review):

(1) We agree that experiments based solely on nifedipine treatment cannot fully exclude potential off-target effects. To address this limitation, we plan to perform additional experiments that rescue the mis-rearrangement of cells by applying mechanical forces.

(2) We agree that more elaborate analyses of cell proliferation and apoptosis are needed. In the revised manuscript, we will incorporate additional analyses using appropriate markers and methods suitable for developing gut tissue.

(3) In Figure 2, we had already shown an increased the frequency of peristaltic contractions (30 s intervals, Fig. 2i, j, k, n). This did not result in a significant increase in elongation or widening compared to the control condition (120 s intervals). This suggests that the effect of peristalsis on elongation may reach a plateau at a certain frequency. We will revise the manuscript to clarify this interpretation and discuss its implications.

(4) We appreciate this important comment and have addressed the issue of novelty and positioning in the General response shown above.

Reference

Yang, Y. et al. Ciliary Hedgehog signaling patterns the digestive system to generate mechanical forces driving elongation. Nat. Commun. 12, 7186 (2021).

Khalipina, D., Kaga, Y., Dacher, N. & Chevalier, N. R. Smooth muscle contractility causes the gut to grow anisotropically. J. R. Soc. Interface 16, 20190484 (2019).

Are we sure about attaching the management accounts (from Companies House)? The format is long and boring

23 or 24; I've asked for the latest number on #Team_Ops on Slack. When we have the correct number please update it across the website and the pdf version

our official numbers for running costs are 16%. In 2026/2027, we probably can use the new accounting categories and do better maths of delivery vs real overheads.

And the presentation seems to say we don't deliver 100% of programme. Maybe 87% (or 82%) cost on programme delivery? Sth like that

5.7 or 5.8m is the 2026 budget The correct data is on the financial master (tab "dashboard"). I sent the link and screenshot on Slack too

I don't think the progress bars add much value here. So perhaps only the numbers are good enough

what is the shared logic? :)

We dont need the top sponsor (organization).

I don't know whether we need the companies (organization) tab. Will think more about this.

We have a different categorization of type of company (which should be called organization actually). There are 8 company types in the organisations table:

Type Pharmaceutical/Biotechnology <br /> Academic Institution/Hospita Other Type Diagnostic Clinical Research Organization (CRO) Investor Government/Regulatory Institution

The columns in both subtabs: 'Drugs in Developement' and 'Drugs Discontinued / No Activity Reported' should have the following columns: Drug,Organization, Target, Action, Modality, Phase, Clinical Trial ID. I am not sure about the order yet though.

Near 'Drugs in Developement' in the right there should be another subtab called ' Drugs Discontinued / No Activity Reported'

The name of the tab can change to 'Development Landscape'.

I dont think we need this tab at all. We can only show UniProt ID in the main page as a clickable entity.

We don't have a function for the target. We have a description but its not good and better not be shown. Thus no need for function. All the info that we have about targets is here: https://datalake.bioloupe.com/admin/targets/683

explore

This source follows a woman who was first stalked online at age 12, documenting how the harassment persisted into her adult life despite her efforts to stop it. The piece highlights how publicly available personal information makes it easy for stalkers to continuously locate and target their victims. It also shows how platforms and law enforcement were slow to respond, leaving victims with little protection. This connects directly to the chapter's discussion of how online features designed for connection can be exploited for sustained harassment.

I feel kind of bad for the woman making the food for neighbors. She just wanted to be kind and do something nice and in return she got a bunch of people telling her that she was doing it for attention. I understand people may have allergies but I think it is the gesture and the thought that matters.

Reading the article about 4chan and its effect on mainstream media was very eyeopening. What is the intrinsic motivation and end goal the users of 4chan have? Is it illegal to spread knowledge or hate in the way that they do or just heavily looked down upon?

I wonder why doxing is such a big thing-> well, I don't really. I know that its meant to do harm on the other party, but I find the why behind it such an interesting thing. Online interactions between people, especially ones that are fueled with anger, dehumanize people. This combined with a sense of anominity allows people to target, and expose, the private information of the person they are mad at. If these people were arguing in real life, I doubt these interactions would go the same way. Its honestly a bit sad, and to me a reminder of why you don't want to get sucked into social media discourse. Its fake, it incentivizes anger, and though it has positives, it can be endangering. I've heard of more anti-dox laws coming in, but the problem may be so rampant that it doesn't matter. I even wonder what measures could be done by government and individual to protect it. If it was easier to individuals to hide their data, people would absolutely do so. Should the government create better protections for this data?

eLife Assessment

This valuable study builds a novel auditory-motor paradigm to investigate how the brain learns associations between movements and their auditory consequences. Solid evidence is provided for early ERPs (50-100 ms latency) reflecting violations of established key-pitch mappings. The writing, however, could be streamlined to better emphasize the paper's key contribution, and some statistical analyses might be improved.

Reviewer #1 (Public review):

Summary:

Zhang et al. report on an ambitious study that investigates multiple aspects of the neural and behavioral underpinnings of auditory-motor surprisal in the context of an auditory-motor learning paradigm (piano keyboard). Using an intricate design comprising several sub-parts and control procedures, they report that early ERPs (50-100 ms latency) reflect violations of established key-pitch mappings.

Strengths:

This is a carefully devised and executed study. The paradigm is quite intricate and, at the same time, addresses multiple aspects of auditory-motor learning, and does so in a rigorous way.

Weaknesses:

Perhaps because of the exhaustive approach, it is sometimes difficult to follow which parts of the experimental design the results come from; there are some questions regarding appropriate statistical methods, the inclusion/treatment of musical background in participants, and the nature (latency & extent) of the identified neural components that detect auditory-motor violations.

Reviewer #2 (Public review):

Summary:

Zhang et al. report an EEG study (n=18) of participants playing a keyboard where the correspondence between keys and pitches is varied to introduce sensory-motor mismatches (discrepancies between sensory inputs and expected sensory consequences of motor commands). They find that the auditory N100 amplitude is enhanced for the initial keystroke following a mapping switch but rapidly attenuates for subsequent keystrokes (showing rapid updating of the forward model), whereas the motor-related P50 amplitude only differentiates trained versus untrained mappings after 30 minutes of goal-directed practice (potentially showing timescales of inverse model updating). Using parallel univariate and mTRF decoding analyses, they conclude that forward models (mapping action to predicted sound) update almost instantly to track short-term context, while inverse models (mapping sound to motor commands) update slowly and require extended, targeted practice.


Strengths

(1) Methodological innovation:<br /> The study utilizes an interesting, continuous auditory-motor paradigm that moves beyond standard trial-by-trial oddball designs, offering a more ecologically valid measure of trial-to-trial adaptation.

(2) Analytical elegance and rigor:<br /> The combination of traditional univariate ERP analyses with multivariate temporal response function (mTRF) decoding is elegant, allowing the authors to successfully dissociate overlapping auditory and motor variance streams.

(3) The dissociation between the rapid adaptation of the N100 forward model and the slower adaptation of the P50 inverse model is interesting.

Weaknesses

(1) Confounded passive listening baseline:<br /> The passive listening control condition lacks an orthogonal behavioural task (e.g., an occasional oddball detection task). Active playing inherently necessitates focused attention on auditory feedback to monitor performance, whereas passive playback does not. The globally weaker stimulus-evoked pattern at electrode Fz during passive listening strongly suggests that the absence of an N100 effect in this condition may simply reflect a lower state of attention, rather than isolating the absence of a motor-driven forward prediction, in particular because the pure sensory suprisal was also enhanced for "firsts" notes, so this could also lead to stronger N1, but this effect may be masked.

(2) Overclaimed theoretical novelty:<br /> The conceptual framing leans excessively on the authors' specific "MirrorNet" framework, presenting foundational, decades-old tenets of the motor control literature (i.e., unsupervised exploration for forward models vs. supervised skill acquisition for inverse models; Wolpert, Jordan, both in the nineties) as their own novel "conjectures." This theory-heavy introduction obscures the paper's actual empirical contribution to the design and the interesting question regarding the distinct temporal adaptation scales of forward versus inverse models. I think some rewriting can improve the paper.

(3) Misplaced surprisal terminology:<br /> In a similar vein, I find the use of the term "auditory-motor surprisal" more theoretical grandstanding than actually useful. The significance statement claims to "extend this principle from sensory processing" but in fact, the concept of sensory motor unexpectedness is again a staple of the forward motor literature. Moreover, nowhere in the paper do they actually estimate sensorimotor surprisal. While the authors compute surprisal for their auditory baseline using IDyOM, their central sensorimotor analysis relies entirely on a simple categorical mismatch (first vs. subsequent keystrokes). The phenomenon can equally be referred to by its established nomenclature-"sensorimotor mismatch" or "sensory motor unexpectedness".

(4) Incremental conceptual advance regarding the N100:<br /> The paper frames the N100 finding as a major discovery, but as far as I know, the attenuation of the auditory N1 to self-generated sounds via accurate motor prediction-and its enhancement during sensorimotor mismatch - is one of the most heavily documented phenomena in the auditory-motor literature (e.g. Timm et al., 2013; Bendixen et al, 2012; 2013). As far as I'm concerned, the authors should clarify that the novelty lies in the novel, elegant design that provides a new way to correct for non-sensory-specific motor-induced attenuation, and characterizing the distinct adaptation timescales of forward versus inverse models  -- not in demonstrating N100 modulation by sensorimotor mismatch, which is well-documented, AFAIC.

eLife Assessment

This useful study asked whether the behaviour of motor units from a hand muscle changed across the two mechanical actions it performs. The authors used high-density intramuscular electrodes to record the activity of several motor units and reported changes in motor unit recruitment order across tasks that were not dependent on motor unit properties, suggesting differential spinal contributions to the two actions. However, the evidence supporting their main claims is incomplete, and some of the conclusions are based on unsubstantiated assumptions: the authors should correct several key analyses and temper claims that are not directly backed up by their data.

Reviewer #1 (Public review):

Summary:

Osswald and colleagues aim to show how motor units of the first dorsal interosseous (FDI) are flexibly recruited across two functionally different movements: index finger abduction and index finger flexion. They motivate this by arguing that FDI is the prime mover in abduction but acts as a synergist in flexion, alongside flexor digitorum profundus (FDP) and flexor digitorum superficialis (FDS) as the prime movers. This is a worthwhile question because it speaks to how descending neural inputs to the spinal cord flexibly control movement.

The authors claim that recruitment order and recruitment threshold of FDI motor units differ between abduction and flexion, and that beta-band intramuscular coherence is reduced when FDI acts as a synergist. However, there are significant methodological concerns that undermine the results and conclusions.

Strengths:

The study certainly aims to address a central question in motor neuroscience - how flexible recruitment of motor units occurs across movements where the same muscle changes its functional role. They correctly identify the FDI as a multi-functional muscle and use intramuscular high-density EMG arrays to record several motor units simultaneously, which is a major technical strength. They also track individual motor units between conditions and, therefore, have generated a potentially valuable dataset for studying spinal motor control across different movements.

Weaknesses:

The key limitation comes from the authors' interpretation of "neural drive" to FDI. The authors acknowledge that global EMG during flexion is smaller than that during abduction (for the same force), and surmise that the FDI receives different amounts of neural drive between these two movements, which is a potential confound for their analyses. To match the neural drive (i.e., global EMG), the authors ask participants to generate the same global EMG in flexion as in abduction; the forces generated by FDI are significantly different (2-3N for abduction and 1-8-6.2 for flexion). From this, they find changes in recruitment order, recruitment threshold, and beta coherence. However, different FDI motor units (and different muscle fibres) are active during abduction versus flexion. Using global EMG as a proxy for neural drive ignores this spatial separation of EMG generation during abduction and flexion, such that some amount of global EMG generated by one part of FDI (during abduction) is considered the same (from a neural drive perspective) as the same amount of EMG generated by a completely different part of FDI (during flexion). But these two global EMGs (during abduction and flexion) are not biologically equivalent because they are generated by different motor units and muscle fibres. Consequently, neural drive during flexion and abduction is not equivalent, which makes biological interpretation less clear. Furthermore, it is difficult to tell if abduction-versus-flexion differences are due to task role (prime mover vs synergist) or differences in force/mechanical demands, multi-muscle coordination, and spatial sampling limits of intramuscular recordings.

As mentioned, we think that the question asked is a very interesting one and framed appropriately to investigate the behaviour of motor units during prime mover and synergist roles. Simultaneously recording the prime movers for index flexion (FDP and FDS) would significantly improve the completeness of the study and allow for multi-muscle comparisons that are more relevant to how the motor system resolves prime mover vs synergist roles.

The authors use motor unit action potential as a proxy for motor unit size. This is not suitable because muscle fibres closer to the electrode will appear larger, independent of their true size. We advise that the authors remove analyses pertaining to motor unit size if it cannot be accurately measured.

Finally, several mechanistic interpretations in the discussion (e.g., spinal interneuronal suppression, reduced corticospinal input, proprioceptive mechanisms) read as more speculative than the current data can support without added controls or citations.

