Reviewer #1 (Public review):

Summary:

Dorrego-Rivas et al. investigated two different DA neurons and their neurotransmitter release properties in the main olfactory bulb. They found that the two different DA neurons in mostly glomerular layers have different morphologies as well as electrophysiological properties. The anaxonic DA neurons are able to self-inhibit but the axon-bearing ones are not. The findings are interesting and important to increase the understanding both of the synaptic transmissions in the main olfactory bulb and the DA neuron diversity. However, there are some major questions that the authors need to address to support their conclusions.

(1) It is known that there are two types of DA neurons in the glomerular layer with different diameters and capacitances (Kosaka and Kosaka, 2008; Pignatelli et al., 2005; Angela Pignatelli and Ottorino Belluzzi, 2017). In this manuscript, the authors need to articulate better which layer the imaging and ephys recordings took place, all glomerular layers or with an exception. Meanwhile, they have to report the electrophysiological properties of their recordings, including capacitances, input resistance, etc.

(2) It is understandable that recording the DA neurons in the glomerular layer is not easy. However, the authors still need to increase their n's and repeat the experiments at least three times to make their conclusion more solid. For example (but not limited to), Fig 3B, n=2 cells from 1 mouse. Fig.4G, the recording only has 3 cells.

(3) The statistics also use pseudoreplicates. It might be better to present the biology replicates, too.

(4) In Figure 4D, the authors report the values in the manuscript. It is recommended to make a bar graph to be more intuitive.

(5) In Figure 4F and G, although the data with three cells suggest no phenotype, the kinetics looked different. So, the authors might need to explore that aside from increasing the n.

(6) Similarly, for Figure 4I and J, L and M, it is better to present and analyze it like F and G, instead of showing only the after-antagonist effect.

Comments on revisions:

In the rebuttal, the authors argued that it had been extremely hard to obtain recordings stable enough for before-and-after effects on the same cell. Alternatively, they could perform the before-and-after comparison on different cells.

Author response:

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

Reviewer #1 (Public review):

This Reviewer was positive about the study, stating ‘The findings are interesting and important to increase the understanding both of the synaptic transmissions in the main olfactory bulb and the DA neuron diversity.’ They provided a number of helpful suggestions for improving the paper, which we have incorporated as follows:

(1) It is known that there are two types of DA neurons in the glomerular layer with different diameters and capacitances (Kosaka and Kosaka, 2008; Pignatelli et al., 2005; Angela Pignatelli and Ottorino Belluzzi, 2017). In this manuscript, the authors need to articulate better which layer the imaging and ephys recordings took place, all glomerular layers or with an exception. Meanwhile, they have to report the electrophysiological properties of their recordings, including capacitances, input resistance, etc.

We thank the Reviewer for this clarification. Indeed, the two dopaminergic cell types we study here correspond directly to the subtypes previously identified based on cell size. Our previous work showed that axon-bearing OB DA neurons have significantly larger somas than their anaxonic neighbours (Galliano et al. 2018), and we replicate this important result in the present study (Figure 3D). In terms of electrophysiological correlates of cell size, we now provide full details of passive membrane properties in the new Supplementary Figure 4, as requested. Axon-bearing DA neurons have significantly lower input resistance and show a non-significant trend towards higher cell capacitance. Both features are entirely consistent with the larger soma size in this subtype. We apologise for the oversight in not fully describing previous categorisations of OB DA neurons, and have now added this information and the appropriate citations to the Introduction (lines 56 to 59 of the revised manuscript). 

In terms of cell location, all cells in this study were located in the OB glomerular layer. We sampled the entire glomerular layer in all experiments, including the glomerular/EPL border where the majority of axon-bearing neurons are located (Galliano et al. 2018). This is now clarified in the Materials and Methods section (lines 535 to 537 and 614 to 616 of the revised manuscript).

(2) It is understandable that recording the DA neurons in the glomerular layer is not easy. However, the authors still need to increase their n's and repeat the experiments at least three times to make their conclusion more solid. For example (but not limited to), Fig 3B, n=2 cells from 1 mouse. Fig.4G, the recording only has 3 cells.

Despite the acknowledged difficulty of these experiments, we have now added substantial extra data to the study as requested. We have increased the number of cells and animals to further support the following findings:

Fig 3B: we now have n=5 cells from N=3 mice. We have created a new Supplementary Figure 1 to show all the examples.

Figure 4G: we now have n=6 cells from N=4 mice.

Figure 5G: we now have n=3 cells from N=3 mice.

The new data now provide stronger support for our original conclusions. In the case of auto-evoked inhibition after the application of D1 and D2 receptor antagonists, a nonsignificant trend in the data suggests that, while dopamine is clearly not necessary for the response, it may play a small part in its strength. We have now included this consideration in the Results section (lines 256 to 264 of the revised manuscript).

(3) The statistics also use pseudoreplicates. It might be better to present the biology replicates, too.

Indeed, in a study focused on the structural and functional properties of individual neurons, we performed all comparisons with cell as the unit of analysis. This did often (though not always) involve obtaining multiple data points from individual mice, but in these low-throughput experiments n was never hugely bigger than N. The potential impact of pseudoreplicates and their associated within-animal correlations was therefore low. We checked this in response to the Reviewer’s comment by running parallel nested analyses for all comparisons that returned significant differences in the original submission. These are the cases in which we would be most concerned about potential false positive results arising from intra-animal correlations, which nested tests specifically take into account (Aarts et al., 2013). In every instance we found that the nested tests also reported significant differences between anaxonic and axonbearing cell types, thus fully validating our original statistical approach. We now report this in the relevant section of the Materials and Methods (lines 686 to 691 of the revised manuscript).

(4) In Figure 4D, the authors report the values in the manuscript. It is recommended to make a bar graph to be more intuitive.

This plot does already exist in the original manuscript. We originally describe these data to support the observation that an auto-evoked inhibition effect exists in anaxonic neurons (corresponding to now lines 240 to 245 of the revised manuscript). We then show them visually in their entirety when we compare them to the lack of response in axon-bearing neurons, depicted in Figure 5C. We still believe that this order of presentation is most appropriate for the flow of information in the paper, so have maintained it in our revised submission.

(5) In Figure 4F and G, although the data with three cells suggest no phenotype, the kinetics looked different. So, the authors might need to explore that aside from increasing the n.

We thank the Reviewer for this suggestion. To quantify potential changes in the autoevoked inhibition response kinetics, we fitted single exponential functions and compared changes in the rate constant (k; Methods, lines 650 to 652 of the revised manuscript). Overall, we observed no consistent or significant change in rate constant values after adding DA receptor antagonists. This finding is now reported in the Results section (lines 260 to 263 of the revised manuscript) and shown in a new Supplementary Figure 3.

(6) Similarly, for Figure 4I and J, L and M, it is better to present and analyze it like F and G, instead of showing only the after-antagonist effect.

We agree that the ideal scenario would have been to perform the experiments in Figure 4J and 4M the same way as those in Figure 4G, with a before vs after comparison. Unfortunately, however, this was not practically possible. 

When attempting to apply carbenoxelone to already-patched cells, we found that this drug highly disrupted the overall health and stability of our recordings immediately after its application. This is consistent with previous reports of similar issues with this compound (e.g. Connors 2012, Epilepsy Currents; Tovar et al., 2009, Journal of Neurophysiology). After many such attempts, the total yield of this experiment was one single cell from one animal. Even so, as shown in the traces below, we were able to show that the auto-evoked inhibition response was not eliminated in this specific case:

Author response image 1.

Traces of an AEI response recorded before (magenta) and after (green) the application of carbenoxolone (n=1 cell from N=1 mouse).

In light of these issues, we instead followed published protocols in applying the carbenoxolone directly in the bath without prior recording for 20 minutes (following Samailova et al., 2003, Journal of Neurochemistry) and ran the protocol after that time. Given that our main question was to ask whether gap junctions were strictly necessary for the presence of any auto-evoked inhibition response, our positive findings in these experiments still allowed us to draw clear conclusions.

In contrast, the issue with the NKCC1 antagonist bumetanide was time. As acknowledged by this Reviewer, obtaining and maintaining high-quality patch recordings from OB DA neurons is technically challenging. Bumetanide is a slow-acting drug when used to modify neuronal chloride concentrations, because in addition to the time it takes to reach the neurons and effectively block NKCC1, the intracellular levels of chloride subsequently change slowly. Studies using this drug in slice physiology experiments typically use an incubation time of at least 20 minutes (e.g. Huberfeld et al., 2007, Journal of Neuroscience), which was incompatible with productive data collection in OB DA neurons. Again, after many unsuccessful efforts, we were forced instead to include bumetanide in the bath without prior recording for 20-30 minutes. As with the carbenoxolone experiment, our goal here was to establish whether autoevoked inhibition was in any way retained in the presence of this drug, so our positive result again allowed us to draw clear conclusions.

Reviewer #1 (Recommendations for the authors):

(1) I suggest the authors reconsider the terminology. For example, they use "strikingly" in their title. The manuscript reported two different transmitter release strategies but not the mechanisms, and the word "strikingly" is not professional, either.

We appreciate the Reviewer’s attention to clarity and tone in the manuscript title, and have nevertheless decided to retain the original wording. The almost all-or-nothing differences between closely related cell types shown in structural and functional properties here (Figures 3F & 5C) are pronounced, extremely clear and easily spotted – all properties appropriate for the word ‘striking.’ In addition, we note that the use of this term is not at all unprofessional, with a PubMed search for ‘strikingly’ in the title of publications returning over 200 hits.

(2) Similarly, almost all confocal scopes are 3D because images can be taken at stacks. So "3D confocal" is misleading.

We understand that this is misleading. We have now replaced the sentence ‘Example snapshot of a 3D confocal stack of…’ by ‘Example confocal images of…’ in all the figure legends that apply.

(3) It is recommended to present the data in bar graphs with data dots instead of showing the numbers in the manuscript directly.

We agree entirely, and now present data plots for all comparisons reported in the study (Supplementary Figures 2, 4 and 5).

Reviewer #2 (Recommendations for the authors):

(1) Several experiments report notably small sample sizes, such as in Figures 3B and 5G, where data from only 2 cells derived from 1-2 mice are presented. Figures 4E-G also report the experimental result only from 3 cells derived from 3 mice. To enhance the statistical robustness and reliability of the findings, these experiments should be replicated with larger sample sizes.

As per our response to Reviewer 1’s comment #2 above, and to directly address the concern that some evidence was ‘incomplete’, we have now added significant extra data and analysis to this revised submission (Figures 4 and 5; and Supplementary Figure 1). We believe that this has further enhanced the robustness and reliability of our findings, as requested.

(2) The authors utilize vGAT-Cre for Figures 1-3 and DAT-tdTomato for Figures 4-5, raising concerns about consistency in targeting the same population of dopaminergic neurons. It remains unclear whether all OB DA neurons express vGAT and release GABA. Clarification and additional evidence are needed to confirm whether the same neuronal population was studied across these experiments.

Although we indeed used different mouse lines to investigate structural and functional aspects of transmitter release, we can be very confident that both approaches allowed us to study the same two distinct DA cell types being compared in this paper. Existing data to support this position are already clear and strong, so in this revision we have focused on the Reviewer’s suggestion to clarify the approaches we chose.