Reviewer #2 (Public review):

In this study, the authors examine whether the structure of motor unit (MU) recruitment and firing varies across movement directions in the human first dorsal interosseous (FDI) muscle. While task-dependent changes in MU recruitment have been reported previously (e.g., Thomas et al. 1986), these findings were largely based on recordings from a limited number of isolated single motor units. By applying high-density intramuscular electromyography and decomposition techniques, the authors demonstrate similar phenomena at the level of larger MU populations, thereby providing a useful consolidation of prior observations. In addition, they show that recruitment thresholds shift across tasks while the inverse relationship between discharge rate and recruitment threshold (the "onion-skin" organization) is preserved, suggesting that the overall structure of inputs to the motoneuron pool remains stable despite changes in recruitment order. Furthermore, by analyzing intramuscular coherence across MU firing, the authors attempt to characterize differences in the extent of synchronization among frequency components of neural inputs between abduction and flexion of the index finger. In particular, they report reduced beta-band coherence during flexion compared to abduction, indicating decreased synchronization in this frequency range (13-30Hz). This observation is noteworthy, as it points to potential differences in the neural inputs underlying these task-dependent changes.

A key strength of the study is that it extends prior work on task-dependent MU recruitment to larger populations using state-of-the-art recording and decomposition approaches. This represents a meaningful technical and conceptual advance over earlier studies limited to small numbers of units. The finding that recruitment shifts between flexion and abduction occur consistently across MUs, independent of motor unit size, further strengthens the robustness and generality of the observed phenomenon. Together, these results provide convincing evidence that MU recruitment is not strictly fixed by a rigid size principle across functional contexts and thus make a valuable contribution to the literature on motor control.

However, several aspects of the mechanistic interpretation are less well supported. The authors interpret their findings as reflecting a "redistribution" of net excitatory input to the motoneuron pool across tasks. While this is a plausible interpretation of the observed changes in recruitment thresholds and recruitment order, it is not directly demonstrated by the analyses presented. The current data do not clearly distinguish redistribution of inputs from alternative explanations, such as task-dependent modulation of shared versus independent inputs, or changes in the effective gain of existing pathways. As such, the evidence for a specific redistribution of input remains incomplete.

The interpretation of the intramuscular coherence analysis represents a further key weakness. By computing frequency-specific coherence across MUs during abduction (as a prime mover) and flexion (as a synergist), the authors report reduced beta-band coherence during flexion and interpret this as evidence for attenuated corticospinal input and increased involvement of spinal circuits. However, the relationship between changes in downstream coherence and the magnitude of upstream neural drive is not well established. Coherence reflects the synchronization of inputs rather than their net strength, and therefore, a reduction in coherence cannot be directly interpreted as a decrease in input from a specific source. Moreover, coherence measures alone do not permit identification of the origin of the inputs, and thus do not provide sufficient evidence to attribute the observed differences to descending or spinal pathways. While the difference between tasks is clear and potentially informative, the mechanistic interpretation appears overstated and should be treated more cautiously.

A related issue concerns the interpretation of the preserved RT-DR relationship. While this finding supports the presence of a stable common input structure across tasks, the additional claim that proprioceptive feedback contributes significantly to maintaining this organization is not clearly justified by the presented data. No direct evidence is provided to dissociate afferent from descending inputs, and the absence of task-dependent differences in lower-frequency coherence further limits support for this interpretation. As such, the proposed role of proprioceptive feedback appears speculative.

Overall, the authors successfully achieve their primary aim of demonstrating task-dependent flexibility in MU recruitment at the population level, and the results provide useful empirical support for this phenomenon using modern techniques. The study is likely to be of interest to researchers in motor control and neuromuscular physiology, particularly given the increasing relevance of MU-level analyses in both basic and applied contexts. However, the broader mechanistic conclusions regarding the nature and origin of the underlying neural inputs are not fully supported by the data and would benefit from more cautious interpretation or additional experimental evidence.

This question raises a difficult issue because determining whether harassment is ever "justified" requires someone to make a moral judgment on behalf of the entire platform. Even if a person has done something widely considered wrong, mass online harassment tends to escalate far beyond any proportional response. Platforms would also need a consistent standard for making these calls, which seems nearly impossible given how differently people interpret what deserves punishment.

A lot of the times, Instagram won't let an account be banned unless there is many people doing it. Instagram "claims" to review certain posts to make sure people aren't just reporting it for no reason but there are instances where Instagram ignores it.. I think they should be more careful and actually review things.

There are two ways to relate with a piece of media – through space, and through time. Time is the conventional one. Narrative tells events sequentially, creating story. But space is a more visual, unstructured medium. There is no right way to enter a painting. This is like the cardinal ordering of the real number line against the relative ordering of the complex number space.

Reminds me of the Garden of Forking Paths by Borges. Also, this is an attitude that might help completionists and perfectionists look at their obsession with "finishing things" in a different way – you can never truly complete anything, only arrive at it in a way different from how you arrived at it the first time. As Venkatesh Rao says in this Ribbonfarms article (https://ribbonfarm.com/2016/05/26/how-to-take-your-brain-off-road/), some pieces of reading are so alien that the first time you read them, you are only learning how to read them. Also explains why good works, like classics, reward rereading more than reading. You never arrive at a classic the same way twice. Like the saying "No man steps into the same river twice."

диаграмма: Перед ConnectionStatusEvent нужно показать какое-то событие сессии. Например, перечеркнутая стрелка от клиента к серверу с надписью Disconnect.

не показываем внутренние объекты, это внешняя документация

диаграмма: 1. Показываем от клиента к WCS /v3/call/startup 2. Показываем от WCS к PBX стрелку с подписью Call established 3. Дальше остается POST в сторону бэкенда и т.д.

не показываем внутренние объекты, это внешняя документация

не показываем внутренние объекты, это внешняя документация

L’Agence NOOR : Éthique, Narration et Engagement dans le Photojournalisme Contemporain

Résumé Exécutif

Ce document synthétise les échanges d'une table ronde tenue à la Bibliothèque nationale de France (BnF) avec quatre photographes de l'agence NOOR : Olga Kravets, Francesco Zizola, Bénédicte Kurzen et Sanne De Wilde.

Fondée en 2007 sous forme de coopérative, NOOR se distingue par un engagement profond dans le temps long et une approche critique des enjeux sociétaux mondiaux.

Face à l'effondrement du modèle économique de la presse traditionnelle et à la prolifération des images amateurs et algorithmiques, ces auteurs revendiquent un passage du statut de "photojournaliste" à celui de "conteur d'histoires".

Leur travail se caractérise par une hybridation des médiums (vidéo, archives, peinture), une exploration des dimensions mythologiques et symboliques du réel, et une volonté farouche de préserver une mémoire collective.

L'agence met également l'accent sur la transmission à travers des programmes éducatifs gratuits, affirmant que la survie de la profession réside dans la singularité du regard de l'auteur et le développement du sens critique du spectateur.

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1. L’Identité et l’Éthique de l’Agence NOOR

L'agence NOOR (qui signifie "lumière" en arabe) a été lancée officiellement en septembre 2007 au festival Visa pour l'Image.

• Structure et Composition : Organisée en coopérative sur le modèle de Magnum, l'agence compte 14 photographes, dont 7 femmes (une parité quasi parfaite). Elle gère également le fonds de son cofondateur disparu, Stanley Greene.

• Contexte de Création : NOOR est née dans un paysage médiatique où les codes du photoreportage étaient bouleversés par l'émergence du témoignage amateur.

L'agence a choisi de privilégier un regard "circonstancié, détaillé, critique et interprétatif".

• Lignes de Force : Les membres sont unis par un désir d'engagement sur le long cours autour de questions critiques :

  • Le changement climatique.

• La surconsommation.

• Les migrations forcées.

• La montée de l'autoritarisme.

• Philosophie de l'Image : Fidèles à la devise de Stanley Greene, "certaines choses ont simplement besoin d'être vues", les photographes de NOOR cherchent à éclairer l'actualité brûlante avec un recul critique nécessaire.

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2. Analyses Individuelles : Entre Engagement et Expérimentation

Chaque photographe apporte une perspective unique qui illustre la diversité des écritures au sein de l'agence.

Olga Kravets : Documenter l'oppression et la résistance

Ressortissante russe vivant en France, Olga Kravets se concentre sur les mécanismes de répression dans son pays d'origine.

• Projet "Plus de terreur qu'Allah" : Un travail de dix ans sur les Russes convertis à l'islam, une communauté doublement persécutée (par l'État et par une opposition souvent islamophobe).

• Le passage au documentaire : Elle utilise la photographie et le film pour raconter des histoires de vies brisées, d'exils forcés et de méthodes oppressives (surveillance des réseaux sociaux, arrestations arbitraires).

• Responsabilité de l'auteur : Elle souligne l'importance d'être responsable de chaque mot et de chaque image pour protéger ses sujets contre toute réutilisation hors contexte.

Francesco Zizola : La métaphore et l'esthétique du réel

Francesco Zizola a fait évoluer sa pratique vers un langage visuel hybride, mêlant images fixes et films.

• Remise en cause de l'objectivité : Il soutient qu'il est impossible de saisir le sens de la réalité sans un filtre culturel.

Pour lui, il existe une "vérité esthétique" atteignable par le style et l'imagination.

• Symbolisme et Noir et Blanc : Il utilise le noir et blanc pour détacher le sujet de l'instantanéité et l'élever au rang de métaphore (ex: les migrations en Méditerranée dans Mare Omnis).

• Interaction physique : Ses projets récents explorent la relation entre l'homme et la nature, utilisant des formats monumentaux pour créer une expérience physique pour le spectateur.

Bénédicte Kurzen : La photographie comme archive et mémoire

Basée au Nigeria depuis une dizaine d'années, elle s'inscrit dans une tradition de photojournalisme engagé tout en revendiquant une subjectivité assumée.

• Temps long et immersion : Son travail sur les crises ethnico-religieuses au Nigeria ou les seigneurs de guerre au Congo nécessite une présence durable pour capter les "traces" des événements.

• Fonction mémorielle : Elle considère la photographie documentaire comme un outil de traçabilité, créant une mémoire collective là où le passage du temps efface les preuves directes de violence.

Sanne De Wilde : L'expérimentation et l'altérité

D'origine belge et issue d'une formation en beaux-arts, Sanne De Wilde utilise la photographie comme une stratégie visuelle pour traduire des réalités invisibles.

• "Land of the Color Blind" : Utilisation de l'infrarouge et de la peinture pour rendre compte de la vision de communautés atteintes d'achromatopsie.

• Interprétation vs Reproduction : Elle refuse la notion de réalité fixe et privilégie une approche "magique" et interprétative du monde.

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3. Collaboration et Narration Mythologique

L'un des traits distinctifs de NOOR est la capacité de ses membres à co-construire des projets complexes.

Le projet "Land of Ibeji"

Bénédicte Kurzen et Sanne De Wilde ont collaboré sur la figure mythologique des jumeaux au Nigeria. | Aspect | Caractéristiques du projet | | :--- | :--- | | Point de départ | La réalité sociale et les croyances traditionnelles (Yoruba) liées aux jumeaux. | | Langage visuel | Utilisation de la dualité (jour/nuit), de la ressemblance physique et de l'imaginaire. | | Hybridation | Intégration d'archives, de peintures et de bandes sonores pour une expérience immersive. | | Objectif | Passer de l'anthropologie à une interprétation universelle de la gémellité. |

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4. Évolution de la Profession et Modèles Économiques

La table ronde a mis en lumière les mutations profondes du secteur.

• L'effondrement de la presse traditionnelle : Les photographes ne se définissent plus seulement comme des photographes de presse. Ils doivent diversifier leurs sources de financement (fondations, ONG, bourses, institutions culturelles comme la BnF).

• De l'image-témoignage à l'image-auteur : "Tout le monde est photographe, mais tout le monde n'est pas auteur."

La plus-value du professionnel réside dans l'intelligence, l'émotion et la construction narrative derrière l'objectif.

• Le défi technologique : Francesco Zizola souligne la menace des images créées par algorithmes. Face à cela, le photographe doit redevenir un "historien de la réalité" et un penseur visuel.

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5. Éducation et Transmission : Une Mission Prioritaire

NOOR accorde une importance capitale à l'éducation à l'image.

• Accessibilité : L'agence organise des ateliers (workshops) gratuits pour les participants grâce à des partenariats (notamment avec Nikon).

• Pédagogie du doute : Face à la saturation d'images et aux fake news, les photographes estiment qu'il est crucial d'apprendre au spectateur — dès le plus jeune âge — à décoder les images, à identifier l'auteur et à mobiliser un recul critique.

• Partage de savoir : La relation entre les photographes établis et les nouvelles générations n'est pas vue comme une relation maître-élève, mais comme une collaboration inspirante.

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Citations Clés

"Certaines choses ont simplement besoin d'être vues." — Stanley Greene (citée par la commissaire d'exposition)

"Le noir et blanc n'existe pas dans la réalité... c'est une manière de mettre au premier plan le niveau symbolique du sujet." — Francesco Zizola

"Nous sommes des conteurs d'histoires... nous créons de la mémoire plus que de la preuve." — Bénédicte Kurzen

"Il faut susciter le doute... c'est l'unique façon de procéder pour comprendre le monde." — Francesco Zizola

Version 3 of this preprint has been peer-reviewed and recommended by Peer Community in Mathematical and Computational Biology.<br /> See the peer reviews and the recommendation.