First, it is well characterised that in mouse and many other species all OB DA neurons are also GABAergic. This has been demonstrated comprehensively at the level of neurochemical identity and in terms of dopamine/GABA co-release, and is true across both small-soma/anaxonic and large-soma/axon-bearing subclasses (Kosaka & Kosaka 2008; 2016; Maher & Westbrook 2008; Borisovska et al., 2013; Vaaga et al., 2016; Liu et al. 2013). To specifically confirm vGAT expression, we have also now provided additional single-cell RNAseq data and immunohistochemical label in a revised Figure 1 (see also Panzanelli et al., 2007, now referenced in the paper, who confirmed endogenous vGAT colocalisation in TH-positive OB neurons). Most importantly, by using vGAT-cre mice here we were able to obtain sufficient numbers of both anaxonic and axon-bearing DA neurons among the vGAT-cre-expressing OB population. We could unambiguously identify these cells as dopaminergic because of their expression of TH protein which, due to the absence of noradrenergic neurons in the OB, is a specific and comprehensive marker for dopaminergic cells in this brain region (Hokfelt et al., 1975; Rosser et al., 1986; Kosaka & Kosaka 2016). Crucially, both axon-bearing and anaxonic OB DA subtypes strongly express TH (Galliano et al., 2018, 2021). We have now added additional text to the relevant Results section (lines 99 to 108 of the revised manuscript) to clarify these reasons for studying vGAT-cre mice here.

We were also able to clearly identify and sample both subtypes of OB DA neuron using DAT-tdT mice. Our previous published work has thoroughly characterised this exact mouse line at the exact ages studied in the present paper (Galliano et al., 2018; Byrne et al., 2022). We know that DAT-tdT mice provide rather specific label for TH-expressing OB DA neurons (75% co-localisation; Byrne et al., 2022), but most importantly we know which non-DA neurons are labelled in this mouse line and how to avoid them. All nonTH-expressing but tdT-positive cells in juvenile DAT-tdT mice are small, dimly fluorescent and weakly spiking neurons of the calretinin-expressing glomerular subtype (Byrne et al., 2022). These cells are easily detected during physiological recordings, and were excluded from our study here. This information is now provided in the relevant Methods section (lines 616 to 619 of the revised manuscript, also referenced in lines 236 to 240 of the results section), and we apologise for its previous omission. Finally, we have shown both structurally and functionally that both axon-bearing and anaxonic OB DA subtypes are labelled in DAT-tdT mice (Galliano et al., 2018, Tufo et al., 2025; present study). Overall, these additional clarifications firmly establish that the same neuronal populations were indeed studied across our experiments.

(3) The low TH+ signal in Figure 1D raises questions regarding the successful targeting of OB DA neurons. Further validation, such as additional staining, is required to ensure that the targeted neurons are accurately identified.

As noted in our response to the previous comment, TH is a specific marker for dopaminergic neurons in the mouse OB, and is widely used for this purpose. Labelling for TH in our tissue is extremely reliable, and in fact gives such strong signal that we were forced to reduce the primary antibody concentration to 1:50,000 to prevent bleedthrough into other acquisition channels. Even at this concentration it was extremely straightforward to unambiguously identify TH-positive cells based on somatic immunofluorescence. We recognise, however, that the original example image in Figure 1D was not sufficiently clear, and have now provided a new example which illustrates the TH-based identification of these cells much more effectively. 

(4) Estimating the total number of dopaminergic neurons in the olfactory bulb, along with the relative proportions of anaxonic and axon-bearing neuron subtypes, would provide valuable context for the study. Presenting such data is crucial to underscore the biological significance of the findings.

This information has already been well characterised in previous studies. Total dopaminergic cell number in the OB is ~90,000 (Maclean & Shipley, 1988; Panzanelli et al., 2007; Parrish-Aungst et al., 2007). In terms of proportions, anaxonic neurons make up the vast majority of these cells, with axon-bearing neurons representing only ~2.5% of all OB dopaminergic neurons at P28 (Galliano et al., 2018). Of course, the relatively low number of the axon-bearing subtype does not preclude its having a potentially large influence on glomerular networks and sensory processing, as demonstrated by multiple studies showing the functional effects of inter-glomerular inhibition (Kosaka & Kosaka, 2008; Liu et al., 2013; Whitesell et al., 2013; Banerjee et al., 2015). This information has now been added to the Introduction (line 47 and lines 59 to 62 of the revised manuscript).

(5) The authors report that in-utero injection was performed based on the premise that the two subclasses of dopaminergic neurons in the olfactory bulb are generated during embryonic development. However, it remains unclear whether in-utero injection is essential for distinguishing between these two subclasses. While the manuscript references a relevant study, the explanation provided is insufficient. A more detailed justification for employing in-utero injection would enhance the manuscript's clarity and methodological rigor.

We apologise for the lack of clarity in explaining the approach. In utero injection is not absolutely essential for distinguishing between the two subclasses, but it does have two major advantages. 1) Because infection happens before cells migrate to their final positions, it produces sparse labelling which permits later unambiguous identification of individual cells’ processes; and 2) Because both subclasses are generated embryonically (compared to the postnatal production of only anaxonic DA neurons), it allows effective targeting of both cell types. We have now expanded the relevant section of the Results to explain the rationale for our approach in more detail (lines 109 to 116 of the revised manuscript).

(6) In Figures 1A and 4A, it appears that data from previously published studies were utilized to illustrate the differential mRNA expression in dopaminergic neurons of the olfactory bulb. However, the Methods section and the manuscript lack a detailed description of how these dopaminergic neurons were classified or analyzed. Given that these figures contribute to the primary dataset, providing additional explanation and context is essential to ensure clarity of the findings.

We apologise for the lack of clarity. We have now extended the part of the methods referring to the RNAseq data analysis (lines 666 to 678 of the revised manuscript). 

(7) In Figure 2C, anaxonic dopamine neurons display considerable variability in the number of neurotransmitter release sites, with some neurons exhibiting sparse sites while others exhibit numerous sites. The authors should address the potential biological or methodological reasons for this variability and discuss its significance.

We thank the Reviewer for highlighting this feature of our data. We have now outlined potential methodological reasons for the variability, whilst also acknowledging that it is consistent with previous reports of presynaptic site distributions in these cells (Kiyokage et al., 2017; Results, lines 169 to 172 of the revised manuscript). We have also added a brief discussion of the potential biological significance (Discussion, lines 446 to 450).

(8) In the images used to differentiate anaxonic and axon-bearing neurons, the soma, axons, and dendrites are intermixed, making it difficult to distinguish structures specific to each subclass. Employing subclass-specific labeling or sparse labeling techniques could enhance clarity and accuracy in identifying these structures.

Distinguishing these structures is indeed difficult, and was the main reason we used viral label to produce sparse labelling (see response to comment #5 above). In all cases we were extremely careful, including cells only when we could be absolutely certain of their anaxonic or axon-bearing identity, and could also be certain of the continuity of all processes. Crucially, while the 2D representations we show in our figures may suggest a degree of intermixing, we performed all analyses on 3D image stacks, significantly improving our ability to accurately assign structures to individual cells. We have now added extra descriptions of this approach in the relevant Methods section (lines 546 to 548 of the revised manuscript).

(9) In Figure 3, the soma area and synaptophysin puncta density are compared between axon-bearing and anaxonic neurons. However, the figure only presents representative images of axon-bearing neurons. To ensure a fair and accurate comparison, representative images of both neuron subtypes should be included.

The original figures did include example images of puncta density (or lack of puncta) in both cell types (Figure 2B and Figure 3E). For soma area, we have now included representative images of axon-bearing and anaxonic neurons with an indication of soma area measurement in a new Supplementary Figure 2A.

(10) In Figure 4B, the authors state that gephyrin and synaptophysin puncta are in 'very close proximity.' However, it is unclear whether this proximity is sufficient to suggest the possibility of self-inhibition. Quantifying the distance between gephyrin and synaptophysin puncta would provide critical evidence to support this claim. Additionally, analyzing the distribution and proportion of gephyrinsynaptophysin pairs in close proximity would offer further clarity and strengthen the interpretation of these findings.

We thank the Reviewer for raising this issue. We entirely agree that the example image previously shown did not constitute sufficient evidence to claim either close proximity of gephyrin and synaptophysin puncta, nor the possibility of self-inhibition. We are not in a position to perform a full quantitative analysis of these spatial distributions, nor do we think this is necessary given previous direct evidence for auto-evoked inhibition in OB dopaminergic cells (Smith and Jahr, 2002; Murphy et al., 2005; Maher and Westbrook, 2008; Borisovska et al., 2013) and our own demonstration of this phenomenon in anaxonic neurons (Figure 4). We have therefore removed the image and the reference to it in the text. 

(11) In Figures 4J and 4M, the effects of the drugs are presented without a direct comparison to the control group (baseline control?). Including these baseline control data is essential to provide a clear context for interpreting the drug effects and to validate the conclusions drawn from these experiments.

We appreciate the Reviewer’s attention to this important point. As this concern was also raised by Reviewer 1 (their point #6), we have provided a detailed response fully addressing it in our replies to Reviewer 1 above. 

(12) In Lines 342-344, the authors claim that VMAT2 staining is notoriously difficult. However, several studies (e.g., Weihe et al., 2006; Cliburn et al., 2017) have successfully utilized VMAT2 staining. Moreover, Zhang et al., 2015 - a reference cited by the authors - demonstrates that a specific VMAT2 antibody effectively detects VMAT2. Providing evidence of VMAT2 expression in OB DA neurons would substantiate the claim that these neurons are GABA-co-releasing DA neurons and strengthen the study's conclusions.

As noted in response to this Reviewer’s comment #2 above, there is clear published evidence that OB DA neurons are GABA- and dopamine-releasing cells. These cells are also known to express VMAT2 (Cave et al., 2010; Borisovska et al., 2013; Vergaña-Vera et al., 2015). We do not therefore believe that additional evidence of VMAT2 expression is necessary to strengthen our study’s conclusions. We did make every effort to label VMAT2-positive release sites in our neurons, but unfortunately all commercially available antibodies were ineffective. The successful staining highlighted by the Reviewer was either performed in the context of virally driven overexpression (Zhang et al., 2015) or was obtained using custom-produced antibodies (Weihe et al., 2006; Cliburn et al., 2017). We have now modified the Discussion text to provide more clarification of these points (lines 393 to 395 of the revised manuscript).

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

This paper investigates the physical mechanisms underlying cell intercalation, which then enables collective cell flows in confluent epithelia. The authors show that T1 transitions (the topological transitions responsible for cell intercalation) correspond to the unbinding of groups of hexatic topological defects. Defect unbinding, and hence cell intercalation and collective cell flows, are possible when active stresses in the tissue are extensile. This result helps to rationalize the observation that many epithelial cell layers have been found to exhibit extensile active nematic behavior.

Strengths

The authors obtain their results based on a combination of active hexanematic hydrodynamics and a multiphase field (MPF) model for epithelial layers, whose connection is a strength of the paper. With the hydrodynamic approach, the authors find the active flow fields produced around hexatic topological defects, which can drive defect unbinding. Using the MPF simulations, the authors show that T1 transitions tend to localize close to hexatic topological defects.

We are grateful to Reviewer #1, for appreciating and highlighting the strengths of work.

Weaknesses

Citations are sometimes not comprehensive. Cases of contractile behavior found in collective cell flows, which would seemingly contradict some of the authors’ conclusions, are not discussed.

I encourage the authors to address the comments and questions below.

We are thankful to Reviewer #1, for their questions and comments. We have addressed them point by point below, and have amended the manuscript accordingly.

(1) In Equation 1, what do the authors mean by the cluster’s size ℓ? How is this quantity defined? The calculations in the Methods suggest that ℓ indicates the distance between the p-atic defects and the center of the T1 cell cluster, but this is not clearly defined.

We are thank Reviewer #1 for their question. We define the cluster size as the initial distance between the center of the quadrupole and any defect (see Methods). In a primary cell cluster, where cells themselves are the defects, the cluster’s size is the distance between the center of the central junction and the center of any cell in the cluster. Hence, this is half the diameter of an cell which, for example in a typical, confluent MDCK epithelial monolayer, would be about 10µm. We have added this clarification in the definition of the cluster size, above Eq. (1).