💻/thinkpad/🧊/me/📓/2026/5/1

http://bafybeievd2adaexnbbodz5gqf5in6efs5yvjw4ufiiqbt3g2vbkhy5agke.ipfs.localhost:8080/_indy_0_index.html

save HTML to File System then upload to IPFS

Here I saved html to IPFS using save and then upload to IPFS Desktop

The nice thing about it that it generates and inde_files folder with the hyp.is/ambed.js in a matching folder

Now that is great because it means

that annotation wil be loaded real fast as it is loaded locally

since we are on IPFS

there is no cost to have the same file the embed.js file everywhere

Once you choose the phase, is there a need for a phase column? Probably phase column should exist only under 'All' tab.

there should be a column called 'Clinical trials' and it should show the NCT ID of the clinical trial where the drug is being studied.

It would be nice to include the technology as well as a column. Also there might be 3 targets as well for one drug.

We dont have this data but it would be nice to collect it, the name of the metric should be market cap/book value<br /> to be consistent with other metrics.

Same for Marketed assets, the number already appears in the Pipeline by Phase as approved drugs.

Market Cap is already included in the financials tab. Thus no need for it to be in the main page.

How Team Trump Helps Hide How Fast He’s Aging<br /> by [[Ellie Quinlan Houghtaling]] in The New Republic<br /> accessed on 2026-05-18T23:54:37

trail.blazed-?pehnomenology+personal.nowledge

reading Polanyi Chapter

wanted to see if phenomenology is discussed at all

does not seem so

very much

creative dislexia forces google search to consult AI

checked in the index

Phenomenology is not in the index for Personal Knowledge

only saving the phenomena n 307

c.f. 146

nurture or nature?

is pacifistic behavior learned or inborn?

is pacifism a result of made order or natural order?

the practice of writing?

Outside of looking at just EEG activity and cognitive development, behavioral and emotional disruptions can occur as a consequence of being raised in institutions. If not corrected early these issues can continue persist long-term and might contribute to the development of more severe disorders in the future.

This demonstrates how getting children out of institutions early can play a huge role in affecting their developmental trajectory

This is the key EEG finding. Greater alpha and lower theta power indicate more mature cortical regions and shows that family‑based care can shift the long‑term trajectory of brain activity after severe early deprivation.

Call back to the BEIP! This follows up with the children that were in this project and provides further findings that can be used to better understand the neurobiological consequences of being raised in an institution.

This clearly defines the characteristics that make institutions detrimental to the socioemotional development of children. These conditions are are in contrast to what might be seen in a health family system.

This means a person's sense of who they are is deeply tied to the groups they belong to.

Archetypal (adjective) describes something that serves as the perfect, quintessential example of a particular thing, or something that acts as the original model or pattern from which other, similar things are derived.

milgram shock sq3r

Stanley Milgram, Yale 1963 an experiment focusing on the conflict between obedience to authority and personal conscience

experiement, yale, 1963 justification genocide, nuremburg war criminals holocaust following orders from superiors

aim: how far to obey instructions

advertised for participants, paired with another person, it was not random assignment of shocker (teacher) and shockee (learner). participant was always teacher, and Milgram's confederates (pretending to be a participant) was always student pretending to feel pain at the shocks 65% to 450 volts for wrong answers. conclusion, ordinary people follow authority to killing innocent persons

agency theory Milgram 1974: autonomous state - directs own actions, takes responsibility. agentic state - allows others to direct, passes responsibility

variations: uniform, change of location, two teacher, touch proxzimity, social support, absent experiementer,

critical eval; lab conditions not real life. biased sample all men. volunteer personality

ethical issues - deception shocking real person not a fellow. protection of participants potential psychological harm. debrief participants fully and follow up. right to withdrawal

this is what we are doing with the OPEN Annex Co-Create Workshop - worth to put a artwork label with description next to the piece

decommission - what happens if we want to take down the artwork?

"... are bootstrap ... corrected .. " Is this the start of a question?

or is the intended sentence ... "... boostrap estimates ARE corrected?

This paper showed how if Lynx1 or VPA can reopen social plasticity in adult mice, then targeted interventions along with positive social experiences could, potentially re-wire social behavior after adverse early experiences. However, the study never tested whether early social deprivation (e.g., isolation during the sensitive period) shifts the timing of when that window opens or closes.

Interesting finding! VPA's effects go beyond simply just reopening the critical period but increasing the sensitivity to the entire social hierarchy system revealing how reopening critical periods can have broader effects than expected.

This points to the additional neural mechanisms involved in dominance status. By measuring c-Fos, the authors show that the same brain regions previously shown to regulate dominance rank are more activated in Lynx1 KO mice following loss.

Interesting how genetic influences like Lynx1 in this case play a role in re-opening of a critical period. Its likely that Lynx1 plays a role in a number of cortical areas and the effects of this knockout on those would be interesting to see.

Interesting finding! One social loss experience was enough to reshape an adolescent mouse's dominance rank within 24 hour but the adult mice given the identical experience showed no change.

This is the gap in research these authors are trying to fill; studying the prefrontal cortex which regulates social behavior, decision making, emotional regulation, etc. through the lens of experience-dependent development.

Sure, but participants too necessitate this language to partake in future designing. That is, they need to learn, they require practice, awareness, validation... help dismantling capitalist myths!

When you get yourself dirty, like Simone de Beauviour, you see how hardcore this is, and how understanding you have to get.

Yes, but watch out not to displace the responsibility of change from social regulations, to individual consumers (addicts, sometimes stuck in discriminatory religions).

Meaning that it must not be "any utopia", it must be a fact-based, historical memory contextualised, and locally-globally contextualised one. It must draw from the ecological validity and past idiosyncrasies of the specific place in which it is being applied, while considering how to organically reproduce it providing a timely fast enough proposal that avoids tipping points, while being exhaustive enough to prevent corruption, thus managing ETTO.

For this, we must not push ourselves hierarchically, but rather understand that democratic emancipatory change that leaves as few people as possible behind, is a process of collective learning, which will have sidesteps, and retries, like someone trying to stop an addiction. It won't be a linear progression, always improving and never failing, like the supermorality and supercoherence that right wingers impose on bureaucracy and the revolutionary alternatives.

Herein lies the difficulty with studying this critical period. Humans are unique in their socioemotional development and how its studied has to take into account broader aspects of human development.

In terms of trying to study a specific discrete process within socioemotional development, fear learning and threat or safety detection might be something to explore because the circuitry involved is conserved across species.

Call back to the The Bucharest Early Intervention Project! They point out how (even though the study was useful and informative), you cannot really isolate if both early deprivation or total deprivation were the reason for the negative outcomes observed.

The authors are pointing out how trying to ask "is there a critical period for development" is too broad of a question because it is a highly complex system like language. Instead it might be more useful to study discrete processes within socioemotional development (e.g. fear conditioning, reward processing,).

Here again the authors identify what past researchers have done in observing how early adversity can lead to poor outcomes and correlating that with a critical period. But correlation between timing and outcome is not the same as a developmental window causing the outcome.

The authors in this paper are making a distinction between simply showing that early experiences matter versus showing that the timing of these experiences influence the outcome which is critical when trying to define a critical period.

Could directionality be added independently here? I don't have an immediate use-case, but if this the current skeleton encodes structure of a graph, might as well add space allowing for directed graphs.

Honestly, I do believe that crowd harassment is necessary sometimes. If someone with extreme persuasion or pull on society does something severely wrong, I believe it is the publics responsibility to call them out for that. I do think there is a line that shouldn't be crossed like telling someone to die or of anything of extreme nature.

These bot hate farms are really intersting. It really reminds me of that star wars filmed that had like over 50% of the hate online originate from Russian bots. What are the incentives behind it? I think especially for that star wars one, it really is interesting. Especially as the public perception of that film is AWFUL, but how much of that is real opinion and how much of that is botting? Was the film so bad? I didn't really enjoy it, but it somewhat didnt deserve ALL the hate that it got. I wonder in the future how much bots will have an influence on public opinion- and what the implications of it are. And also like, how much of the internet today (like youtube comment sections) are real people with real opinions?

Kirsti Marohn, “In Minn., Gulf Shrimpers Meet Farmers Trying to Save Their Catch,” MPR News, August 28, 2018, https://www.mprnews.org/story/2018/08/28/gulf-shrimpers-meet-midwest-farmers

“Photograph of Dean Blanchard at Seafood Dock.” Gulf of Mexico Oil Spill Blog. Accessed April 29, 2026. https://gulfofmexicooilspillblog.wordpress.com/category/gulf-of-mexico-oil-spill-blog/louisiana/grand-isle/dean-blanchard-seafood-inc/

Marohn, Kirsti. “In Minn., Gulf Shrimpers Meet Farmers Trying to Save Their Catch.” MPR News, August 28, 2018. https://www.mprnews.org/story/2018/08/28/gulf-shrimpers-meet-midwest-farmers.

Huang, L., L. A. Nieland, J. K. Craig, and M. D. Smith. “Measuring Welfare Losses from Hypoxia: The Case of North Carolina Brown Shrimp.” Marine Resource Economics 27, no. 1 (2012): 3–23. https://ir.library.oregonstate.edu/downloads/6h440t39c.

Huang, L., M. D. Smith, and J. K. Craig. “Quantifying the Economic Effects of Hypoxia on a Shrimp Fishery.” Marine and Coastal Fisheries 2, no. 1 (2010): 232–248. https://doi.org/10.1577/C09-019.1

This is one of the most compelling aspects of the manuscript to me. Gaining broad adoption really depends on lowering costs, reducing failure points, reducing the need for specialized equipment, and, importantly, speed.

Are these efficiencies the results of a single experiment or averages of multiple experiments? How much experiment to experiment variation would you expect to see?

You have listed 6 strains as being tested, but only have data for 4A-. Is data for the remaining strains going to be included as supplemental data in the final pub? Are there any differences in transformation efficiency between strains?

“Agriculture Policy for Agricultural and Rural Development,” Fall 2024. Course materials. Brian Fleury, “Flood Control on the Mississippi River.” Tulane University, Environmental Biology course materials. Accessed April 28, 2026. https://environmentalbiology.tulane.edu.

S. S. Rabotyagov et al., “The Economics of Dead Zones: Causes, Impacts, Policy Challenges, and a Model of the Gulf of Mexico Hypoxic Zone,” Review of Environmental Economics and Policy 8, no. 1 (January 1, 2014): 58–79, https://doi.org/10.1093/reep/ret024.

Agricultural Adjustment Act of 1933, Pub. L. No. 73-10, 48 Stat. 31 (1933); Sergey S. Rabotyagov et al., “The Economics of Dead Zones: Causes, Impacts, Policy Challenges, and a Model of the Gulf of Mexico Hypoxic Zone,” Review of Environmental Economics and Policy 8, no. 1 (2014): 61–63, https://doi.org/10.1093/reep/ret024

specifically list the types of objects somewhere in this section

Federal Water Pollution Control Act Amendments of 1972 (Clean Water Act), Pub. L. No. 92-500, 86 Stat. 816 (1972), § 402(l)(1); Food Security Act of 1985, as amended by the Federal Agriculture Improvement and Reform Act of 1996, Pub. L. No. 104-127, 110 Stat. 888 (1996); National Agricultural Law Center, “Conservation Provisions in the Farm Bill: Expanded Discussion,” accessed April 28, 2026, https://nationalaglawcenter.org

Agricultural Adjustment Act of 1933, Pub. L. No. 73-10, 48 Stat. 31 (1933); Sergey S. Rabotyagov et al., “The Economics of Dead Zones: Causes, Impacts, Policy Challenges, and a Model of the Gulf of Mexico Hypoxic Zone,” Review of Environmental Economics and Policy 8, no. 1 (2014): 61–63, https://doi.org/10.1093/reep/ret024

Act of June 28, 1879, ch. 43, 21 Stat. 37; U.S. Army Corps of Engineers, Mississippi River Commission, “History of the Mississippi River Commission,” accessed April 28, 2026, https://www.mvd.usace.army.mil/About/Mississippi-River-Commission-MRC/History/ ; John M. Barry, Rising Tide: The Great Mississippi Flood of 1927 and How It Changed America (New York: Simon & Schuster, 1997).

Swampland Act of 1849, ch. 87, 9 Stat. 252 (March 2, 1849); Swamp Land Act of 1850, ch. 84, 9 Stat. 519 (September 28, 1850); Act of March 12, 1860, ch. 5, 12 Stat. 3; Library of Congress, “Draining America,” Worlds Revealed: Geography and Maps at the Library of Congress, February 5, 2024, https://blogs.loc.gov/maps/2024/02/draining-america/

US. 2022. “Dead Zone in the Gulf of Mexico.” Noaa.gov. 2022. https://oceantoday.noaa.gov/deadzonegulf/welcome.html.

“Mississippi River Basin Nutrient Runoff Information Resources.” 2024. Noaa.gov. 2024. https://www.noaa.gov/regional-collaboration-network/regions-central/mississippi-river-basin-nutrient-runoff-information-resources.