(2) The multiphase field model was developed and reviewed already, before the Loewe et al. 2020 paper that the authors cite. Earlier papers include Camley et al. PNAS 2014, Palmieri et al. Sci. Rep. 2015, Mueller et al. PRL 2019, and Peyret et al. Biophys. J. 2019, as reviewed in Alert and Trepat. Annu. Rev. Condens. Matter Phys. 2020.

We thank the referee for their suggestion to incorporate further MPF literature. We have done so in the amended manuscript.

(3) At what time lag is the mean-squared displacement in Figure 3f calculated? How does the choice of a lag time affect these data and the resulting conclusions?

The scatter plot in Fig. 3f was constructed by dividing the system into square subregions of size ∆ℓ = 35 l.u., each containing approximately 4 cells. For each subregion, we analyzed a time window of ∆t = 25 × 10³ iterations, measuring both the normalized mean square displacement of cells (relative to the subregion area ∆ℓ²) and the average defect density. The normalized displacement is calculated as m.s.d. , where t∗ denotes the start time of the observation window. We chose the time window ∆t used to compute the mean square displacement to match the characteristic duration of T1 events and defect lifetimes in our simulations. Observation times much longer (∆t > 35 × 10³) than the typical T1 event duration would cause the two sets of data points to merge into a single group, suggesting no correlation between cell motility and defect density beyond defect life-time.

(4) The authors argue that their results provide an explanation for the extensile behavior of cell layers. However, there are also examples of contractile behavior, such as in Duclos et al., Nat. Phys., 2017 and in P´erez-Gonz´alez et al., Nat. Phys., 2019. In both cases, collective cell flows were observed, which in principle require cell intercalations. How would these observations be rationalized with the theory proposed in this paper? Can these experiments and the theory be reconciled?

The contractile or extensile nature of stress in epithelia depends crucially on the specific tissue type and its biological context. Different cell populations, depending on their position along the epithelial/mesenchymal spectrum, can exhibit either contractile or extensile behaviors. Our theory applies to tissues where hexatic order dominates at the cellular scale, particularly in confluent systems where neighbor exchanges occur primarily through T1 transitions. In contrast, the systems studied by Duclos et al., Nat. Phys. (2018) and Perez-Gonzalez et al. (Nat. Phys., 2019) exhibit nematic order at the cellular level, meaning their dynamics are governed by fundamentally different mechanisms. Since our framework is derived for hexatic-dominated tissues, it does not directly apply to those cases, though a hybrid hexanematic descriptions previously developed by some of the authors in Armengol-Collado et al. eLife 13:e86400 (2024) could help reconcile these observations. In general, a key distinction must be made between the contractility of individual cells and the extensile/contractile nature of the collective force network. To illustrate this, consider a cell exerting a 6- fold symmetric force distribution: each vertex force arises from an imbalance in junctional tensions with neighboring cells, which are themselves contractile due to actomyosin activity. However, the resulting vertex forces can be either contractile or extensile depending on network geometry and tension distribution. This is captured in our coarse-grained description [see Armengol-Collado et al. eLife 13:e86400 (2024)], where the active stress emerges from higher-order moments of cellular forces. Specifically, the deviatoric part of the hexatic active stress tensor , where is the cell radius, the number cell density and the intensity of cellular tension. The negative sign of the coefficient of the active stress shows that the active stress is extensile—consistently with observations in various epithelial systems (e.g., Saw et al., Nature 2017; Blanch-Mercader et al., Phys. Rev. Lett. 2018). Finally, we note that the connection between cellular-scale forces and large-scale extensility has been rationalized in other contexts, such as active nematics (Balasubramaniam et al., Nat. Mater. 2021).

Reviewer #2 (Public Review):

This paper studies the role of hexatic defects in the collective migration of epithelia. The authors emphasize that epithelial migration is driven by cell intercalation events and not just isolated T1 events, and analyze this through the lens of hexatic topological defects. Finally, the authors study the effect of active and passive forces on the dynamics of hexatic defects using analytical results, and numerical results in both continuum and phase-field models.

The results are very interesting and highlight new ways of studying epithelial cell migration through the analysis of the binding and unbinding of hexatic defects.

We are grateful to Reviewer #2, for their interest and for emphasizing the novelty of our work.

Strengths

(1) The authors convincingly argue that intercalation events are responsible for collective cell migration, and that these events are accompanied by the formation and unbinding of hexatic topological defects.

(2) The authors clearly explain the dynamics of hexatic defects during T1 transitions, and demonstrate the importance of active and passive forces during cell migration.

(3) The paper thoroughly studies the T1 transition through the viewpoint of hexatic defects. A continuum model approach to study T1 transitions in cell layers is novel and can lead to valuable new insights.

We thank the Reviewer for their kind and supporting words, and for highlighting the clarity, persuasiveness, and thoroughness.

Weaknesses

(1) The authors could expand on the dynamics of existing hexatic defects during epithelial cell migration, in addition to how they are created during T1 transitions.

We thank the referee for their comment. The detailed analysis of dislocation-pair unbinding modes and their statistical impact on the transition to collective migration is comprehensively addressed in our subsequent work Puggioni et al., arXiv:2502.09554. In the present study, we focus specifically on the fundamental mechanism enabling dislocation unbinding: active extensile stresses generate flows that drive dislocation pairs apart, while passive elastic stresses tend to pull them together (Krommydas et al., Phys. Rev. Lett. 2023; Armengol- Collado et al., arXiv:2502.13104). When active forces dominate over passive restoring forces, the dislocations unbind. This represents a crucial distinction from classical Berezinskii–Kosterlitz–Thouless or Kosterlitz–Thouless–Halperin–Nelson–Youn transitions, where thermal fluctuations drive defect unbinding. In our system, the process is fundamentally activity-driven. Nevertheless, the resulting state - characterized by unbound defects and collective migration - bears strong analogy to the melting transition in equilibrium systems. We emphasize that the dynamics of passive defects has been previously examined in Krommydas et al., Phys. Rev. Lett. 2023. A discussion of these aspects can be found in the Appendix “Numerical simulations of defect annihilation and unbinding”.

(2) The different terms in the MPF model used to study cell layer dynamics are not fully justified. In particular, it is not clear why the model includes self-propulsion and rotational diffusion in addition to nematic and hexatic stresses, and how these quantities are related to each other.

We thank the referee for their comment. The MPF model’s terms (e.g., self-propulsion, rotational diffusion), reflect the stochastic, deformable nature of cells as active droplets migrating with near-constant speed. We emphasize that self-propulsion is the only non-equilibrium mechanism in our model — no additional active stresses (nematic or hexatic) are imposed. We have clarified this point in the revised manuscript and expanded our discussion of the MPF model.

(3) The authors could provide some physical intuition on what an active extensile or contractile term in the hexatic order parameter means, and how this is related to extensility and contractility in active nematics and/or for cell layers.

We thank the referee for their comment. As we explain in the reply to comment [4] of Reviewer #1, the contractile or extensile nature of stress in epithelia depends crucially on the specific tissue type and its biological context. Different cell populations, depending on their position along the epithelial/mesenchymal spectrum, can exhibit either contractile or extensile behaviors. Our theory applies to tissues where hexatic order dominates at the cellular scale, particularly in confluent systems where neighbor exchanges occur primarily through T1 transitions. In contrast, the systems studied by Duclos et al., Nat. Phys. (2018) and Perez-Gonzalez et al. (Nat. Phys., 2019) exhibit nematic order at the cellular level, meaning their dynamics are governed by fundamentally different mechanisms. Since our framework is derived for hexatic-dominated tissues, it does not directly apply to those cases, though a hybrid hexanematic descriptions previously developed by some of the authors in Armengol-Collado et al. eLife 13:e86400 (2024) could help reconcile these observations. In general, a key distinction must be made between the contractility of individual cells and the extensile/contractile nature of the collective force network. To illustrate this, consider a cell exerting a 6-fold symmetric force distribution: each vertex force arises from an imbalance in junctional tensions with neighboring cells, which are themselves contractile due to actomyosin activity. However, the resulting vertex forces can be either contractile or extensile depending on network geometry and tension distribution. This is captured in our coarse-grained description [see Armengol-Collado et al. eLife 13:e86400 (2024)], where the active stress emerges from higher-order moments of cellular forces. Specifically, the deviatoric part of the hexatic active stress tensor , where is the cell radius, the number cell density and the intensity of cellular tension. The negative sign of the coefficient of the active stress shows that the active stress is extensile—consistently with observations in various epithelial systems (e.g., Saw et al., Nature 2017; Blanch-Mercader et al., Phys. Rev. Lett. 2018). Finally, we note that the connection between cellular-scale forces and large-scale extensility has been rationalized in other contexts, such as active nematics (Balasubramaniam et al., Nat. Mater. 2021).

Recommendations for the Authors: Reviewer #2 (Recommendations for the Authors):

(1) The authors point out that hexatic topological defects are produced in quadrupoles (L109). Does this also mean that these defects can be annihilated only in quadrupoles as well? In the same vein, are hexatic defects always bound in pairs, as suggested by the schematics, or is it possible to observe an isolated hexatic defect?

We thank the referee for their question. Hexatic disclinations (the defect monopoles discussed in this work), much like electrons and positrons, can annihilate in any number of neutral charge configuration (dipole, quadrupole, octupole, etc.). Unbinding a pair of hexatic disinclination, however, costs much more energy than unbinding a quadrupole to dipoles. Hence isolated defects appear in abundance only in late, fully disordered phase, where the system has completely “melted”. For more details on how defect unbinding modes affect tissue dynamics, please see our subsequent work Puggioni et al., arXiv:2502.09554.

(2) Could you clarify if the flows described in Figures 2(a)-(b), panel (i) are driven by a passive backflow term without activity? Could you compare the magnitudes of these flows compared to the typical active terms?

We thank the referee for their question. In panel 2(b) there is only passive backflow. In 2(a) instead, both terms are included, and are in a regime of parameters where the active flow overcomes the active flow (and hence the active force overcomes the passive force as delineated in the discussions section). In turn, the magnitude of the passive flows, is studied in detail in our previous work Krommydas et al., (Phys. Rev. Lett. 2023).

(3) Could you clarify how the continuum hexatic model and MPF model are related to each other? What are the similarities and differences in the dynamics of these models?

We thank the referee for this insightful question. A key point of our work is precisely that the continuum hexatic model and the MPF (Multi-Phase Field) model are distinct in nature.

The MPF model is an established agent-based framework used to simulate tissue dynamics at the cellular level. It captures individual cell behaviors and interactions through phase-field variables. In our work, we use the MPF model as a benchmark to extract statistical features of tissue dynamics, such as defect motion and orientational correlations. In contrast, our continuum hexatic model is a coarse-grained hydrodynamic theory that describes the dynamics of orientational order in active tissues. It is built on symmetry principles and conservation laws, and it does not rely on microscopic cell-level details. Instead, it captures the collective behavior of the system through a hexatic order parameter and its coupling to flow and activity.

Despite their conceptual differences, the MPF model and our hydrodynamic theory exhibit similar statistical features. This agreement—also observed in the independent study by Jain et al. (Phys. Rev. Res. 2024)—provides strong support for the validity and generality of our continuum description.

(4) When multiple references by the same author and year are cited using alphabets, the second alphabet is not in bold e.g. Giomi et al., 2022b, a in Line 75, and others.

We are grateful to the referee carefully going through the manuscript and pointing out these typos. We have corrected them in the amended manuscript.