Agricultural Adjustment Act of 1933, Pub. L. No. 73-10, 48 Stat. 31 (1933); Sergey S. Rabotyagov et al., “The Economics of Dead Zones: Causes, Impacts, Policy Challenges, and a Model of the Gulf of Mexico Hypoxic Zone,” Review of Environmental Economics and Policy 8, no. 1 (2014): 61–63, https://doi.org/10.1093/reep/ret024

Rob Nixon, Slow Violence and the Environmentalism of the Poor (Harvard University Press, 2011), https://doi.org/10.2307/j.ctt2jbsgw.

S. S. Rabotyagov et al., “The Economics of Dead Zones: Causes, Impacts, Policy Challenges, and a Model of the Gulf of Mexico Hypoxic Zone,” Review of Environmental Economics and Policy 8, no. 1 (January 1, 2014): 58–79, https://doi.org/10.1093/reep/ret024.

I'd like to revise this wording from my somewhat hastily written beliefs form submission to the following:

CM has broad and extensive potential for animal welfare benefit. The risks in/path to meaningful market adoption creates uncertainty as to extent of realized benefit on a given timeline, certainly, but we are past the point in which we question if this can be achieved. This benefit profile (impact x likelihood) is massively favorable when taken on the long term view because of just how significant the impact potential is. And despite significant investments to date, albeit largely to private companies, I would argue CM is relatively neglected as compared to investment profiles seen for other emerging technology spaces with comparable potential benefit impacts to society.

• The Fallacy of Faster Processing: Companies mistake faster individual tasks for faster overall production. While tools like LLMs can generate a boilerplate codebase in seconds, the overall development cycle remains bottlenecked by human review, architecture design, testing, and deployment.
• The "Checking" Overhead: Automated code generation shifts the developer's role from writing to auditing. Reading, understanding, and debugging AI-generated code often takes more cognitive effort and time than writing it from scratch, as developers must hunt for subtle hallucinated bugs.
• Quality and Maintenance Debt: Speeding up the initial creation phase leads to a mountain of undocumented, low-context code. This causes long-term maintenance issues, increases technical debt, and can drastically slow down future feature development.
• Process vs. Execution: Business bottlenecks are rarely caused by the speed of typing code; they are rooted in shifting requirements, communication gaps, and organizational bureaucracy. AI does not fix these foundational process issues.

Hacker News Discussion

• Shift in Cognitive Load: Several commenters agree that AI changes the bottleneck from "writing code" to "reviewing code." They point out that reviewing code is a fundamentally harder cognitive task because you have to reverse-engineer intent, making the overall process feel more exhausting.
• The "Junior Dev" Analogy: A prominent sentiment is that current AI behaves like an incredibly fast but highly unreliable junior developer. It can write 1,000 lines of code in seconds, but a senior engineer still needs to spend significant time verifying it for security, architectural fit, and edge cases.
• Where AI Actually Succeeds: Users note that AI does speed up specific, isolated processes—such as writing boilerplate code, generating regex, translating syntax between languages, or acting as an interactive documentation search tool.
• The Danger of Code Inflation: Commenters express concern that because code is now "free" to generate, codebases will balloon in size unnecessarily. This explosion of text makes the entire system harder for humans to maintain, ultimately slowing down software evolution.

Summary of AI Subscription Time Bomb for Enterprise

• Industry-Wide Loss-Leaders: Major AI labs (OpenAI, Anthropic, Google) are heavily subsidizing their subscription services to lock in enterprise users. They are absorbing massive compute costs to build market dependency.
• The Revenue vs. Cost Disconnect: Flat-rate consumer and team plans costing around $20 per month offer intensive access to premium models. Heavy knowledge-worker workloads can run up $200–$400 per month in actual API-equivalent usage, resulting in catastrophic unit economics for providers.
• Agentic Workloads Breaking the Model: The shift from simple conversational chatbots to autonomous agentic workflows (e.g., Claude Code, concurrent agent teams) has caused token consumption to skyrocket. Flat-fee business models cannot sustain this level of compute demand, forcing providers like GitHub Copilot to pivot to usage-based billing starting June 1, 2026.
• Enterprise Budget Exposure: Thousands of companies have built load-bearing workflows on top of subsidized AI tools without tracking consumption costs. When pricing inevitably corrects to reflect true infrastructure costs, organizations will face massive, unbudgeted cost increases.
• The IPO Catalyst: With both OpenAI and Anthropic preparing for IPOs, the public markets will demand healthy profit margins rather than venture-capital-subsidized losses. This pressure will accelerate the transition toward usage caps, price hikes, or consumption-based billing models.

Hacker News Discussion

• The Rise of Competent Local Models: A primary consensus among many developers is that open-weight, local models (such as Qwen 3.6, Gemma 4) have advanced dramatically. Many tech-savvy users find that running these models locally on consumer hardware like an M-series MacBook Pro or Nvidia RTX 4090 handles tasks with roughly 75% or more of the capability of frontier cloud models, making paid subscriptions less appealing.
• The Gap Between Local and Frontier Models: Commenters remain sharply divided on how far local models lag behind closed cloud giants like OpenAI and Anthropic. Estimates range from a 6-to-18-month delay to a persistent structural gap, with some users pointing out that benchmark scores are often inflated and that massive cloud infrastructure remains necessary for true frontier intelligence and high-speed token generation.
• Shared Infrastructure vs. Local Computing: Critics of the local-first outlook argue that running giant frontier models at full utilization on dedicated hosted hardware will always be more cost-efficient at scale than running hardware locally, once pricing model corrections settle down.
• Privacy and Control: The discussion highlights that on-premise and local execution provide immense value for businesses and individuals due to full privacy, lack of censorship, and protection against future "enshittification" or price spikes by large tech providers.

1. Understand the organization system that we use for the RMC vault.
2. Learn the difference between circulation and reserve equipment.
3. Learn how to return piece of equipment to the correct location

Maybe put on the flip cards?

The RMC has 2 groups of equipment available for checkout. 1. Circulation Equipment: Circulation equipment are the pieces of equipment that student, faculty, and staff do NOT need training or prior permission on in order to check out. They simply need to come to the RMC desk with their ID in hand. This equipment tends to be smaller and cheaper than reserve equipment. You can find what items are categorized as circulation equipment on the RMC website. 2. Reserve Equipment: Reserve equipment are the pieces of equipment that require trainings or prior permission to check out. This includes high-end cameras, light kits, 3D printers, large PA set ups and more. You can find bookings for these equipment and trainings on the RMC website.

Swap for the Vault organization system - which should've been covered earlier in this lesson

Take out this section

The RMC is more than just a study space - it hosts many media production studios. There are four studios housed at the Robertson Media Center. They are the Audio Studio, Video Studio, Photography/Animation Studio, and 3D printing Studio.

Educational Technology Librarians help you with your media projects?

Write up about Rachel and Josh - their specialities, what projects they help with, when to reach out to them vs when RMC staff should be the helpers

get rid of this section

Where should they go for help? 1. A student needs to be trained on a high-end camera to take graduation pictures. - RMC Website 2. A professor wants to digitize his old projector slide deck. - Digital Media Lab 3. A professor's USB-C charger broke, and he needs to borrow one for the next few hours. - RMC Desk 4. A student band wants to record a single and needs to book a time in the audio studio. - RMC Website 5. A student needs to use photoshop to edit her final project for her photography class. - Digital Media Lab 6. A student wants to check out a small lav mic to record a TikTok for their CIO. - RMC Desk

get rid of this section

get rid of this section

True / False

1. The video and audio studios require prior training to book (False)
2. You can book trainings on the RMC Website (true)
3. The high-end reserve equipment and the 3D printers require prior trianing to book (true)
4. To check anything out at the RMC desk, you must complete prior trainings (false)
5. The RMC website has a publicly available list of the equipment available for checkout (true)
6. You can book times the audio and video studios on the RMC website (true)

If a patron asks for help digitizing their VHS, where would you direct them?

A) The Digital Media Lab. We have many various of digitizing machines in the DML. (correct) B) The Video studio. The video studio is equipped to support digitization of all video materials. C) N/A. We do not have VHS digitizing machines.

Scenario: A professor comes into the RMC and tells you about a video project she wants to create. She wants the video to be high quality, but doesn't know exactly where to start.

Where should the professor go to get information on high tech reserve equipment? A) Search for cameras on the Virgo website B) At the RMC desk, because she is a professor, she does NOT need training on equipment C) The RMC Website. The professor can find bookings for high-end camera training (Correct)

get rid of this question

Where can patrons find all of the studios and equipment hosted by the RMC? A) Virgo B) RMC Website (correct)

get rid of this section

add a section about RMC resources:

At the Robertson Media Center we have high-end cameras, high-end lighting kits, 3D printing, microphones for podcasting and music recording, multiple studios, and so much more!

consider new branding for IndyWiki to become an

indy0.net.work.ed.wiki

or indranet.work.ed.wiki

the interpersonal interplanetary over web networked wiki for networked mutual learning symmathesy

design to work across and between Networked Wikis and existing networked Thinking Tools

provide a local-first personal web arhive and interpersonal connectivity

and the abiity to have threaded conversations across the web and connecting existing netowrked wikis and Tools for Thought tool that

As a vitreoretinal surgeon and managing partner of an independent physician owned retina practice, I want to raise several concerns about this study that I believe are material to its interpretation and that the retina community deserves to consider carefully.

1. Conflict of Interest and Data Provenance The data for this study was drawn exclusively from the RCA EMR database. RCA is an MSO in which Cencora holds approximately 85% ownership as of January 2025. Cencora also owns Besse Medical, one of the primary US distributors of Pavblu. The study was funded by Amgen. The presenting author is a disclosed paid Amgen consultant.

Every party involved in the design, data provision, funding, and presentation of this study has a direct financial interest in Pavblu adoption. The structural nature of these relationships, where the data source, the distributor, and the funder are financially interconnected through Cencora and Amgen, goes beyond a standard COI disclosure. These relationships raise questions about the independence of the study that should be explicitly acknowledged and discussed in the manuscript.

1. Reason for Switching: A Critical Missing Variable 91% of the study population switched from prior anti-VEGF therapy to aflibercept-ayyh. The manuscript acknowledges that reasons for switching were not captured. This is the most important missing variable in the study. In independent physician owned practices, switching decisions typically reflect individual clinical judgment based on disease activity, treatment response, and patient need. In a large MSO operating under centralized drug purchasing arrangements, switching decisions may additionally reflect institutional formulary policies or organizational priorities. The relative contribution of clinical versus institutional factors to the switching decisions observed in this study is unknown and unknowable from the data presented. A study designed to describe real world clinical adoption of a new biosimilar should be able to characterize the basis on which prescribing decisions were made. The inability to do so is a fundamental limitation that should be prominently disclosed rather than noted as a minor caveat.

2. ASP Trajectory, and Missing Economic Analysis In a buy and bill specialty, economics may drive prescribing decisions especially in larger practices. This study contains no economic analysis. Independent review of publicly available CMS Medicare Part B ASP data shows that aflibercept-ayyh entered the CMS payment system on April 1, 2025 at an ASP identical to reference aflibercept at $795.861 per mg. In its first full quarter the ASP rose to $869.084 per mg while reference aflibercept declined to $790.037 per mg. As of Q2 2026 aflibercept-ayyh is priced at $821.396 per mg versus $731.885 per mg for reference aflibercept. Aflibercept-ayyh has never been priced below the reference product since entering the CMS payment system. The retina biosimilar market has demonstrated a consistent and observable pattern in which products maintain ASPs above their reference products during periods of concentrated corporate network adoption supported by rebate arrangements. During these managed periods the reported ASP does not reflect the true net price being experienced by large corporate customers. Under Section 1847A of the Social Security Act, rebates must be reported in the quarter they are earned, not the quarter in which they are paid. When deferral arrangements expire and rebates are reported, ASPs have corrected suddenly and dramatically in this market. Independent practices making formulary decisions during the managed ASP period are doing so on the basis of pricing data that may not reflect the true market price. A real world study that omits this economic context does not provide the complete picture that independent practices need to make informed decisions.

3. Formulation Disparity and Patient Safety Equity Aflibercept-ayyh received FDA approval on August 23, 2024 in two formulations simultaneously. FDA review documents confirm that Amgen submitted separate labeling designating the single dose vial specifically as the physician sample formulation and the prefilled syringe as the commercial trade product. This was a deliberate product design decision made from the date of approval.

These two formulations carry meaningfully different preparation pathways and documented safety profiles. Published systematic review data reports consistently lower endophthalmitis rates with prefilled syringes compared to vials for intravitreal aflibercept, 0.01 to 0.02% versus 0.02 to 0.06%, attributed to reduced handling steps and contamination risk during preparation. This formulation disparity is compounded by Amgen’s own patient support program. The Amgen SupportPlus Co-Pay Program is explicitly limited to patients with commercial insurance and is expressly unavailable to patients whose costs are covered by Medicare, Medicaid, or any other federal or state healthcare program. The retina patient population is predominantly elderly and Medicare dependent. Those patients cannot access Amgen’s co-pay support and are most likely to depend on manufacturer provided samples. Amgen directs Medicare and Medicaid patients to independent third party nonprofit assistance programs over which it states it has no control and for which it takes no responsibility.