Reviewer #3 (Public Review):

In this manuscript, the authors discuss epithelial tissue fluidity from a theoretical perspective. They focus on the description of topological transitions whereby cells change neighbors (T1 transitions). They explain how such transitions can be described by following the fate of hexatic defects. They first focus on a single T1 transition and the surrounding cells using a hydrodynamic model of active hexatics. They show that successful T1 intercalations, which promote tissue fluidity, require a sufficiently large extensile hexatic activity in the neighborhood of the cells attempting a T1 transition. If such activity is contractile or not sufficiently extensile, the T1 is reversed, hexatic defects annihilate, and the epithelial network configuration is unchanged. They then describe a large epithelium, using a phase field model to describe cells. They show a correlation between T1 events and hexatic defects unbinding, and identify two populations of T1 cells: one performing T1 cycles (failed T1), and not contributing to tissue migration, and one performing T1 intercalation (successful T1) and leading to the collective cell migration.

Strengths

The manuscript is scientifically sound, and the variety of numerical and analytical tools they use is impressive. The approach and results are very interesting and highlight the relevance of hexatic order parameters and their defects in describing tissue dynamics.

We thank the Reviewer for recognizing the scientific soundness of the manuscript, the breadth of numerical and analytical tools employed, as well as their interest in our work.

Weaknesses

(1) Goal and message of the paper. (a) In my opinion, the article is mainly theoretical and should be presented as such. For instance, their conclusions and the consequences of their analysis in terms of biology are not extremely convincing, although they would be sufficient for a theory paper oriented to physicists or biophysicists. The choice of journal and potential readership should be considered, and I am wondering whether the paper structure should be re-organized, in order to have side-by-side the methods and the results, for instance (see also below).

We thank the referee for their criticism. In response, we have made an effort to reword certain parts of the manuscript. As with any theoretical study, the biological implications of our work can only be fully assessed through experimental validation — a prospect we look forward to. Nevertheless, we have submitted our work to the subsection of Physics of Life, which we believe is perfectly suited to our content.

(b) Currently, the two main results sections are somewhat disconnected, because they use different numerical models, and because the second section only marginally uses the results from the first section to identify/distinguish T1.

We thank the referee, for their comment. In the second section we are using statistics from the MPF model, to support the analytical and numerical findings of our hydrodynamic theory of cell intercalation. In the time between our submission, further qualitative evidence have been brought to light in the work of Jain et al. (Phys. Rev. Res. 2024).

(2) Quite surprisingly, the authors use a cell-based model to describe the macroscopic tissuescale behavior, and a hydrodynamic model to describe the cell-based events. In particular, their hydrodynamic description (the active hexatic model) is supposed to be a coarse-grained description, valid to capture the mesoscopic physics, and yet, they use it to describe cellscale events (T1 transitions). For instance, what is the meaning of the velocity field they are discussing in Figure 2? This makes me question the validity of the results of their first part.

We thank the referee for their comment. There are many excellent discrete models of epithelial tissues in the literature (e.g., Bi et al., Phys. Rev. X 2016; Pasupalak et al., Soft Matter 2020; Graner et al., Phys. Rev. Lett. 1992), each capturing essential biological features such as cell division, apoptosis and sorting. While these models have provided invaluable insights, our work takes a different approach by developing a continuum theory aimed at describing epithelial dynamics at two levels: (1) mesoscopic intercalation events and (2) macroscopic collective migration. Crucially, our goal is not to replicate a specific discrete model — which would risk constructing a “model of a model” — but rather to derive a hydrodynamic description of tissue dynamics grounded in symmetry principles and conservation laws. Along this logic, the velocity field in our theory should be interpreted as an Eulerian (continuum) velocity, representing the coarse-grained flow of the tissue rather than the Lagrangian motion of individual cells. This distinction is central to our framework, which operates at scales where cellular details are averaged out, yet retains the essential physics of hexatic order and active stresses. We validate our predictions against the Multiphase Field (MPF) model. [We thank Reviewer 1 for their suggestion to incorporate further MPF literature.] Furthermore, Jain et al. (Phys. Rev. Res. 2024) have used the MPF to predict flow patterns around T1 transitions and obtained results compatible with those of our hydrodynamic theory. From this comparison we can conclude that both the MPF and our theory are able to capture the same aspect of cell intercalation in epithelial layer. This, however, does not imply that other discrete models of epithelia can reproduce this aspect too, nor that our theory is specifically tailored to the MPF model. We have clarified these points in the revised manuscript and expanded our discussion of the MPF model.

(3) The quality of the numerical results presented in the second part (phase field model) could be improved. (a) In terms of analysis of the defects. It seems that they have all the tools to compare their cell-resolved simulations and their predictions about how a T1 event translates into defects unbinding. However, their analysis in Figure 3e is relatively minimal: it shows a correlation between T1 cells and defects. But it says nothing about the structure and evolution of the defects, which, according to their first section, should be quite precise.

We thank the referee for their comment. Further qualitative evidence have been brought to light in the work of Jain et al. (Phys. Rev. Res. 2024), were the exact flow pattern predicted by our hydrodynamic theory is obtained, in the MPF, around cells undergoing T1 rearrangements.

(b) In terms of clarity of the presentation. For instance, in Figure 3f, they plot the mean-square displacement as a function of a defect density. I thought that MSD was a time-dependent quantity: they must therefore consider MSD at a given time, or averaged over time. They should be explicit about what their definition of this quantity is.

We thank the referee for raising this point. As clarified in our response to Reviewer 1, point 3, the mean square displacement (MSD) plotted in Fig. 3f is computed over a fixed time window of ∆t = 25×103 iterations, chosen to match the typical duration of T1 events and defect lifetimes. [See also reply to Reviewer #1, point (3).] The MSD is normalized by the subregion area and averaged over time within each window. We have now made this explicit in the amended version of the manuscript.

(c) In terms of statistics. For instance, Figure 3g is used to study the role of rotational diffusion on the average time between T1s. The error bars in this figure are huge and make their claims hardly supported. Their claim of a ”monotonic decay” of the average time between intercalations is also not fully supported given their statistics.

We appreciate the Reviewer’s comment regarding the statistical robustness of Fig. 3g. While we acknowledge that the error bars are substantial – reflecting the inherent variability in cell intercalation dynamics – the yellow curve does exhibit a consistent downward trend in the average time between T1 transitions as rotational diffusion increases. This monotonic decrease is visible across the entire range of variation of the rotational diffusion Dr, and is statistically supported when considering the trend over independent simulations. To address this concern, we have revised the main text to adjusted the wording: instead of stating that “the former is a monotonically decreasing function of Dr,” we now write that “the former displays a decreasing trend with Dr,” which better reflects the statistical variability while preserving the observed behavior.

Reviewer #3 (Recommendations for the Authors):

(1) Section 1 is difficult to follow due to multiple reasons: early but delayed definitions, unclear use of T1 intercalation vs. T1 cycles, disconnected figures and unclear simulation descriptions. We recommend including simulation setup details earlier and restructuring the flow of arguments.

We thank the referee for their comment. We have made an effort in rewording and clarifying things in our amended manuscript. We are slightly confused by what they mean by “early but delayed definitions”, if they could clarify, we would be happy to amend the position and phrasing of these definitions accordingly.

(2) It could be useful to have an additional figure early on defining schematically hexatic defects and an illustration showing an epithelium (or a simulation), similar to what the authors have produced in some of their other publications on this topic.

We thank the referee for their comment. Figures 3c and 3d show what a hexatic defect looks like in a simulation of the epithelium. Following the referee’s recommendation, we have added a note in the caption of figure 3, citing our work were we show the same defects in MDCK epithelial monolayers (Armengol et al., Nat. Phys. 2023).

(3) Minor points and typos:

Line 88: the bond between vertices shrinks, not the vertices.

Figure 1: the 1/6 is displayed as 1 6 (fraction bar missing).

Line 232: “and order” → “one/an order”.

Line 237: Fig. 3g) → Fig. 3g

Line 298: ”nu” and ”v” hard to distinguish in eLife font.

Methods: define all notation clearly (e.g., tensor product exponent, D/Dt in Eq. 3c).

Methods: ”cell orientation, coarse-graining and topological defects” section is difficult to follow, schematic would help.

Line 457 onward: unclear how panels (ii-iv) of Fig. 2ab are obtained.

Line 480 onward: not referenced in main text.

Figure 2: “avalancHe” typo.

Figure 2 caption: “cell intercalaTION” typo.

Movies are neither referenced nor explained.

Figure 5 and 6 are not referenced in the main text.

We thank the referee for their detailed read of the paper. We have corrected all typos.

Document d'Information : Peut-on Réinventer les Lumières ?

Synthèse

Ce document d'information synthétise les arguments et les thèmes clés abordés lors de la séance de clôture du cycle "Peut-on réinventer les Lumières ?", organisée par l'Institut d'Études Avancées de Paris.

Les interventions de Francis Wolf et Céline Spector, deux philosophes éminents, ont convergé vers une défense robuste et nuancée de l'universalisme, tout en examinant de manière critique les objections contemporaines, notamment celles issues des courants identitaires et postcoloniaux.

L'argument central, porté par Francis Wolf, est que l'humanité forme une communauté morale unique, fondée sur des droits et des devoirs réciproques.

Il déconstruit méthodiquement les critiques affirmant que les valeurs universelles ne sont qu'un masque pour la domination occidentale.

En distinguant l'origine d'une idée de sa portée et en s'appuyant sur des exemples concrets de luttes pour la démocratie et la liberté à travers le monde (Printemps arabes, Iran), il soutient que l'universalisme est un outil d'émancipation essentiel. Il insiste sur la distinction fondamentale entre l'universel, qui garantit la diversité, et l'uniforme, qui la nie.

Céline Spector prolonge cette analyse en se concentrant sur les critiques postcoloniales des droits de l'homme.

Elle en systématise les principaux arguments (ethnocentrisme, fiction idéologique, outil de colonisation) tout en soulignant les paradoxes inhérents au concept de droits humains dès son origine.

Son propos, en accord avec celui de Wolf, vise à réaffirmer la pertinence de cet héritage des Lumières face à ces objections.

La discussion a ensuite exploré plusieurs concepts connexes, dont la notion de "pluriversel" (jugée contradictoire ou maladroite), l'existence de précédents non-occidentaux aux droits humains (la Charte du Mandé de 1236), et la tension persistante entre l'idéal universel et son application souvent défaillante ("deux poids, deux mesures").

Enfin, le débat s'est ouvert sur les défis contemporains, tels que les droits de la nature face à la crise environnementale et le rôle de l'héritage des Lumières dans la construction d'une Europe capable de résister aux dynamiques impériales.

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Contexte de l'Événement

La discussion s'est tenue dans le cadre de la séance de clôture du cycle de conférences de l'IEA de Paris, présidé par Betina Laville, sur le thème "Peut-on réinventer les Lumières ?".

L'objectif était de conclure une année de réflexion sur la place de l'universel dans un monde qualifié de "fracturé" et de plus en plus contestataire envers l'héritage intellectuel européen.

Les deux intervenants principaux étaient :

• Francis Wolf : Philosophe, professeur émérite à l'École Normale Supérieure, spécialiste de philosophie antique et auteur de travaux significatifs sur l'humanisme et l'universalisme, notamment Plaidoyer pour l'universel.

• Céline Spector : Philosophe, professeure à Sorbonne Université, spécialiste des Lumières (en particulier Montesquieu et Rousseau) et des questions européennes, auteure de No Demos. Souveraineté et démocratie à l'épreuve de l'Europe.

Le Plaidoyer pour l'Universalisme de Francis Wolf

Francis Wolf a structuré son intervention comme une défense des valeurs universelles, qu'il définit à travers une thèse fondatrice : "l'humanité forme une communauté morale de droit et de devoirs réciproques".

Il se concentre principalement sur la réfutation des critiques qui jugent cet universalisme excessif, au profit de communautés morales restreintes ("infrahumaines").

Les Critiques de l'Universalisme

Wolf identifie deux grands courants critiques contemporains de l'universalisme :

1. Les idéologies "de droite" : Nationalistes, racistes et xénophobes, elles nient l'existence de l'Homme en général pour n'admettre que des communautés de "semblables" ("nous" contre "eux").

Cette vision, selon Wolf, est en pleine résurgence et se manifeste par le piétinement du droit international (depuis l'invasion de l'Ukraine), la remise en cause du droit des réfugiés (accords de Genève) et la montée des politiques discriminatoires ou d'épuration ethnique.

2. Les idéologies identitaristes "de gauche" : Symétriques aux premières, elles reprennent des arguments hérités d'un "marxisme simplifié" selon lesquels toute prétention à l'universalité est un leurre masquant la domination.

Réfutation des Arguments Anti-Universalistes

Wolf examine et réfute systématiquement plusieurs arguments récurrents contre les valeurs universelles.

Argument Critique

Réfutation par Francis Wolf

1. Aucune lutte ne peut se faire au nom de l'universel, car elle défend toujours des victimes particulières.

Si les combats pour des minorités oublient qu'ils visent l'égalité pour tous, ils trahissent leur propre cause.

Les colonisés n'ont pas lutté pour devenir colonisateurs, mais pour abolir le colonialisme.

2. L'universel se présente comme neutre, mais ne l'est jamais ; il nie les relations de domination.

Bien que l'universel soit parfois utilisé pour nier les injustices, il n'est pas nécessaire de se définir uniquement "en tant que" (femme, colonisé, etc.).

Les identités sont métissées, fluides et non des essences réifiées.

3. L'expérience des souffrances particulières est incommunicable et il n'y a pas de lieu neutre pour juger.

Une injustice ne concerne pas que la victime ou le coupable, mais la communauté morale entière. Sans un "tiers lieu" permettant de juger, il n'y a plus de justice, seulement des vengeances. Toute souffrance a une dimension communicable.

4. L'universel n'est que le masque des intérêts dominants.

Cet argument, bien que souvent justifié par l'histoire (colonisation, guerre d'Irak), n'est pas généralisable.

Les pires entreprises de domination (génocides) n'ont pas besoin de ce prétexte et se font au nom d'identités essentialisées ("sous-hommes", "bêtes nuisibles").

5. Tout universel est en fait particulier ; c'est un autre nom de l'Occident.

Concéder qu'un universel naît dans un contexte particulier n'en limite pas la portée. L'algèbre, née en Perse, n'est pas une science "iranienne".

La démocratie et les droits humains sont réclamés par les peuples en lutte partout dans le monde (Printemps arabes, Hong Kong, Iran), et leurs despotes les rejettent en les qualifiant de "valeurs occidentales".

Prétendre que l'Occident a seul inventé les droits humains est une "illusion occidentaliste" (Amartya Sen).

La Vertu Émancipatrice de l'Universel

Pour conclure, Wolf affirme que l'universalisme conserve sa force émancipatrice.

Il pose la question : qui est le véritable ethnocentriste ?

Celui qui croit en l'existence de consciences critiques dans toutes les cultures, ou celui qui essentialise les autres cultures en leur déniant cette capacité critique ?

Il distingue enfin l'universel de l'uniforme. Loin d'effacer les particularités, les valeurs universelles (laïcité, liberté d'opinion, tolérance) sont la condition de leur coexistence.

Elles constituent un "universel de second niveau", formel, qui garantit la diversité.

La Critique Postcoloniale des Droits de l'Homme selon Céline Spector

Céline Spector se déclare en "profond accord" avec Francis Wolf et concentre son propos sur la critique spécifique des droits de l'homme par les études postcoloniales et décoloniales.

Les Paradoxes Originels des Droits de l'Homme

Dès leur proclamation aux États-Unis (1776) et en France (1789), les droits de l'homme présentent des paradoxes fondamentaux :

• Ils sont à la fois évidents et advenus (nés de révolutions).

• Ils sont à la fois naturels et historiques.

• Ils sont à la fois innés et civiques.

• Ils sont à la fois universels et situés.

Ces paradoxes ont nourri les critiques (marxistes, féministes) qui y voyaient une hypocrisie, notamment en raison de l'exclusion des femmes, des esclaves et d'autres minorités.

Les Cinq Piliers de la Critique Postcoloniale

Spector résume la critique postcoloniale des droits de l'homme en cinq arguments principaux :

1. Ils ne sont pas universels mais occidentaux, protégeant uniquement les citoyens d'Europe.

2. Ce sont des fictions idéologiques ayant servi à justifier la "mission civilisatrice" de la colonisation.

3. Ils sont associés à une conception de la raison qui exclut les peuples "sauvages" ou "barbares", jugés incapables d'y accéder.

4. La liste des droits est arbitraire et abusive, notamment l'inclusion du droit de propriété qui a servi à exproprier les peuples nomades.

5. Ce sont les droits des colons et de leurs complices, qui n'avaient aucune volonté politique de mettre fin au pillage des colonies ou à l'esclavage.

Tout en reconnaissant la nécessité de prendre en compte ces critiques pour révéler les "tensions inhérentes aux Lumières", l'approche de Céline Spector vise à formuler des objections à cette vision, rejoignant ainsi la défense de l'universalisme de Francis Wolf.

Thèmes Clés de la Discussion

La période d'échange avec le public a permis d'approfondir plusieurs thématiques.

Le Concept de "Pluriversel"

Interrogés sur cette notion issue des théories décoloniales, les deux intervenants expriment leur scepticisme :

• Francis Wolf y voit soit une contradiction dans les termes, soit une simple reformulation du fait que l'universel est toujours perçu depuis un point de vue culturel particulier, sans pour autant y être prisonnier.

• Céline Spector, citant la définition du Dictionnaire décolonial, le décrit comme une "critique radicale de l'universalisme".

Elle considère ce concept comme une "tentative maladroite" de la part d'auteurs (Ramon Grosfoguel, Walter Mignolo, etc.) qui se retrouvent dans une impasse existentielle : vouloir lutter pour les droits sans utiliser l'outil des droits universels.

Précédents Historiques et Application du Droit

• La Charte du Mandé (1236) : Cette charte, issue de l'empire du Mali, est évoquée comme un possible précédent africain à la reconnaissance de valeurs universelles, telles que l'égalité entre ethnies et religions, et la participation des femmes au gouvernement.

• Le "Deux Poids, Deux Mesures" : Un participant soulève le problème du "double standard" dans l'application du droit international.

Céline Spector reconnaît la légitimité de cette critique mais met en garde contre une indignation qui dévalorise les institutions internationales (ONU, CPI), les rendant fragiles et poussant les puissances hégémoniques à simplement les quitter.

Universalité, Environnement et Europe

• Droits de la Nature : La question d'un "droit à l'environnement" est soulevée comme un défi majeur pour réinventer les Lumières.

La discussion porte sur la tension entre les droits humains et les "droits de la nature", un concept de plus en plus débattu juridiquement (ex: le fleuve Whanganui en Nouvelle-Zélande, la lagune Mar Menor en Espagne).

Ce débat interroge la centralité de l'homme dans la définition de l'environnement.

• L'Héritage des Lumières pour l'Europe : Céline Spector propose de voir dans l'héritage de Montesquieu, et spécifiquement son modèle de "République fédérative", un outil puissant pour penser la résistance des démocraties face à la résurgence des empires.

Francis Wolf abonde en ce sens, soulignant que la construction européenne illustre la primauté du demos (communauté politique) sur l'ethnos (communauté préexistante), un principe également au cœur de la résistance ukrainienne.

• Les "Lumières Noires" : Ce terme, associé à Curtis Yarvin, est décrit comme un "usage complètement perverti" des Lumières, désignant une technocratie oligarchique où une élite numérique domine des citoyens dépossédés de leurs droits.

C'est l'antithèse même de l'idéal des Lumières.

Author response:

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

Reviewer #1 (Public review)

Summary:

This study by Park and colleagues uses longitudinal saliva viral load data from two cohorts (one in the US and one in Japan from a clinical trial) in the pre-vaccine era to subset viral shedding kinetics and then use machine learning to attempt to identify clinical correlates of different shedding patterns. The stratification method identifies three separate shedding patterns discriminated by peak viral load, shedding duration, and clearance slope. The authors also assess micro-RNAs as potential biomarkers of severity but do not identify any clear relationships with viral kinetics.

Strengths:

The cohorts are well developed, the mathematical model appears to capture shedding kinetics fairly well, the clustering seems generally appropriate, and the machine learning analysis is a sensible, albeit exploratory approach. The micro-RNA analysis is interesting and novel.

Weaknesses:

The conclusions of the paper are somewhat supported by the data but there are certain limitations that are notable and make the study's findings of only limited relevance to current COVID-19 epidemiology and clinical conditions.

We sincerely appreciate the reviewer’s thoughtful and constructive comments, which have been invaluable in improving the quality of our study. We have carefully revised the manuscript to address all points raised.

(1) The study only included previously uninfected, unvaccinated individuals without the omicron variant. It has been well documented that vaccination and prior infection both predict shorter duration shedding. Therefore, the study results are no longer relevant to current COVID-19 conditions. This is not at all the authors' fault but rather a difficult reality of much retrospective COVID research.

Thank you for your comment. We agree with the review’s comment that some of our results could not provide insight into the current COVID-19 conditions since most people have either already been infected with COVID-19 or have been vaccinated. We revised our manuscript to discuss this (page 22, lines 364-368). Nevertheless, we believe it is novel that we have extensively investigated the relationship between viral shedding patterns in saliva and a wide range of clinical and microRNA data, and that developing a method to do so remains important. This is important for providing insight into early responses to novel emerging viral diseases in the future. Therefore, we still believe that our findings are valuable.

(2) The target cell model, which appears to fit the data fairly well, has clear mechanistic limitations. Specifically, if such a high proportion of cells were to get infected, then the disease would be extremely severe in all cases. The authors could specify that this model was selected for ease of use and to allow clustering, rather than to provide mechanistic insight. It would be useful to list the AIC scores of this model when compared to the model by Ke.

Thank you for your feedback and suggestion regarding our mathematical model. As the reviewer pointed out, in this study, we adopted a simple model (target cell-limited model) to focus on reconstruction of viral dynamics and stratification of shedding patterns rather than exploring the mechanism of viral infection in detail. Nevertheless, we believe that the target cell-limited model provides reasonable reconstructed viral dynamics as it has been used in many previous studies. We revised manuscript to clarify this point (page 10, lines 139-144). Also, we revised our manuscript to provide more detailed description of the model comparison along with information about AIC (page 10, lines 130-135).

(3) Line 104: I don't follow why including both datasets would allow one model to work better than the other. This requires more explanation. I am also not convinced that non-linear mixed effects approaches can really be used to infer early model kinetics in individuals from one cohort by using late viral load kinetics in another (and vice versa). The approach seems better for making populationlevel estimates when there is such a high amount of missing data.

Thank you for your feedback. We recognized that our explanation was insufficient by your comment. We intended to describe that, rather than comparing performance of the two models, data fitting can be performed with same level for both models by including both datasets. We revised the manuscript to clarify this point (page 10, lines 135-139).

Additionally, we agree that nonlinear mixed effects models are a useful approach for performing population-level estimates of missing data. On the other hand, in addition, the nonlinear mixed effects model has the advantage of making the reasonable parameter estimation for each individual with not enough data points by considering the distribution of parameters of other individuals. Paying attention to these advantages, we adopted a nonlinear mixed effects model in our study. We also revised the manuscript to clarify this (page 27, lines 472-483).

(4) Along these lines, the three clusters appear to show uniform expansion slopes whereas the NBA cohort, a much larger cohort that captured early and late viral loads in most individuals, shows substantial variability in viral expansion slopes. In Figure 2D: the upslope seems extraordinarily rapid relative to other cohorts. I calculate a viral doubling time of roughly 1.5 hours. It would be helpful to understand how reliable of an estimate this is and also how much variability was observed among individuals.