The result is a documented and foreseeable chain. Medicare and Medicaid patients are excluded from Amgen’s co-pay program (however, this is not unique to Amgen - all co-pay assistance programs generally assist commercial patients), are most dependent on manufacturer samples, and manufacturer samples are designated as single dose vials carrying a higher preparation burden and documented higher infection risk than the prefilled syringe received by commercially insured patients. The manuscript does not track formulation type as a variable, does not analyze it as a potential safety confounder, and does not address this disparity or its implications for the most financially vulnerable patients in the retina population.

1. A Note on the Broader Pattern The retina community should consider this study in the context of the commercial pattern it represents. Biosimilar manufacturers have repeatedly commissioned real world studies using data from corporate practice networks with which they have direct financial relationships, during periods of managed ASP supported by rebate deferral arrangements, to validate adoption that may be driven by institutional formulary decisions rather than independent clinical judgment. When those commercial arrangements unwind, as they consistently have in the retina biosimilar market, ASPs correct, networks transition to the next available product, and the cycle begins again.

This pattern normalizes commercially driven adoption as clinical evidence and leaves independent physician owned practices without the transparent and unbiased evidence they need to make informed formulary decisions. The patients most affected are disproportionately elderly, Medicare dependent, and reliant on manufacturer samples. They bear the downstream consequences of formulary decisions made on the basis of commercially managed evidence. The solution is not to suppress sponsored research. It is to hold it to standards that make it credible. Independent data sources. Genuine conflict of interest management. Economic outcomes alongside clinical outcomes. Transparent disclosure of rebate structures and their impact on reported ASP. Explicit discussion of patient safety equity implications when product design creates differential risk across patient populations. Until those standards are consistently applied the retina community should interpret sponsored biosimilar real world studies with appropriate skepticism regardless of which product they validate or which network’s data they use.

A Call for Independent Study The retina community would benefit from an independent study, not funded by the manufacturer, not using data from a distributor affiliated network, and not presented by paid consultants, that examines clinical and economic outcomes across a representative sample of US retina practices including independent physician owned practices.

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

Learn more at Review Commons

Reply to the reviewers

Reviewer #1

Summary: This manuscript has presented a high-throughput fluorescence recovery after photobleaching (HiT-FRAP) platform to screen genes affecting the dynamics of the nucleolar scaffold nucleophosmin (NPM1). The platform included the siRNA-based screening of 65 RNA helicases, 9 phylogenetically related helicase pairs, and 290 ribosomal proteins along with selected assembly factors. These factors were classified as those accelerating or decelerating NPM1 dynamics based on the t1/2 measurements. Combined with nucleolar morphological changes, the authors identified that depletion of early-stage (A-F) and later-stage (G-H) LSU assembly factors resulted in different nucleolar phenotypes, suggesting the pre-ribosome assembly can impact nucleolar morphology. Further exploring the potential mechanis m suggested that the NPM1's intrinsically disordered region (IDR) contributed to the nucleolar organization and dynamics.

Together, this well-designed study uncovered that the ribosome assembly, both the early and late ribosomal precursors can influence biophysical properties of the nucleolus. Below please find our concerns for the authors to consider to strengthen the major conclusions.

Major comments:

The main conclusion that NPM1's biophysical states directly impact its interaction strength with ribosome intermediates (and thereby nucleolar dynamics) should be further strengthened as listed below:

1). Given the nucleolus's complexity, an additional GC factor, or/and one more marker of other nucleolar regions, should be examined to substantiate the proposed impact of LSU-associated factors on nucleolar morphology (Figures 3, 4).

We thank the reviewer for this very important point. We have now included representative images for representative hits in major phenotypic clusters co-stained for SURF6, another GC marker, which shows similar localization patterns as NPM1 (Fig. S4B). For other nucleolar subcompartments, we have included images obtained from a cell line harboring endogenously tagged FBL-mNeonGreen (a marker for the DFC) for representative hits (Fig. S4A). We see a similar overall distribution of the DFC within the GC (i.e. DFCs distribute to fill the area of the disrupted GC), confirming our screen results. We look forward to further examining the changes in nucleolar subcompartment architecture in future work.

As additional support, we note that we probed NOG2, NOP53, and NOP2 in our IF results, all of which are GC-localized factors. We see a very similar distribution for these factors in our hits as for NPM1 (see Fig. S8D). In addition, FISH data for pre-rRNA precursors show similar morphological patterns as NPM1, further confirming our results (Fig. S7). We have noted this in text and have also included representative images in supplement.

2). Additional experiments are needed to support the proposed model that ribosomal intermediates, especially the pre-LSU complexes could determine nucleolar biophysical properties through the interaction with NPM1. Their direct interaction by biochemical assays should be provided. Also, when analyzing the interaction with other nucleolar factors, the authors should provide data that show NPM1 mutant expression levels were comparable to endogenous levels (Figures 4, 6).

We agree that directly probing NPM1's interactions with LSU precursors is critical to supporting our model, and we have addressed this through several complementary biochemical approaches. First, we performed immunoprecipitation of tagged NPM1 (NPM1-mScarlet, IP-ed using RFP-trap agarose) and assessed interaction with pre-LSU rRNA transcripts via Northern blot (Fig. 5D). We find that NPM1 interacts strongly with the 32S pre-rRNA. Second, we performed sucrose gradient sedimentation and find that NPM1 preferentially co-migrates with pre-60S complexes (Fig. 5B). Together with previous reports of NPM1-pre-LSU interactions, these data provide direct biochemical support for the proposed interaction.

To test whether interaction strength with pre-LSUs could regulate NPM1 dynamics, we next asked whether our NPM1 mutants that differ in their dynamics in turn interact differentially with pre-LSU complexes. Using co-IP Northern blot for ITS2 and sucrose co-sedimentation, we find that NPM1 mA3 pulls down more 32S and co-sediments more robustly with pre-60S complexes, while NPM1 mB2 shows reduced association (Fig. 5D, E; Fig. S10F, G). These data support that the strength of the NPM1-pre-LSU interaction is a determinant of NPM1 exchange dynamics, and, by extension, of nucleolar biophysical properties.

Exogenous mutant NPM1 is expressed at approximately 10% of endogenous levels (Fig. S10A). We address this in two ways. First, all interaction comparisons are made between WT and mutant exogenous constructs, not against endogenous NPM1, controlling for expression level differences. Second, we observe similar effects on interactions both in the presence of endogenous NPM1 and in null backgrounds, indicating that the differences we detect reflect NPM1 mutation, not expression level.

3). Northern Blotting should be done to dissect which pre-rRNA intermediates interact with NPM1 and contribute to the nucleolar dynamics (Figures 4B, D, F). These additional experiments should be feasible within a reasonable timeframe.

We agree with the reviewer and have performed northern blots for major hits in our different nucleolar phenotypes, and results reinforce what we see by FISH and qPCR (Fig. S6B). Briefly, depletion of the “RNA Exosome” hit SKIV2L2 results in smearing of pre-rRNA precursors that harbor both ITS1 and ITS2 and an accumulation of the 12S, in keeping with its role in end-processing of these transcripts. For “Other” hit PHF5A, we see an enrichment for 47S/45S/41S species, consistent with an early precursor stall. Notably, we do not see this phenotype for depletion of “Other” hit CNOT1, which suggests multiple processing defects may lead to a similar nucleolar phenotype. Treatment with PolI inhibitor CX5461 shows a depletion in ITS1 containing transcripts, and minimal impact on ITS2-containing transcripts, similar to FISH results. Lastly, depletion of “LSU” hits NOP53 and RPF2 leads to accumulation of the 32S and 12S species, in keeping with accumulation of abortive pre-LSUs.

In addition, the authors should provide the code and the hardware control procedures for HiT-FRAP to ensure reproducibility.

We thank the reviewer for this thoughtful suggestion. We have made our software available on GitHub (https://github.com/jess-sheu/colony_blob_bleacher) and archived on Zenodo

(https://doi.org/10.5281/zenodo.20275447).

According to the authors' statement, all the experiments are adequately replicated, and the statistical analysis is adequate.

Minor comments:

To enhance clarity and focus, consider the following:

1). Simplifying the HiT-FRAP screening section (Fig. 1-3) would emphasize the significant findings.

We have simplified text throughout to better highlight significant findings.

2). Expanding analysis and experimental validation could help to solidify the interdependency between rRNA / ribosome precursors and the NPM1- driven nucleolar dynamics (Fig. 4-5). Indeed, additional experiments suggested above in the major concerns should be supplemented here.

We have performed additional experiments to demonstrate the interdependency between ribosomal precursors and their interaction with NPM1 in shaping nucleolar dynamics, as described above.

Reviewer #1 (Significance (Required)):

This work has established a powerful toolkit, named HiT-FRAP, to identify factors involved in the organization and regulation of the membrane-less nucleolus, which will be useful for understanding the complexity not only the nucleolus, but likely other condensates in cells in the future. Using this platform and with the Granular Component (GC)-localized NPM1 as an indicator of nucleolar morphology, the authors found that the biophysical properties of the nucleolus are sensitive to the ordered assembly of ribosomes, in particular the LSU maturation steps at the GC. This finding is important as it suggests the interdependency between the dynamic rRNA processing and the functional assembly and morphology of the nucleolus. Further studies are warranted to analyze the dynamics of other nucleolar constituents, particularly those localized at other sub-nucleolar regions, to fully depict how exactly the nucleolar function is coordinated with its biophysical properties.

Reviewer #2

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Summary: The nucleolus is a multiphase biomolecular condensate whose primary function is ribosome biogenesis. There are mounting evidences that the material state of condensates is important for their function. Here the authors have probed how the material property of the nucleolus responds to inhibitions of ribosome biogenesis.

They have assessed nucleolar dynamics (molecular diffusivity) of a nucleolar protein, NPM1, by fluorescence recovery after photobleaching (FRAP). NPM1 is a protein that labels the periphery of the nucleolus (the so-called granular component, GC). (The nucleolus has 3 main subcompartments: the internal fibrillar centers, the middle dense fibrillar components, and the GC).

One of the main findings of the work is that inhibition of late steps of ribosome biogenesis increases fluidity (faster recovery of NPM1), while inhibition of earlier (and inhibition of mRNA processing -but see below) rather increases rigidification (slower recovery). They then attempt to correlate what is interpreted as biophysical changes to pre-ribosomal intermediates and interaction with NPM1.

Practically, the authors have produced reporter cell lines (HeLa) expressing stably (CRISPR engineering) mono or bi-allelic fluorescent version of NPM1; they have developed a powerful platform to conduct high throughout FRAP (this is really good); they have calibrated their system, initially with basic perturbations (ATP depletion, proteasome inhibition, etc), and then they focused on a family of trans-acting factors: the helicases, investigating systematically their effect on NPM1 recovery. They then extended their initial candidate-based screen to additional factors (using STRING interactions). This is nice and useful. Later in the work, they include in their analysis additional (morphological) features of nucleoli to cluster functionally their hits, as was done earlier by others in similar works. Finally, using recently published structural data (CryoEM), they attempt to correlate groups in the cluster with particular pre-ribosomal species. This part is less advanced and weaker than the initial part of the paper (screens and FRAP measurements).

Major comments:

-A major comment is with the compositional analysis of precursor intermediates that should be better defined. The stage assignment of particles is not quite as good as the screening part of the paper. At the RNA level, the authors provided FISH, as histograms of quantifications (see e.g. Fig 4D, and Fig SS6E). It would be necessary to show images, and to perform biochemistry. At the protein level, the authors provide immunostaining, but it does not really prove the detected protein is part of a particle,..

We thank the reviewer for this important critique. We have taken several steps to address both the stage assignment and biochemical characterization concerns.

Regarding stage assignment: We have consolidated our LSU phenotypic clusters (previously LSU1 and LSU2) into a single "late pre-LSU" group based on their shared features and proximity in PCA space. We want to be clear that this consolidation is intended to more accurately represent what our data can support: the screen reliably identifies factors whose perturbation produces a coherent late LSU assembly phenotype, and we do not wish to overstate the resolution of state assignment from imaging data alone. Sub-cluster distinctions are retained in supplementary materials for transparency. We have revised language throughout to reflect this framing.

Regarding biochemical characterization of intermediates: We have now performed Northern blots on strong hits within our phenotypic groups (Fig. S6B). For LSU cluster hits, we observe accumulation of the 32S and 12S species, indicating a stall in ITS2 processing, which is directly consistent with our ITS2 FISH results and confirms that the RNA-level phenotypes reflect genuine pre-rRNA processing defects rather than indirect effects. For "Other" group factor PHF5A, we observe 47/45/41S accumulation consistent with an early processing stall. We have also added representative FISH images to Fig. S7 to allow direct visual assessment of RNA-level phenotypes.

Regarding protein-level particle assignment: We agree that IF alone cannot establish that assembly factors are incorporated into discrete pre-ribosomal particles rather than existing as free factors. To more directly test whether the LSU cluster phenotypes reflect accumulation of genuine pre-ribosomal particles rather than mislocalized free factors we used NOP53 knockdown as a representative LSU cluster perturbation and, similar to RPF2 knockdown, see an accumulation of ITS2 and NOG2 in the nucleolus by FISH and IF (Fig. 4E). We then performed nuclear sucrose gradient fractionation and found that NOG2 co-migrates with the LSU peak and does not enrich in soluble fractions (Fig. 4F-H), supporting the interpretation that late pre-LSU particles accumulate in the nucleolus upon disruption of LSU cluster genes. Importantly, we also observe a strong decrease in co-sedimentation of NPM1 with the LSU peak upon depletion of NOP53 (Fig. 4G,H). This result, together with the Northern blot and FISH data, provides biochemical and cell biological evidence that the nucleolar phenotypes we identified by HiT-FRAP are associated with accumulation of late LSU assembly intermediates.