We appreciate your detailed feedback on the estimated up-slope of viral dynamics. As the reviewer noted, the pattern differs from that observed in the NBA cohort, which may be due to their measurement of viral load from upper respiratory tract swabs. In our estimation, the mean and standard deviation of the doubling time (defined as ln2/(𝛽𝑇₀𝑝𝑐⁻¹ − 𝛿)) were 1.44 hours and 0.49 hours, respectively. Although direct validation of these values is challenging, several previous studies, including our own, have reported that viral loads in saliva increase more rapidly than in the upper respiratory tract swabs, reaching their peak sooner. Thus, we believe that our findings are consistent with those of previous studies. We revised our manuscript to discuss this point with additional references (page 20, lines 303-311).

(5) A key issue is that a lack of heterogeneity in the cohort may be driving a lack of differences between the groups. Table 1 shows that Sp02 values and lab values that all look normal. All infections were mild. This may make identifying biomarkers quite challenging.

Thank you for your comment regarding heterogeneity in the cohort. Although the NFV cohort was designed for COVID-19 patients who were either mild or asymptomatic, we have addressed this point and revised the manuscript to discuss it (page 21, lines 334-337).

(6) Figure 3A: many of the clinical variables such as basophil count, Cl, and protein have very low pre-test probability of correlating with virologic outcome.

Thank you for your comment regarding some clinical information we used in our study. We revised our manuscript to discuss this point (page 21, lines 337-338).

(7) A key omission appears to be micoRNA from pre and early-infection time points. It would be helpful to understand whether microRNA levels at least differed between the two collection timepoints and whether certain microRNAs are dynamic during infection.

Thank you for your comment regarding the collection of micro-RNA data. As suggested by the reviewer, we compared micro-RNA levels between two time points using pairwise t-tests and Mann-Whitney U tests with FDR correction. As a result, no micro-RNA showed a statistically significant difference. This suggests that micro-RNA levels remain relatively stable during the course of infection, at least for mild or asymptomatic infection, and may therefore serve as a biomarker independent of sampling time. We have revised the manuscript to include this information (page 17, lines 259-262).

(8) The discussion could use a more thorough description of how viral kinetics differ in saliva versus nasal swabs and how this work complements other modeling studies in the field.

We appreciate the reviewer’s thoughtful feedback. As suggested, we have added a discussion comparing our findings with studies that analyzed viral dynamics using nasal swabs, thereby highlighting the differences between viral dynamics in saliva and in the upper respiratory tract. To ensure a fair and rigorous comparison, we referred to studies that employed the same mathematical model (i.e., Eqs.(1-2)). Accordingly, we revised the manuscript and included additional references (page 20, lines 303-311).

Furthermore, we clarified the significance of our study in two key aspects. First, it provides a detailed analysis of viral dynamics in saliva, reinforcing our previous findings from a single cohort by extending them across multiple cohorts. Second, this study uniquely examines whether viral dynamics in saliva can be directly predicted by exploring diverse clinical data and micro-RNAs. Notably, cohorts that have simultaneously collected and reported both viral load and a broad spectrum of clinical data from the same individuals, as in our study, are exceedingly rare. We revised the manuscript to clarify this point (page 20, lines 302-311).

(9) The most predictive potential variables of shedding heterogeneity which pertain to the innate and adaptive immune responses (virus-specific antibody and T cell levels) are not measured or modeled.

Thank you for your comment. We agree that antibody and T cell related markers may serve as the most powerful predictors, as supported by our own study [S. Miyamoto et al., PNAS (2023), ref. 24] as well as previous reports. While this point was already discussed in the manuscript, we have revised the text to make it more explicit (page 21, lines 327-328).

(10) I am curious whether the models infer different peak viral loads, duration, expansion, and clearance slopes between the 2 cohorts based on fitting to different infection stage data.

Thank you for your comment. We compared features between 2 cohorts as reviewer suggested. As a result, a statistically significant difference between the two cohorts (i.e., p-value ≤ 0.05 from the t-test) was observed only at the peak viral load, with overall trends being largely similar. At the peak, the mean value was 7.5 log₁₀ (copies/mL) in the Japan cohort and 8.1 log₁₀ (copies/mL) in the Illinois cohort, with variances of 0.88 and 0.87, respectively, indicating comparable variability.

Reviewer #2 (Public review)

Summary:

This study argues it has found that it has stratified viral kinetics for saliva specimens into three groups by the duration of "viral shedding"; the authors could not identify clinical data or microRNAs that correlate with these three groups.

Strengths:

The question of whether there is a stratification of viral kinetics is interesting.

Weaknesses:

The data underlying this work are not treated rigorously. The work in this manuscript is based on PCR data from two studies, with most of the data coming from a trial of nelfinavir (NFV) that showed no effect on the duration of SARS-CoV-2 PCR positivity. This study had no PCR data before symptom onset, and thus exclusively evaluated viral kinetics at or after peak viral loads. The second study is from the University of Illinois; this data set had sampling prior to infection, so has some ability to report the rate of "upswing." Problems in the analysis here include:

We are grateful to the reviewer for the constructive feedback, which has greatly enhanced the quality of our study. In response, we have carefully revised the manuscript to address all comments.

The PCR Ct data from each study is treated as equivalent and referred to as viral load, without any reports of calibration of platforms or across platforms. Can the authors provide calibration data and justify the direct comparison as well as the use of "viral load" rather than "Ct value"? Can the authors also explain on what basis they treat Ct values in the two studies as identical?

Thank you for your comment regarding description of viral load data. We recognized the lack of explanation for the integration of viral load data by reviewer's comment. We calculated viral load from Ct value using linear regression equations between Ct and viral load for each study's measurement method, respectively. We revised the manuscript to clarify this point in the section of Saliva viral load data in Methods.

The limit of detection for the NFV PCR data was unclear, so the authors assumed it was the same as the University of Illinois study. This seems a big assumption, as PCR platforms can differ substantially. Could the authors do sensitivity analyses around this assumption?

Thank you for your comment regarding the detection limit for viral load data. As reviewer suggested, we conducted sensitivity analysis for assumption of detection limit for the NFV dataset. Specifically, we performed data fitting in the same manner for two scenarios: when the detection limit of NFV PCR was lower (0 log₁₀ copies/mL) or higher (2 log₁₀ copies/mL) than that of the Illinois data (1.08 log₁₀ copies/mL), and compared the results.

As a result, we obtained largely comparable viral dynamics in most cases (Supplementary Fig 6). When comparing the AIC values, we observed that the AIC for the same censoring threshold was 6836, whereas it increased to 7403 under the low censoring threshold and decreased to 6353 under the higher censoring threshold. However, this difference may be attributable to the varying number of data points treated as below the detection limit. Specifically, when the threshold is set higher, more data are treated as below the detection limit, which may result in a more favorable error calculation. To discuss this point, we have added a new figure (Supplementary Fig 6) and revised the manuscript accordingly (page 25, lines 415-418).

The authors refer to PCR positivity as viral shedding, but it is viral RNA detection (very different from shedding live/culturable virus, as shown in the Ke et al. paper). I suggest updating the language throughout the manuscript to be precise on this point.

We appreciate the reviewer’s feedback regarding the terminology used for viral shedding. In response, we have revised all instances of “viral shedding” to “viral RNA detection” throughout the manuscript as suggested.

Eyeballing extended data in Figure 1, a number of the putative long-duration infections appear to be likely cases of viral RNA rebound (for examples, see S01-16 and S01-27). What happens if all the samples that look like rebound are reanalyzed to exclude the late PCR detectable time points that appear after negative PCRs?

We sincerely thank the reviewer for the valuable suggestion. In response, we established a criterion to remove data that appeared to exhibit rebound and subsequently performed data fitting

(see Author response image 1 below). The criterion was defined as: “any data that increase again after reaching the detection limit in two measurements are considered rebound and removed.” As a result, 15 out of 144 cases were excluded due to insufficient usable data, leaving 129 cases for analysis. Using a single detection limit as the criterion would have excluded too many data points, while defining the criterion solely based on the magnitude of increase made it difficult to establish an appropriate “threshold for increase.”

The fitting result indicates that the removal of rebound data may influence the fitting results; however, direct comparison of subsequent analyses, such as clustering, is challenging due to the reduced sample size. Moreover, the results can vary substantially depending on the criterion used to define rebound, and establishing a consistent standard remains difficult. Accordingly, we retained the current analysis and have added a discussion of rebound phenomena in the Discussion section as a limitation (page 22, lines 355-359). We once again sincerely appreciate the reviewer’s insightful and constructive suggestion.

Author response image 1.

Comparison of model fits before and after removing data suspected of rebound. Black dots represent observed measurements, and the black and yellow curves show the fitted viral dynamics for the full dataset and the dataset with rebound data removed, respectively.

There's no report of uncertainty in the model fits. Given the paucity of data for the upslope, there must be large uncertainty in the up-slope and likely in the peak, too, for the NFV data. This uncertainty is ignored in the subsequent analyses. This calls into question the efforts to stratify by the components of the viral kinetics. Could the authors please include analyses of uncertainty in their model fits and propagate this uncertainty through their analyses?

We sincerely appreciate the reviewer’s detailed feedback on model uncertainty. To address this point, we revised Extended Fig 1 (now renumbered as Supplementary Fig 1) to include 95% credible intervals computed using a bootstrap approach. In addition, to examine the potential impact of model uncertainty on stratified analyses, we reconstructed the distance matrix underlying stratification by incorporating feature uncertainty. Specifically, for each individual, we sampled viral dynamics within the credible interval and averaged the resulting feature, and build the distance matrix using it. We then compared this uncertainty-adjusted matrix with the original one using the Mantel test, which showed a strong correlation (r = 0.72, p < 0.001). Given this result, we did not replace the current stratification but revised the manuscript to provide this information through Result and Methods sections (page 11, lines 159-162 and page 28, lines 512-519). Once again, we are deeply grateful for this insightful comment.

The clinical data are reported as a mean across the course of an infection; presumably vital signs and blood test results vary substantially, too, over this duration, so taking a mean without considering the timing of the tests or the dynamics of their results is perplexing. I'm not sure what to recommend here, as the timing and variation in the acquisition of these clinical data are not clear, and I do not have a strong understanding of the basis for the hypothesis the authors are testing.

We appreciate the reviewers' feedback on the clinical data. We recognized that the manuscript lacked description of the handling of clinical data by your comment. In this research, we focused on finding “early predictors” which could provide insight into viral shedding patterns. Thus, we used clinical data measured in the earliest time (date of admission) for each patient. Another reason is that the date of admission is the almost only time point at which complete clinical data without any missing values are available for all participants. We revised our manuscript to clarify this point (page 5, lines 90-95).

It's unclear why microRNAs matter. It would be helpful if the authors could provide more support for their claims that (1) microRNAs play such a substantial role in determining the kinetics of other viruses and (2) they play such an important role in modulating COVID-19 that it's worth exploring the impact of microRNAs on SARS-CoV-2 kinetics. A link to a single review paper seems insufficient justification. What strong experimental evidence is there to support this line of research?

We appreciate the reviewer’s comments regarding microRNA. Based on this feedback, we recognized the need to clarify our rationale for selecting microRNAs as the analyte. The primary reason was that our available specimens were saliva, and microRNAs are among the biomarkers that can be reliably measured in saliva. At the same time, previous studies have reported associations between microRNAs and various diseases, which led us to consider the potential relevance of microRNAs to viral dynamics, beyond their role as general health indicators. To better reflect this context, we have added supporting references (page 17, lines 240-243).