-Another concern is to know if NPM: a GC component located periphery of the condensate and a late assembly factor is an appropriate marker for assessing the effects on nucleolar material state of all (including early and late) inhibitions.

Would factors involved in earlier ribosomal assembly steps, and localized more internally would not be better tools to evaluate change in material states caused by alterations in early steps?

We appreciate this important point and agree that NPM1 reports primarily on GC dynamics. However, we would argue this is a feature rather than a limitation for two reasons.

First, the GC is the terminal assembly compartment through which pre-ribosomal particles must transit before nuclear export. Perturbations to earlier assembly steps, including FC/DFC-localized processes, likely propagate into GC dynamics, because stalled or aberrant particles accumulate in or are excluded from the GC. NPM1 FRAP thus functions as a downstream integrator of upstream assembly status, not only a reporter of GC-proximal events. This interpretation is consistent with our observation that depletion of early factors (and, therefore, depletion of downstream intermediates) do produce detectable NPM1 phenotypes in our screen. Second, the pattern of our screen results supports rather than undermines this logic: the striking enrichment of late LSU factors and near-complete absence of SSU hits is precisely what one would predict if NPM1 reports selectively on pre-LSU flux through the GC. A sensor that reported indiscriminately on all condensate perturbations would not produce this specificity.

We do acknowledge, however, that NPM1 cannot report on material state changes that are compartmentally confined to the FC or DFC and do not propagate outward. Extending this approach to internal markers remains an important future direction. To clarify the scope of our readout, we have revised the text to specify that we are monitoring GC dynamics, and we have added representative images of fibrillarin localization in Supplemental Figure S4A to illustrate the relationship between DFC and GC compartments in our experimental system.

-About the engineered cell lines used for screening by FRAP (Fig 1S): NPM1-mNeonGreen (biallelic with reduced expression of NPM1) and mScarlet (heterozygous): There is a need to characterize pre-rRNA processing in both cell lines to show they are not affected for ribosome biogenesis. This is important information since the entire work is based on these cells.

We have performed a Northern blot across the cell lines used in this paper as compared to their parent cell line and see no substantial difference in rRNA processing. We have included this data as Supplemental Figure 1D.

The screening cells are HeLa cells implying they are not physiologically regulated for p53. Nucleolar surveillance is a key regulatory surveillance loop triggered by ribosome biogenesis inhibitions leading to p53 stabilisation. How could this affect this work? Should key findings be confirmed in diploid p53 positive cells?

We acknowledge that our choice of HeLa cells limits our ability to distinguish cell-type-specific responses from more universal mechanisms and have added an explicit discussion of cell choice in the main text. To begin exploring the impact of p53, we performed gene depletions for representative hits across phenotypic clusters in untransformed, diploid hTERT-RPE cells that were lentivirally-transduced with NPM1-mScarlet and assessed nucleolar morphological phenotypes at smaller scale (Figure S6C, Supplementary Text). At baseline, RPE cells show more and smaller nucleoli than HeLa cells, which may reflect a difference in basal nucleolar assembly and, potentially, ribosome biogenesis, in keeping with previous observations that transformed cells rely more heavily on ribosome biogenesis than non-transformed.

Upon gene depletion, we found that hits from the "RNA exosome" cluster shows a different phenotype than seen in HeLa cells, where we observe less size difference and a marked decrease in eccentricity, which may reflect a p53 or cell type specific response. Depletion of the “Other” cluster gene PHF5A results in a milder though qualitatively similar phenotype as seen in HeLa cells, with nucleolar rounding and an increase in NPM1 intensity. Depletion of “LSU”-associated hits in RPE cells very robustly replicated most of the nucleolar features we observed in HeLa, which suggest that these are likely generalizable responses to LSU disruption. We have included this data in Supplementary Figure 5C. We note that we did not directly test whether p53 is stabilized upon depletion of our hits in RPE cells, and whether p53 activation feeds back on condensate dynamics remains an open area for future work. However, the concordance of LSU-associated phenotypes across HeLa and RPE cells, which differ substantially in p53 status, transformation state, and baseline nucleolar architecture, supports the generalizability of our core findings.

-About factor depletion, e.g. helicases, it's important to consider direct versus indirect effects on ribosome biogenesis, the timeline of depletion should be well described in the paper. Apparently, most factors, including the helicases were depleted for 72 hours, this is very long considering most of them play important roles in essential processes for cell homeostasis implying severely reduced growth at the time of capture (and the possibility of indirect effects).

We thank the reviewer for this important point. To directly address depletion timeline, we performed time courses for strong hits and monitored nucleolar morphology at 24 and 48 hour intervals (now included in Fig. S3D). Morphological changes begin to emerge by 48 hours across phenotypic classes; for the RPF2 LSU phenotype specifically, nucleolar expansion and decreased NPM1 intensity are detectable as early as 24 hours, inconsistent with a general stress response and more consistent with a direct downstream consequence of LSU assembly disruption. Moreover, despite all targeted genes being essential for homeostasis, phenotypic profiles are cluster-specific and associated with multiple genes of coherent function, which suggests that observed impacts are downstream of specific pathway inhibition rather than a general cellular stress response.

-Another cause of concern is that some perturbations (factor depletion) affect very deeply nucleolar structure/morphology (eg uL2 depletion shown in Fig 2C); how easy/difficult was it to control/make sure that a correct area was obliterated in the FRAP experiment using the (remarkable) data-adaptive approach. For cases where the nucleolus was deeply affected how did you check that a significant nucleolar area had been selected for analysis? It would be good to describe this in the text.

We manually ensured our segmentation protocol accurately captured nucleoli, defined by higher intensity regions of NPM1, for all depletion cases during screen development. As this is the key factor in ensuring where the bleach point is, most bleaches, even in disrupted cases, bleached the nucleolar interior. To address this point, we have included figures in the supplement (Fig. S4D) that show bleaching time courses for select highly disrupted hits uL2 and eL39.

• Fig 6C, interaction of NPM1 constructs with pre-ribosomes: the authors have tested interaction with select nucleolar proteins (NOP53, NOP2, NOG2, and uL2), which is not the same as preribosomes.

It would be important to see the interactions with precursors (Fig S9C, now histograms) please show the actual data, this was tested by qPCR, please show classical northern blots as RTqPCR have shown their limits in such applications.

Indeed, we cannot distinguish between assembly factors/ribosomal proteins that are associated with NPM1 in their latent, non-pre-LSU bound state versus those that are part of a developing ribosome. We have addressed this gap in several ways. Firstly, we have performed IP-northern blots for tagged NPM1-mutants, as suggested, and find that the mA3 mutant co-IPs more 32S than WT, while the mB2 binds less (Fig. 5D). We also performed sucrose gradient analysis of pre-ribosomal complexes and find that the mA3 mutant co-sediments more with the pre-60S peak, while mB2 co-sediments less (Fig. 5E). These findings are consistent with in vitro findings in the field that B2 mediates interactions with rRNA, while A3 occludes B2 through intramolecular interactions. Collectively with our co-IP western data, we believe the evidence strongly suggests that NPM1 mutants interact differentially with pre-LSU complexes.

-Minor comments:

-The effects of mRNA processing disruption on nucleolar dynamics could be (is most likely) very indirect (the so-called "slow hits"). The respective time course of inhibitions is important to describe.

We direct the reviewer to our response above for other phenotypes. For our "slow hit" / "Other" cluster, we also used the splicing inhibitor PladB as an orthogonal approach. Strikingly, nucleolar rounding was detectable within less than one hour of treatment, well before any general cell health effects would be expected, while dynamics changes required approximately 24 hours — suggesting that morphological and biophysical responses are kinetically separable and that the early morphological response is directly downstream of splicing inhibition. We have included a representative rounding timecourse in Fig. S8E.

Reviewer #2 (Significance (Required)):

-General assessment: strengths and limitations

Strengths: -The automated platform for high throughput FRAP\

-The authors develop a potentially interesting model where they attempt to connect rigidification/fluidity of a condensate to its function in assembly of large ribonucleoprotein complexes. -The manuscript reads very well; it has been prepared with great care (figures). Some complicated concepts are explained very well (Introduction/Discussion). Limitations: -particle stage assignment based on FISH and immunostaining only. The authors have not demonstrated that the LSU1 cluster = state F and LSU2 cluster = states G/H

-Advance: -Technological advance, high throughput FRAP, a powerful platform to interrogate macromolecular diffusivity.

-Several nucleolar screens have been conducted in the past (but at steady-state, not using FRAP), in these works textural and morphological features were used together with dimensionality reduction techniques to define functional clusters of genes that impact the homeostasis of the nucleolus. Often these references are cited but it could be useful to expand a bit on some of the earlier findings to bring the new ones in perspective. Some clusters (typically, the transcriptional cluster that disrupts the nucleolus; and the late binder ribosomal proteins) have been well identified before.

-Audience: Cell biologists, scientists involved in ribosome biogenesis research, scientists with an interest in helicases. The growing condensate community.

-Describe your expertise: ribosome biogenesis, structure-function relationships in the nucleolus, technological development in microscopy.

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

Summary: The authors use high throughput FRAP (HiT-FRAP) in arrayed genetic screens of HeLa cells expressing nucleophosmin (NPM1)-fluorescent protein variants to monitor the biophysical properties of the nucleolus in response to genetic perturbations. HiT-FRAP uses a data adaptive imaging strategy to automatically identify and photobleach fluorescently labeled organelles in living cells and acquire movies for FRAP. Quantitative analysis of FRAP curves include t1/2 and mobile fraction. NPM1 was monitored since it is an important nucleolar scaffolding protein that is thought to interact with many pre-ribosome intermediates.

The authors depleted 65 RNA helicases (+ 9 pairs) with siRNA and found that 15 of them either increased or decreased t1/2. Knockdowns were confirmed with western blotting. RNA helicase knockdowns with faster NPM1 diffusion were associated with large subunit (LSU) assembly. Most RNA helicase knockdowns with slower NPM1 diffusion were associated with early rRNA processing via the small subunit (SSU) intermediate. The authors screened an additional 290 gene depletions of many ribosomal proteins and assembly factors. With this expanded set of perturbations, they categorized nucleoli based on four morphological features in addition to t1/2 and mobile fraction. Using principal component analysis (PCA), the authors identified clusters of genes with similar effects on NPM1 dynamics and nucleolar morphology. From this secondary screen, the majority exhibited slower NPM1 dynamics. The knockdowns associated with faster NPM1 dynamics were associated with LSU assembly, similar to the helicase experiments. The authors further analyzed several mutants of NPM1 to elucidate the likely interactions between the scaffolding protein and ribosome biogenesis factors. The accumulation of early ribosomal intermediates were associated with decreases in NPM1 dynamics, and accumulation of late intermediates led to increased NPM1 dynamics. The findings established a link between the biophysical properties of the nucleolus and the stages of ribosome biogenesis.

Major comments:

• The claims are supported by experimentation.
• No additional experiments requested.
• The experiments are adequately replicated, and statistical analysis is sufficient. • Methods are very detailed, which should facilitate reproducibility. Minor comments:

• Prior studies are referenced appropriately.

• A bit more coverage of background on the nucleolar scaffolding protein, nucleophosmin (NPM1) would be helpful in the introduction, perhaps in favor of the details on ribosome biogenesis o Paragraph 2 could be shorter or placed elsewhere

We thank the reviewer for this suggestion and have now included some background on NPM1 in the introduction and have shortened paragraph 2.

• Figures

o In Figures 2 - 5: explicitly state in the figure caption what dotted lines are encircling (entire cell?)

We have now included this in the figure captions (they encircle the nucleus).

o In Figures 2 - 5: explicitly state what the mp-inferno LUT intensity in the images is quantitating (amount of NPM1?)

We have now included this in the figure captions (NPM1/mScarlet intensity).

o Figure 7: more detail in the figure caption

We have now expanded our model figure caption.

• The paper is quite dense with a lot of nice work, discussing many different genetic perturbations. It feels a bit overwhelming, and I think the biological significance gets somewhat lost in the presentation of all the data. Perhaps some of the presentation of results can be moved to the supplement in favor of a "leaner" main text. Currently, there are only figures in the supplement, but I feel that some of the text that is not central to the key conclusions can be moved to the supplement. I found myself getting a bit bogged down and having to re-read several times to catch the takeaway messages. Some of the clarifying statements that are found in the discussion section can be moved to the results section. In short, some reorganization would help with readability. One suggestion is to move the Inhibition of rRNA transcription or the RNA exosome leads to nucleolar fragmentation and/or the Perturbation of mRNA processing pathways results in slowed NPM1 dynamics and accumulation of rRNA precursors in the nucleolus to the supplement.

We thank the reviewer for this helpful suggestion. Due to this and other reviewers, we have now simplified discussion of phenotypic groups, including combining the “LSU” phenotypes into a single group and discussing LSU1/2 in the supplementary text. In addition, while we have chosen to keep the “rRNA transcription/exosome” and “Other” descriptions in the main text, they have been condensed and included in one main section with the other ribosome biogenesis phenotypes to highlight this key takeaway. Remaining discussion of phenotypes is now in supplemental text, as suggested.

Reviewer #3 (Significance (Required)):

• General Assessment: The main claim of the paper is that nucleolar phenotype (measured by morphology and NPM1 diffusivity) is correlated with stages in ribosome assembly - i.e. the stage of ribosome assembly determines the biophysical properties of the nucleolus. A strength of the study is the wide range of genetic perturbations tested enabled by the high throughput FRAP. With FRAP, I do worry a bit about using t1/2 as the sole dynamic measurement, but it is not a deal breaker. The authors introduce morphology as another way to characterize the nucleoli. • The claims are well supported by extensive experiments and data. The experiments are well designed, and proper controls were conducted. To validate the method, the authors used perturbations of NPM1 dynamics from the literature including ATP depletion, blocking glycolysis and oxidative phosphorylation, inhibition with MG132, and treatment with sodium arsenite. They observed slower NPM1 diffusivity under all validation conditions. • Advance: The authors have introduced a high-throughput technique for extracting diffusivity with FRAP, yielding a lot of data, but I think the paper suffers a bit in trying to present so much data in the main text. The mechanistic biological insights are compelling but get a bit overshadowed. Improved organization can help the messages come across more clearly. • To my knowledge, there is not a similar study in the literature as the detailed mechanisms of ribosome biogenesis are not well studied. • Audience: The audience for this manuscript seems to be biophysical researchers, thought there may be broader interest due to the wide screening of genetic perturbations. • Expertise: I have evaluated this manuscript from the perspective of a single-molecule biophysicist that studies protein-protein interactions between ribosome biogenesis factors. I am not an expert in FRAP, but I use FCS.

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

Learn more at Review Commons

Referee #3

Evidence, reproducibility and clarity

Summary:

The authors use high throughput FRAP (HiT-FRAP) in arrayed genetic screens of HeLa cells expressing nucleophosmin (NPM1)-fluorescent protein variants to monitor the biophysical properties of the nucleolus in response to genetic perturbations. HiT-FRAP uses a data adaptive imaging strategy to automatically identify and photobleach fluorescently labeled organelles in living cells and acquire movies for FRAP. Quantitative analysis of FRAP curves include t1/2 and mobile fraction. NPM1 was monitored since it is an important nucleolar scaffolding protein that is thought to interact with many pre-ribosome intermediates.

The authors depleted 65 RNA helicases (+ 9 pairs) with siRNA and found that 15 of them either increased or decreased t1/2. Knockdowns were confirmed with western blotting. RNA helicase knockdowns with faster NPM1 diffusion were associated with large subunit (LSU) assembly. Most RNA helicase knockdowns with slower NPM1 diffusion were associated with early rRNA processing via the small subunit (SSU) intermediate. The authors screened an additional 290 gene depletions of many ribosomal proteins and assembly factors. With this expanded set of perturbations, they categorized nucleoli based on four morphological features in addition to t1/2 and mobile fraction. Using principal component analysis (PCA), the authors identified clusters of genes with similar effects on NPM1 dynamics and nucleolar morphology. From this secondary screen, the majority exhibited slower NPM1 dynamics. The knockdowns associated with faster NPM1 dynamics were associated with LSU assembly, similar to the helicase experiments. The authors further analyzed several mutants of NPM1 to elucidate the likely interactions between the scaffolding protein and ribosome biogenesis factors. The accumulation of early ribosomal intermediates were associated with decreases in NPM1 dynamics, and accumulation of late intermediates led to increased NPM1 dynamics. The findings established a link between the biophysical properties of the nucleolus and the stages of ribosome biogenesis.

Major comments:

• The claims are supported by experimentation.
• No additional experiments requested.
• The experiments are adequately replicated, and statistical analysis is sufficient.
• Methods are very detailed, which should facilitate reproducibility.

Minor comments:

• Prior studies are referenced appropriately.
• A bit more coverage of background on the nucleolar scaffolding protein, nucleophosmin (NPM1) would be helpful in the introduction, perhaps in favor of the details on ribosome biogenesis
• Paragraph 2 could be shorter or placed elsewhere
• Figures
  • In Figures 2 - 5: explicitly state in the figure caption what dotted lines are encircling (entire cell?)
  • In Figures 2 - 5: explicitly state what the mp-inferno LUT intensity in the images is quantitating (amount of NPM1?)
  • Figure 7: more detail in the figure caption
• The paper is quite dense with a lot of nice work, discussing many different genetic perturbations. It feels a bit overwhelming, and I think the biological significance gets somewhat lost in the presentation of all the data. Perhaps some of the presentation of results can be moved to the supplement in favor of a "leaner" main text. Currently, there are only figures in the supplement, but I feel that some of the text that is not central to the key conclusions can be moved to the supplement. I found myself getting a bit bogged down and having to re-read several times to catch the takeaway messages. Some of the clarifying statements that are found in the discussion section can be moved to the results section. In short, some reorganization would help with readability. One suggestion is to move the Inhibition of rRNA transcription or the RNA exosome leads to nucleolar fragmentation and/or the Perturbation of mRNA processing pathways results in slowed NPM1 dynamics and accumulation of rRNA precursors in the nucleolus to the supplement.

Significance

• General Assessment: The main claim of the paper is that nucleolar phenotype (measured by morphology and NPM1 diffusivity) is correlated with stages in ribosome assembly - i.e. the stage of ribosome assembly determines the biophysical properties of the nucleolus. A strength of the study is the wide range of genetic perturbations tested enabled by the high throughput FRAP. With FRAP, I do worry a bit about using t1/2 as the sole dynamic measurement, but it is not a deal breaker. The authors introduce morphology as another way to characterize the nucleoli.
• The claims are well supported by extensive experiments and data. The experiments are well designed, and proper controls were conducted. To validate the method, the authors used perturbations of NPM1 dynamics from the literature including ATP depletion, blocking glycolysis and oxidative phosphorylation, inhibition with MG132, and treatment with sodium arsenite. They observed slower NPM1 diffusivity under all validation conditions.
• Advance: The authors have introduced a high-throughput technique for extracting diffusivity with FRAP, yielding a lot of data, but I think the paper suffers a bit in trying to present so much data in the main text. The mechanistic biological insights are compelling but get a bit overshadowed. Improved organization can help the messages come across more clearly.
• To my knowledge, there is not a similar study in the literature as the detailed mechanisms of ribosome biogenesis are not well studied.
• Audience: The audience for this manuscript seems to be biophysical researchers, thought there may be broader interest due to the wide screening of genetic perturbations.
• Expertise: I have evaluated this manuscript from the perspective of a single-molecule biophysicist that studies protein-protein interactions between ribosome biogenesis factors. I am not an expert in FRAP, but I use FCS.

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

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

Summary:

The nucleolus is a multiphase biomolecular condensate whose primary function is ribosome biogenesis. There are mounting evidences that the material state of condensates is important for their function. Here the authors have probed how the material property of the nucleolus responds to inhibitions of ribosome biogenesis. They have assessed nucleolar dynamics (molecular diffusivity) of a nucleolar protein, NPM1, by fluorescence recovery after photobleaching (FRAP). NPM1 is a protein that labels the periphery of the nucleolus (the so-called granular component, GC). (The nucleolus has 3 main subcompartments: the internal fibrillar centers, the middle dense fibrillar components, and the GC).

One of the main findings of the work is that inhibition of late steps of ribosome biogenesis increases fluidity (faster recovery of NPM1), while inhibition of earlier (and inhibition of mRNA processing -but see below) rather increases rigidification (slower recovery). They then attempt to correlate what is interpreted as biophysical changes to pre-ribosomal intermediates and interaction with NPM1. Practically, the authors have produced reporter cell lines (HeLa) expressing stably (CRISPR engineering) mono or bi-allelic fluorescent version of NPM1; they have developed a powerful platform to conduct high throughout FRAP (this is really good); they have calibrated their system, initially with basic perturbations (ATP depletion, proteasome inhibition, etc), and then they focused on a family of trans-acting factors: the helicases, investigating systematically their effect on NPM1 recovery. They then extended their initial candidate-based screen to additional factors (using STRING interactions). This is nice and useful. Later in the work, they include in their analysis additional (morphological) features of nucleoli to cluster functionally their hits, as was done earlier by others in similar works. Finally, using recently published structural data (CryoEM), they attempt to correlate groups in the cluster with particular pre-ribosomal species. This part is less advanced and weaker than the initial part of the paper (screens and FRAP measurements).

Major comments:

• A major comment is with the compositional analysis of precursor intermediates that should be better defined. The stage assignment of particles is not quite as good as the screening part of the paper.

At the RNA level, the authors provided FISH, as histograms of quantifications (see e.g. Fig 4D, and Fig SS6E). It would be necessary to show images, and to perform biochemistry. At the protein level, the authors provide immunostaining, but it does not really prove the detected protein is part of a particle,.. - Another concern is to know if NPM: a GC component located periphery of the condensate and a late assembly factor is an appropriate marker for assessing the effects on nucleolar material state of all (including early and late) inhibitions. Would factors involved in earlier ribosomal assembly steps, and localized more internally would not be better tools to evaluate change in material states caused by alterations in early steps? - About the engineered cell lines used for screening by FRAP (Fig 1S): NPM1-mNeonGreen (biallelic with reduced expression of NPM1) and mScarlet (heterozygous): There is a need to characterize pre-rRNA processing in both cell lines to show they are not affected for ribosome biogenesis. This is important information since the entire work is based on these cells. The screening cells are HeLa cells implying they are not physiologically regulated for p53. Nucleolar surveillance is a key regulatory surveillance loop triggered by ribosome biogenesis inhibitions leading to p53 stabilisation. How could this affect this work? Should key findings be confirmed in diploid p53 positive cells? - About factor depletion, e.g. helicases, it's important to consider direct versus indirect effects on ribosome biogenesis, the timeline of depletion should be well described in the paper. Apparently, most factors, including the helicases were depleted for 72 hours, this is very long considering most of them play important roles in essential processes for cell homeostasis implying severely reduced growth at the time of capture (and the possibility of indirect effects). - Another cause of concern is that some perturbations (factor depletion) affect very deeply nucleolar structure/morphology (eg uL2 depletion shown in Fig 2C); how easy/difficult was it to control/make sure that a correct area was obliterated in the FRAP experiment using the (remarkable) data-adaptive approach. For cases where the nucleolus was deeply affected how did you check that a significant nucleolar area had been selected for analysis? It would be good to describe this in the text. - Fig 6C, interaction of NPM1 constructs with pre-ribosomes: the authors have tested interaction with select nucleolar proteins (NOP53, NOP2, NOG2, and uL2), which is not the same as preribosomes. It would be important to see the interactions with precursors (Fig S9C, now histograms) please show the actual data, this was tested by qPCR, please show classical northern blots as RTqPCR have shown their limits in such applications.

Minor comments:

• The effects of mRNA processing disruption on nucleolar dynamics could be (is most likely) very indirect (the so-called "slow hits"). The respective time course of inhibitions is important to describe.

Significance

General assessment: strengths and limitations

Strengths:

• The automated platform for high throughput FRAP
• The authors develop a potentially interesting model where they attempt to connect rigidification/fluidity of a condensate to its function in assembly of large ribonucleoprotein complexes.
• The manuscript reads very well; it has been prepared with great care (figures). Some complicated concepts are explained very well (Introduction/Discussion).

Limitations:

• particle stage assignment based on FISH and immunostaining only. The authors have not demonstrated that the LSU1 cluster = state F and LSU2 cluster = states G/H

Advance:

• Technological advance, high throughput FRAP, a powerful platform to interrogate macromolecular diffusivity.
• Several nucleolar screens have been conducted in the past (but at steady-state, not using FRAP), in these works textural and morphological features were used together with dimensionality reduction techniques to define functional clusters of genes that impact the homeostasis of the nucleolus. Often these references are cited but it could be useful to expand a bit on some of the earlier findings to bring the new ones in perspective. Some clusters (typically, the transcriptional cluster that disrupts the nucleolus; and the late binder ribosomal proteins) have been well identified before.

Audience: Cell biologists, scientists involved in ribosome biogenesis research, scientists with an interest in helicases. The growing condensate community.

Describe your expertise: ribosome biogenesis, structure-function relationships in the nucleolus, technological development in microscopy.

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

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

Summary:

This manuscript has presented a high-throughput fluorescence recovery after photobleaching (HiT-FRAP) platform to screen genes affecting the dynamics of the nucleolar scaffold nucleophosmin (NPM1). The platform included the siRNA-based screening of 65 RNA helicases, 9 phylogenetically related helicase pairs, and 290 ribosomal proteins along with selected assembly factors. These factors were classified as those accelerating or decelerating NPM1 dynamics based on the t1/2 measurements. Combined with nucleolar morphological changes, the authors identified that depletion of early-stage (A-F) and later-stage (G-H) LSU assembly factors resulted in different nucleolar phenotypes, suggesting the pre-ribosome assembly can impact nucleolar morphology. Further exploring the potential mechanism suggested that the NPM1's intrinsically disordered region (IDR) contributed to the nucleolar organization and dynamics.