Reviewer #3 (Public review)

The article presents a comprehensive study on the stratification of viral shedding patterns in saliva among COVID-19 patients. The authors analyze longitudinal viral load data from 144 mildly symptomatic patients using a mathematical model, identifying three distinct groups based on the duration of viral shedding. Despite analyzing a wide range of clinical data and micro-RNA expression levels, the study could not find significant predictors for the stratified shedding patterns, highlighting the complexity of SARS-CoV-2 dynamics in saliva. The research underscores the need for identifying biomarkers to improve public health interventions and acknowledges several limitations, including the lack of consideration of recent variants, the sparsity of information before symptom onset, and the focus on symptomatic infections. 

The manuscript is well-written, with the potential for enhanced clarity in explaining statistical methodologies. This work could inform public health strategies and diagnostic testing approaches. However, there is a thorough development of new statistical analysis needed, with major revisions to address the following points:

We sincerely appreciate the thoughtful feedback provided by Reviewer #3, particularly regarding our methodology. In response, we conducted additional analyses and revised the manuscript accordingly. Below, we address the reviewer’s comments point by point.

(1) Patient characterization & selection: Patient immunological status at inclusion (and if it was accessible at the time of infection) may be the strongest predictor for viral shedding in saliva. The authors state that the patients were not previously infected by SARS-COV-2. Was Anti-N antibody testing performed? Were other humoral measurements performed or did everything rely on declaration? From Figure 1A, I do not understand the rationale for excluding asymptomatic patients. Moreover, the mechanistic model can handle patients with only three observations, why are they not included? Finally, the 54 patients without clinical data can be used for the viral dynamics fitting and then discarded for the descriptive analysis. Excluding them can create a bias. All the discarded patients can help the virus dynamics analysis as it is a population approach. Please clarify. In Table 1 the absence of sex covariate is surprising.

We appreciate the detailed feedback from the reviewer regarding patient selection. We relied on the patient's self-declaration to determine the patient's history of COVID-19 infection and revised the manuscript to specify this (page 6, lines 83-84).

In parameter estimation, we used the date of symptom onset for each patient so that we establish a baseline of the time axis as clearly as possible, as we did in our previous works. Accordingly, asymptomatic patients who do not have information on the date of symptom onset were excluded from the analysis. Additionally, in the cohort we analyzed, for patients excluded due to limited number of observations (i.e., less than 3 points), most patients already had a viral load close to the detection limit at the time of the first measurement. This is due to the design of clinical trial, as if a negative result was obtained twice in a row, no further follow-up sampling was performed. These patients were excluded from the analysis because it hard to get reasonable fitting results. Also, we used 54 patients for the viral dynamics fitting and then only used the NFV cohort for clinical data analysis. We acknowledge that our description may have confused readers. We revised our manuscript to clarify these points regarding patient selecting for data fitting (page 6, lines 96-102, page 24, lines 406-407, and page 7, lines 410-412). In addition, we realized, thanks to the reviewer’s comment, that gender information was missing in Table 1. We appreciate this observation and have revised the table to include gender (we used gender in our analysis). 

(2) Exact study timeline for explanatory covariates: I understand the idea of finding « early predictors » of long-lasting viral shedding. I believe it is key and a great question. However, some samples (Figure 4A) seem to be taken at the end of the viral shedding. I am not sure it is really easier to micro-RNA saliva samples than a PCR. So I need to be better convinced of the impact of the possible findings. Generally, the timeline of explanatory covariate is not described in a satisfactory manner in the actual manuscript. Also, the evaluation and inclusion of the daily symptoms in the analysis are unclear to me.

We appreciate the reviewer’s feedback regarding the collection of explanatory variables. As noted, of the two microRNA samples collected from each patient, one was obtained near the end of viral shedding. This was intended to examine potential differences in microRNA levels between the early and late phases of infection. No significant differences were observed between the two time points, and using microRNA from either phase alone or both together did not substantially affect predictive accuracy for stratified groups. Furthermore, microRNA collection was motivated primarily by the expectation that it would be more sensitive to immune responses, rather than by ease of sampling. We have revised the manuscript to clarify these points regarding microRNA (page 17, lines 243-245 and 259-262).

Furthermore, as suggested by the reviewer, we have also strengthened the explanation regarding the collection schedule of clinical information and the use of daily symptoms in the analysis (page 6, lines 90-95, page 14, lines 218-220,).

(3) Early Trajectory Differentiation: The model struggles to differentiate between patients' viral load trajectories in the early phase, with overlapping slopes and indistinguishable viral load peaks observed in Figures 2B, 2C, and 2D. The question arises whether this issue stems from the data, the nature of Covid-19, or the model itself. The authors discuss the scarcity of pre-symptom data, primarily relying on Illinois patients who underwent testing before symptom onset. This contrasts earlier statements on pages 5-6 & 23, where they claim the data captures the full infection dynamics, suggesting sufficient early data for pre-symptom kinetics estimation. The authors need to provide detailed information on the number or timing of patient sample collections during each period.

Thank you for the reviewer’s thoughtful comments. The model used in this study [Eqs.(1-2)] has been employed in numerous prior studies and has successfully identified viral dynamics at the individual level. In this context, we interpret the rapid viral increase observed across participants as attributable to characteristics of SARS-CoV-2 in saliva, an interpretation that has also been reported by multiple previous studies. We have added the relevant references and strengthened the corresponding discussion in the manuscript (page 20, lines 303-311).

We acknowledge that our explanation of how the complementary relationship between the two cohorts contributes to capturing infection dynamics was not sufficiently clear. As described in the manuscript, the Illinois cohort provides pre-symptomatic data, whereas the NFV cohort offers abundant end-phase data, thereby compensating for each other’s missing phases. By jointly analyzing the two cohorts with a nonlinear mixed-effects model, we estimated viral dynamics at the individual-level. This approach first estimates population-level parameters (fixed effects) using data from all participants and then incorporates random effects to account for individual variability, yielding the most plausible parameter values.

Thus, even when early-phase data are lacking in the NFV cohort, information from the Illinois cohort allows us to infer most reasonable dynamics, and the reverse holds true for the end phase. In this context, we argued that combining the two cohorts enables mathematical modeling to capture infection dynamics at the individual level. Recognizing that our earlier description could be misleading, we have carefully reinforced the relevant description (page 27, lines 472-483). In addition, as suggested by the reviewer, we have added information on the number of data samples available for each phase in both cohorts (page 7, lines 106-109).

(4) Conditioning on the future: Conditioning on the future in statistics refers to the problematic situation where an analysis inadvertently relies on information that would not have been available at the time decisions were made or data were collected. This seems to be the case when the authors create micro-RNA data (Figure 4A). First, when the sampling times are is something that needs to be clarified by the authors (for clinical outcomes as well). Second, proper causal inference relies on the assumption that the cause precedes the effect. This conditioning on the future may result in overestimating the model's accuracy. This happens because the model has been exposed to the outcome it's supposed to predict. This could question the - already weak - relation with mir-1846 level.

We appreciate the reviewer’s detailed feedback. As noted in Reply to Comments 2, we collected micro-RNA samples at two time points, near the peak of infection dynamics and at the end stage, and found no significant differences between them. This suggests that micro-RNA levels are not substantially affected by sampling time. Indeed, analyses conducted using samples from the peak, late stage, or both yielded nearly identical results in relation to infection dynamics. To clarify this point, we revised the manuscript by integrating this explanation with our response in Reply to Comments 2 (page 17, lines 259-262). In addition, now we also revised manuscript to clarify sampling times of clinical information and micro-RNA (page 6, lines 90-95).

(5) Mathematical Model Choice Justification and Performance: The paper lacks mention of the practical identifiability of the model (especially for tau regarding the lack of early data information). Moreover, it is expected that the immune effector model will be more useful at the beginning of the infection (for which data are the more parsimonious). Please provide AIC for comparison, saying that they have "equal performance" is not enough. Can you provide at least in a point-by-point response the VPC & convergence assessments?

We appreciate the reviewer’s detailed feedback regarding the mathematical model. We acknowledge the potential concern regarding the practical identifiability of tau (incubation period), particularly given the limited early-phase data. In our analysis, however, the nonlinear mixed-effects model yielded a population-level estimate of 4.13 days, which is similar with previously reported incubation periods for COVID-19. This concordance suggests that our estimate of tau is reasonable despite the scarcity of early data.

For model comparison, first, we have added information on the AIC of the two models to the manuscript as suggested by the reviewer (page 10, lines 130-135). One point we would like to emphasize is that we adopted a simple target cell-limited model in this study, aiming to focus on reconstruction of viral dynamics and stratification of shedding patterns rather than exploring the mechanism of viral infection in detail. Nevertheless, we believe that the target cell-limited model provides reasonable reconstructed viral dynamics as it has been used in many previous studies. We revised manuscript to clarify this (page 10, lines 135-144). 

Furthermore, as suggested, we have added the VPC and convergence assessment results for both models, together with explanatory text, to the manuscript (Supplementary Fig 2, Supplementary Fig 3, and page 10, lines 130-135). In the VPC, the observed 5th, 50th, and 95th percentiles were generally within the corresponding simulated prediction intervals across most time points. Although minor deviations were noted in certain intervals, the overall distribution of the observed data was well captured by the models, supporting their predictive performance (Supplementary Fig 2). In addition, the log-likelihood and SAEM parameter trajectories stabilized after the burn-in phase, confirming appropriate convergence (Supplementary Fig 3).

(6) Selected features of viral shedding: I wonder to what extent the viral shedding area under the curve (AUC) and normalized AUC should be added as selected features.

We sincerely appreciate the reviewer’s valuable suggestion regarding the inclusion of additional features. Following this recommendation, we considered AUC (or normalized AUC) as an additional feature when constructing the distance matrix used for stratification. We then evaluated the similarity between the resulting distance matrix and the original one using the Mantel test, which showed a very high correlation (r = 0.92, p < 0.001). This indicates that incorporating AUC as an additional feature does not substantially alter the distance matrix. Accordingly, we have decided to retain the current stratification analysis, and we sincerely thank the reviewer once again for this interesting suggestion.

(7) Two-step nature of the analysis: First you fit a mechanistic model, then you use the predictions of this model to perform clustering and prediction of groups (unsupervised then supervised). Thus you do not propagate the uncertainty intrinsic to your first estimation through the second step, ie. all the viral load selected features actually have a confidence bound which is ignored. Did you consider a one-step analysis in which your covariates of interest play a direct role in the parameters of the mechanistic model as covariates? To pursue this type of analysis SCM (Johnson et al. Pharm. Res. 1998), COSSAC (Ayral et al. 2021 CPT PsP), or SAMBA ( Prague et al. CPT PsP 2021) methods can be used. Did you consider sampling on the posterior distribution rather than using EBE to avoid shrinkage?

Thank you for the reviewer’s detailed suggestions regarding our analysis. We agree that the current approach does not adequately account for the impact of uncertainty in viral dynamics on the stratified analyses. As a first step, we have revised Extended Data Fig 1 (now renumbered as Supplementary Fig 1) to include 95% credible intervals computed using a bootstrap approach, to present the model-fitting uncertainty more explicitly. Then, to examine the potential impact of model uncertainty on stratified analyses, we reconstructed the distance matrix underlying stratification by incorporating feature uncertainty. Specifically, for each individual, we sampled viral dynamics within the credible interval and averaged the resulting feature, and build the distance matrix using it. We then compared this uncertainty-adjusted matrix with the original one using the Mantel test, which showed a strong correlation (r = 0.72, p < 0.001). Given this result, we did not replace the current stratification but revised the manuscript to provide this information (page 11, lines 159-162 and page 28, 512-519).

Furthermore, we carefully considered the reviewer’s proposed one-step analysis. However, implementation was constrained by data-fitting limitations. Concretely, clinical information is available only in the NFV cohort. Thus, if these variables are to be entered directly as covariates on the parameters, the Illinois cohort cannot be included in the data-fitting process. Yet the NFV cohort lacks any pre-symptomatic observations, so fitting the model to that cohort alone does not permit a reasonable (well-identified/robust) fitting result. While we were unable to implement the suggestion under the current data constraints, we sincerely appreciate the reviewer’s thoughtful and stimulating proposal.