Together, this well-designed study uncovered that the ribosome assembly, both the early and late ribosomal precursors can influence biophysical properties of the nucleolus. Below please find our concerns for the authors to consider to strengthen the major conclusions.

Major comments:

The main conclusion that NPM1's biophysical states directly impact its interaction strength with ribosome intermediates (and thereby nucleolar dynamics) should be further strengthened as listed below:

1. Given the nucleolus's complexity, an additional GC factor, or/and one more marker of other nucleolar regions, should be examined to substantiate the proposed impact of LSU-associated factors on nucleolar morphology (Figures 3, 4).
2. Additional experiments are needed to support the proposed model that ribosomal intermediates, especially the pre-LSU complexes could determine nucleolar biophysical properties through the interaction with NPM1. Their direct interaction by biochemical assays should be provided. Also, when analyzing the interaction with other nucleolar factors, the authors should provide data that show NPM1 mutant expression levels were comparable to endogenous levels (Figures 4, 6).
3. Northern Blotting should be done to dissect which pre-rRNA intermediates interact with NPM1 and contribute to the nucleolar dynamics (Figures 4B, D, F). These additional experiments should be feasible within a reasonable timeframe. In addition, the authors should provide the code and the hardware control procedures for HiT-FRAP to ensure reproducibility. According to the authors' statement, all the experiments are adequately replicated, and the statistical analysis is adequate.

Minor comments:

To enhance clarity and focus, consider the following:

1. Simplifying the HiT-FRAP screening section (Fig. 1-3) would emphasize the significant findings.
2. Expanding analysis and experimental validation could help to solidify the interdependency between rRNA / ribosome precursors and the NPM1- driven nucleolar dynamics (Fig. 4-5). Indeed, additional experiments suggested above in the major concerns should be supplemented here.

Significance

This work has established a powerful toolkit, named HiT-FRAP, to identify factors involved in the organization and regulation of the membrane-less nucleolus, which will be useful for understanding the complexity not only the nucleolus, but likely other condensates in cells in the future. Using this platform and with the Granular Component (GC)-localized NPM1 as an indicator of nucleolar morphology, the authors found that the biophysical properties of the nucleolus are sensitive to the ordered assembly of ribosomes, in particular the LSU maturation steps at the GC. This finding is important as it suggests the interdependency between the dynamic rRNA processing and the functional assembly and morphology of the nucleolus. Further studies are warranted to analyze the dynamics of other nucleolar constituents, particularly those localized at other sub-nucleolar regions, to fully depict how exactly the nucleolar function is coordinated with its biophysical properties.

we should focus more on the eye movement and easy accessibility for 3D printers instead of more AI stuff with LLMs. we should be developing more technology to make it easier to convey our thoughts than have a machine think it for us.

I agree with the author. i heavily doubt that air gestures will replace existing interaction because most people like to have something tangible in their hand. they want an accurate visual anchor does their anchor like a cursor or a finger tip.

I think that Shota is right about air gestures not becoming a replacement for physically swiping or using a mouse, because using air gestures can only be functional for so many things and this method doesn't encompass the full functionalities that physically tapping and swiping do. It seems like developing the technologies to perfect this method would likely be very expensive and it probably would not be worth it in the long run for general usage. Overall, air gestures may present benefits in certain use cases, but they will likely not overtake traditional methods of interaction.

I agree with the article when it says that the LLMs are useful in the many processes, including GUI design and implementation. I feel the way we are going AI is only getting better, and in the future it'll continue implement truly usable GUIs with minimal human intervention by combining them with the reinforcement learning agent-based testing.

With this paper I agree with the examination of user classification this is very important because the data shows that participants who don't perform the task the right away don't know what they are doing and are not understanding the topic well. I can relate that when I do the task the right and correct way first it all makes sense and second you are doing it the correct way and it states in the second paragraph to the paper that what the key findings was and all of the data to it.

Using humans to test GUI's can cause issues as not all humans will perform the various tasks at the same level. It's important to understand what sample size is needed to collect an acurate enough data set. It will be interesting to see in the future how AI can make the testing not just more accurate, but to be able to do it more quickly.

Fitt's law is an extremely important part in marketing as it's used to make the consumers life easier. Easier to read, navigate, and make a decision. A more categorized, less scattered page helps direct the consumer so companies use this tactic to make things less overwhelming.

With this paper I agree with the examination of user classification this is very important because the data shows that participants who don't perform the task the right away don't know what they are doing and are not understanding the topic well. I can relate that when I do the task the right and correct way first it all makes sense and second you are doing it the correct way and it states in the second paragraph to the paper that what the key findings was and all of the data to it.

LY corporations created a dedicated HCI team after they realized that many of their company problems could be solved using HCI methods. This article also explains how researches use models like Fitt's Law to measure how quickly and accurately the users can click or tap the targets on screens.

I agree with this point because I don’t think air gestures will fully replace touchscreens or a mouse. They seem more useful for certain situations where hands-free control is easier, like driving or medical settings. But for tasks that need accuracy, like typing or detailed work, traditional input methods will probably still be better.

when they conducted the data test, I think the most interesting thing is the results. The people who were conducting a small task such as the pinch to the image followed this preliminary task more precise than the main task itself. This is interesting because the main task at hand like data labeling or short-text should be the tasks that are followed to be as precise as they can however this test made it seem like that shorter much easier test of pinching an image resulted in people closely following the intrustions comapred to the larger task.

The researcher used Fitts' law to change how user interfaces can be more practical, less confusing and minimize task time. They did this by estimating the time required to point at targets like icons and hyperlinks. The research also took account how accurately users can perform tasks. This would lead to an innovation that would lead to agents to operate a GUI and perform tasks at scale.

Smart home devices like Amazon Echo already achieves this differently. For sure air gestures can be a great addition but it's already obsolete because Amazon Echo can listen, which majority of the population prefers to use their own voice.

This makes sense in the context of fitts law where we are measuring the time required to point at a target. For example, if we have a button on screen and the user is able to tap the button using air gestures, then the time it takes to hit that button would be slightly faster than tapping the actual screen. This is pretty similar to when the professor showed off vimium to the class where instead of navigating with a cursor, we navigate with the keyboard eliminating time to click a link or url.

One thing I found interesting in the article about Shota Yamanaka was how he focuses on designing technology around real human behavior instead of just making apps look better. I also liked his idea that AI could help improve app interfaces and make them easier for people to use in the future.Bold

Using AI agents to simulate diverse user demographics could revolutionize UX design by making accessibility testing faster and much less expensive than traditional human trials.

Classically, Fitts’ law has been used to estimate the time required to point at targets like icons and hyperlinks. This enables the design of graphical user interfaces (GUIs) that minimize task time.<

Its pretty cool to see leaders of successful companies and initiatives use the laws of UX / UI design in a non-malicious way. in reference to the quote above, Fitts' law was used to benefit workflow by enabling users to navigate to their tools quicker.

Fitts' Law is something we have talked about in class. It says that close and large objects are easier to interact with, and results in the user taking less time on the website. It is very interesting to see this law being applied in the real world. It is expanded here to not only speed, but accuracy. Machine learning is getting faster and more accurate. This will drasctically change business operations, as agents can take over testing and further enchance the product. Overall, there is still so much potential in human-computer interaction.

I agree with this idea and how the GUIs will eventually become fully automated in the near future with close to no human work to be done to create these GUIs. AI is only getting better and over time very soon this will become a real possibility.

That's certainly a way to preemptively counter the Christian meme.

wow. That's why Sunday is the first day of the week in English-speaking countries. But for some reason in Spain it's the last day?

Apparently in Europe in the 19th-20th century, with the push towards a two day "week-end", it got standardized that Sabbath and Sunday are at the end of the week.

Apparently here it just means believers. As opposed to Catholic saints are formally canonized people in Heaven.

and ofc also Jesus

Palpatine-ass line.

I'm mildly disturbed that I know bible references in Star Wars because I memorized many Star Wars lines, as opposed to because I read the bible.

Is Pilate therefore actually innocent? Or does he just want to be?

I just... my whole education was taking notes on a laptop or ipad. Without notifications. with full attention and eye contact maintained with lecturers/interlocutors throughout. It's possible. And a lot better for health in certain ways than printing a physical packet-- lugging around a bunch of textbooks vs. an iPad, for one. I wish we wouldn't metonynically use "screens" for what we really mean-- the theft of our attention by companies vying for our attention through algorithms that are designed to distract us and notifications. It's not screens at all.

why not fix the design of the UI instead of demonizing the hardware of the "screen"?

very confident assertion of causality here. I'd love to hear this author's response to Stolen Focus which looks at the myriad of reasons that our attention spans are collectively plummeting

does Canvas or the reporting institutions sell data to the tech giants? I heard this rumor before- where is it coming from?

Assessment pursues less measurement of "intellect" than more a pursuit of a meaningful education- and better guarantees that someone who enrolls will gain certain life skills (including the affective ones- like finding joy in learning). Which I think the author also wants? But they just want "effectiveness" to not be captured and analyzed at an institutional level? Unsure what the platonic ideal of institutional learning is here.

do not modern teaching techniques, which include offloading logistic work to the LMS, leave room for more imaginative inquiries like these?

why did the author leave off the affective outcome, of developing appreciation and a new source of inspiration? affective outcomes are important too!

I don't think this author understands the art of a good rubric

I'd like to hear about a more instructive alternative.

but in the paper paradigm, an assignment might only exist on someone's hard drive, which is also easily corrupted?

right, the LMS can't measure those... but other SIS can? And "students will fall in love with..." can become a more directly observable learning objective. (or perhaps this one justifies self-reporting). but is the core argument here that LOs = bad because they can't be measured, or shouldn't be measured? How does the author feel so confidently that a class or institution is effective or not, then?

curious about the reference for this?

I think that this is the global trend, but some places have seen an increase in distribution of labor, with more specialized staff experts, like accessibility experts and instructional designers. I think the management decisions to put more work on the faculty is just that-- a management decision. Not a natural consequence of better information systems (like the LMS), but a natural consequence of maximizing profit.

Is the admin assistant doing the work of passing along information really the gold standard?

If the admin assistant's information networking skills can be deferred to digital systems, might this human resource- and person used as social glue- be used for greater purposes, like maybe bringing people together?

Agree with the observation- but this isn't a technology at large problem- this is a capitalism & attention economy problem.

I read this article that talked about a hiker whose phone was dying while he was out on a hike, resulting in him sending one last photo to a friend and explaining that he was lost. This photo then was used to locate the hiker when someone was able to 3D recreate the area he took the photo in, leading to rescuers finding the missing hiker.

I think this article was quite interesting in that it was a perfect case point regarding the weaknesses of crowdsourced information platforms such as Wikipedia. Although it was concerning that congressional staffers were editing articles to remove criticism to begin with, the fact that those edits were publicly traceable later and thus quickly corrected also shows how strong moderation/oversight helps keep this crowdsourcing in check.

This article suggests that technology should offer more than the offline face to face interaction. Users can have more various form of interaction online.

This article is about a missing hiker, Rene Compean. He was lost in a rural area and before his phone died sent a photo to one of his friends asking to be found. The police department decided to publicly ask for help (crowdsourcing). Within a few days Rene was found safe.

I think it's crazy how with so little surroundings people are able to find enough background information to actually locate him.

The WIRED video explains and demonstrates the social etiquette involved when humans and robots work together in office settings. The video uses humor and real-life examples to show that both people and robot users need to communicate respectfully, avoid surprising each other, and adapt to the challenges of technology in the workplace.

The video was super off putting. I am really glad that it's 11 years old and that didnt become reality that is very odd and I cant say I would want to work in a place with robot co workers. The idea of there being a remote worker that just rolls around the office also seems so unproductive. It is interesting to see what people thought the future of remote working would be 10 years ago before we were forced to become adapted at working from home.

I have noticed this same thing whenever I use Twitter, but I never knew it was a true statistic. It seems that when I open Twitter, the algorithm likes to show me the same accounts over and over, despite me not liking any posts. I don't use Twitter frequently nor do I ever like posts since I just am not very active on the site, so it is was weird to see the same accounts posting unfunny / uninteresting things all the time. Made me curious why it was even recommending me these posts in the first place.

Hearing the statistic of most of the tweets on twitter being from the same few people isn't very shocking. However, now I am wondering if 90% of twitter is being interacted with using bots then what percentage of real people are actually consistently interacting with twitter. Would the percentage be even lower?

This sentence emphasizes that crowdsourcing is critical to the effective communication. It suggests how communication between a lot of people can be properly done depending on various situations.

I think platforms are definitely in a position to promote accurate and constructive contributions while simultaneously limiting the visibility of misinformation and mob behavior. They could be utilizing, for example, features like stronger moderation by reducing engagement-driven amplification, which could work to help encourage healthier crowdsourcing.

I feel as though I probably have unintentionally participated in crowdsourcing online, specifically on platforms such as TikTok, especially as someone who typically engages with the content I watch. However, I don't particularly mind if this is the case.