(8) Need for advanced statistical methods: The analysis is characterized by a lack of power. This can indeed come from the sample size that is characterized by the number of data available in the study. However, I believe the power could be increased using more advanced statistical methods. At least it is worth a try. First considering the unsupervised clustering, summarizing the viral shedding trajectories with features collapses longitudinal information. I wonder if the R package « LongituRF » (and associated method) could help, see Capitaine et al. 2020 SMMR. Another interesting tool to investigate could be latent class models R package « lcmm » (and associated method), see ProustLima et al. 2017 J. Stat. Softwares. But the latter may be more far-reached.

Thank you for the reviewer’s thoughtful suggestions regarding our unsupervised clustering approach. The R package “LongitiRF” is designed for supervised analysis, requiring a target outcome to guide the calculation of distances between individuals (i.e., between viral dynamics). In our study, however, the goal was purely unsupervised clustering, without any outcome variable, making direct application of “LongitiRF” challenging.

Our current approach (summarizing each dynamic into several interpretable features and then using Random Forest proximities) allows us to construct a distance matrix in an unsupervised manner. Here, the Random Forest is applied in “proximity mode,” focusing on how often dynamics are grouped together in the trees, independent of any target variable. This provides a practical and principled way to capture overall patterns of dynamics while keeping the analysis fully unsupervised.

Regarding the suggestion to use latent class mixed models (R package “lcmm”), we also considered this approach. In our dataset, each subject has dense longitudinal measurements, and at many time points, trajectories are very similar across subjects, resulting in minimal inter-individual differences. Consequently, fitting multi-class latent class mixed models (ng ≥ 2) with random effects or mixture terms is numerically unstable, often producing errors such as non-positive definite covariance matrices or failure to generate valid initial values. Although one could consider using only the time points with the largest differences, this effectively reduces the analysis to a feature-based summary of dynamics. Such an approach closely resembles our current method and contradicts the goal of clustering based on full longitudinal information.

Taken together, although we acknowledge that incorporating more longitudinal information is important, we believe that our current approach provides a practical, stable, and informative solution for capturing heterogeneity in viral dynamics. We would like to once again express our sincere gratitude to the reviewer for this insightful suggestion.

(9) Study intrinsic limitation: All the results cannot be extended to asymptomatic patients and patients infected with recent VOCs. It definitively limits the impact of results and their applicability to public health. However, for me, the novelty of the data analysis techniques used should also be taken into consideration.

We appreciate your positive evaluation of our research approach and acknowledge that, as noted in the Discussion section as our first limitation, our analysis may not provide valid insights into recent VOCs or all populations, including asymptomatic individuals. Nonetheless, we believe it is novel that we extensively investigated the relationship between viral shedding patterns in saliva and a wide range of clinical and micro-RNA data. Our findings contribute to a deeper and more quantitative understanding of heterogeneity in viral dynamics, particularly in saliva samples. To discuss this point, we revised our manuscript (page 22, lines 364-368).

Strengths are:

Unique data and comprehensive analysis.

Novel results on viral shedding.

Weaknesses are:

Limitation of study design.

The need for advanced statistical methodology.

Reviewer #1 (Recommendations For The Authors):

Line 8: In the abstract, it would be helpful to state how stratification occurred.

We thank the reviewer for the feedback, and have revised the manuscript accordingly (page 2, lines 8-11).

Line 31 and discussion: It is important to mention the challenges of using saliva as a specimen type for lab personnel.

We thank the reviewer for the feedback, and have revised the manuscript accordingly (page 3, lines 36-41).

Line 35: change to "upper respiratory tract".

We thank the reviewer for the feedback, and have revised the manuscript accordingly (page 3, line 35).

Line 37: "Saliva" is not a tissue. Please hazard a guess as to which tissue is responsible for saliva shedding and if it overlaps with oral and nasal swabs.

We thank the reviewer for the feedback, and have revised the manuscript accordingly (page 3, lines 42-45).

Line 42, 68: Please explain how understanding saliva shedding dynamics would impact isolation & screening, diagnostics, and treatments. This is not immediately intuitive to me.

We thank the reviewer for the feedback, and have revised the manuscript accordingly (page 3, lines 48-50).

Line 50: It would be helpful to explain why shedding duration is the best stratification variable.

We thank the reviewer for the feedback. We acknowledge that our wording was ambiguous. The clear differences in the viral dynamics patterns pertain to findings observed following the stratification, and we have revised the manuscript to make this explicit (page 4, lines 59-61).

Line 71: Dates should be listed for these studies.

We thank the reviewer for the feedback, and have revised the manuscript accordingly (page 6, lines 85-86).

Reviewer #2 (Recommendations For The Authors):

Please make all code and data available for replication of the analyses.

We appreciate the suggestion. Due to ethical considerations, it is not possible to make all data and code publicly available. We have clearly stated in the manuscript about it (Data availability section in Methods).

Reviewer #3 (Recommendations For The Authors):

Here are minor comments / technical details:

(1) Figure 1B is difficult to understand.

Thank you for the comment. We updated Fig 1B to incorporate more information to aid interpretation.

(2) Did you analyse viral load or the log10 of viral load? The latter is more common. You should consider it. SI Figure 1 please plot in log10 and use a different point shape for censored data. The file quality of this figure should be improved. State in the material and methods if SE with moonlit are computed with linearization or importance sampling.

Thank you for the comment. We conducted our analyses using log10-transformed viral load. Also, we revised Supplementary Fig 1 (now renumbered as Supplementary Fig 4) as suggested. We also added Supplementary Fig 3 and clarified in the Methods that standard errors (SE) were obtained in Monolix from the Fisher information matrix using the linearization method (page 28, lines 498-499).

(3) Table 1 and Figure 3A could be collapsed.

Thank you for the comment, and we carefully considered this suggestion. Table 1 summarizes clinical variables by category, whereas Fig 3A visualizes them ordered by p-value of statistical analysis. Collapsing these into a single table would make it difficult to apprehend both the categorical summaries and the statistical ranking at a glance, thereby reducing readability. We therefore decided to retain the current layout. We appreciate the constructive feedback again. 

(4) Figure 3 legend could be clarified to understand what is 3B and 3C.

We thank the reviewer for the feedback and have reinforced the description accordingly.

(5) Why use AIC instead of BICc?

Thank you for your comment. We also think BICc is a reasonable alternative. However, because our objective is predictive adequacy (reconstruction of viral dynamics), we judged AIC more appropriate. In NLMEM settings, the effective sample size required by BICc is ambiguous, making the penalty somewhat arbitrary. Moreover, since the two models reconstruct very similar dynamics, our conclusions are not sensitive to the choice of criterion.

(6) Bibliography. Most articles are with et al. (which is not standard) and some are with an extended list of names. Provide DOI for all.

We thank the reviewer for the feedback, and have revised the manuscript accordingly.

(7) Extended Table 1&2 - maybe provide a color code to better highlight some lower p-values (if you find any interesting).

We thank the reviewer for the feedback. Since no clinical information and micro-RNAs other than mir-1846 showed low p-values, we highlighted only mir-1846 with color to make it easier to locate.

(8) Please make the replication code available.

We appreciate the suggestion. Due to ethical considerations, it is not possible to make all data and code publicly available. We have clearly stated in the manuscript about it (Data availability section in Methods).

Author response:

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

Reviewer #1 (Public review): 

Summary: 

In this work, van Paassen et al. have studied how CD8 T cell functionality and levels predict HIV DNA decline. The article touches on interesting facets of HIV DNA decay, but ultimately comes across as somewhat hastily done and not convincing due to the major issues. 

(1) The use of only 2 time points to make many claims about longitudinal dynamics is not convincing. For instance, the fact that raw data do not show decay in intact, but do for defective/total, suggests that the present data is underpowered. The authors speculate that rising intact levels could be due to patients who have reservoirs with many proviruses with survival advantages, but this is not the parsimonious explanation vs the data simply being noisy without sufficient longitudinal follow-up. n=12 is fine, or even reasonably good for HIV reservoir studies, but to mitigate these issues would likely require more time points measured per person. 

(1b) Relatedly, the timing of the first time point (6 months) could be causing a number of issues because this is in the ballpark for when the HIV DNA decay decelerates, as shown by many papers. This unfortunate study design means some of these participants may already have stabilized HIV DNA levels, so earlier measurements would help to observe early kinetics, but also later measurements would be critical to be confident about stability. 

The main goal of the present study was to understand the relationship of the HIV-specific CD8 T-cell responses early on ART with the reservoir changes across the subsequent 2.5-year period on suppressive therapy. We have revised the manuscript in order to clarify this.  We chose these time points because the 24 week time point is past the initial steep decline of HIV DNA, which takes place in the first weeks after ART initiation. It is known that HIV DNA continues to decay for years after (Besson, Lalama et al. 2014, Gandhi, McMahon et al. 2017). 

(2) Statistical analysis is frequently not sufficient for the claims being made, such that overinterpretation of the data is problematic in many places. 

(2a) First, though plausible that cd8s influence reservoir decay, much more rigorous statistical analysis would be needed to assert this directionality; this is an association, which could just as well be inverted (reservoir disappearance drives CD8 T cell disappearance). 

To correlate different reservoir measures between themselves and with CD8+ T-cell responses at 24 and 156 weeks, we now performed non-parametric (Spearman) correlation analyses, as they do not require any assumptions about the normal distribution of the independent and dependent variables. Benjamini-Hochberg corrections for multiple comparisons (false discovery rate, 0.25) were included in the analyses and did not change the results. 

Following this comment we would like to note that the association between the T-cell response at 24 weeks and the subsequent decrease in the reservoir cannot be bi-directional (that can only be the case when both variables are measured at the same time point). Therefore, to model the predictive value of T-cell responses measured at 24 weeks for the decrease in the reservoir between 24 and 156 weeks, we fitted generalized linear models (GLM), in which we included age and ART regimen, in addition to three different measures of HIV-specific CD8+ T-cell responses, as explanatory variables, and changes in total, intact, and total defective HIV DNA between 24 and 156 weeks ART as dependent variables.

(2b) Words like "strong" for correlations must be justified by correlation coefficients, and these heat maps indicate many comparisons were made, such that p-values must be corrected appropriately. 

We have now used Spearman correlation analysis, provided correlation coefficients to justify the wording, and adjusted the p-values for multiple comparisons (Fig. 1, Fig 3., Table 2). Benjamini-Hochberg corrections for multiple comparisons (false discovery rate, 0.25) were included in the analyses and did not change the results.  

(3) There is not enough introduction and references to put this work in the context of a large/mature field. The impacts of CD8s in HIV acute infection and HIV reservoirs are both deep fields with a lot of complexity. 

Following this comment we have revised and expanded the introduction to put our work more in the context of the field (CD8s in acute HIV and HIV reservoirs). 

Reviewer #2 (Public review): 

Summary: 

This study investigated the impact of early HIV specific CD8 T cell responses on the viral reservoir size after 24 weeks and 3 years of follow-up in individuals who started ART during acute infection. Viral reservoir quantification showed that total and defective HIV DNA, but not intact, declined significantly between 24 weeks and 3 years post-ART. The authors also showed that functional HIV-specific CD8⁺ T-cell responses persisted over three years and that early CD8⁺ T-cell proliferative capacity was linked to reservoir decline, supporting early immune intervention in the design of curative strategies. 

Strengths: 

The paper is well written, easy to read, and the findings are clearly presented. The study is novel as it demonstrates the effect of HIV specific CD8 T cell responses on different states of the HIV reservoir, that is HIV-DNA (intact and defective), the transcriptionally active and inducible reservoir. Although small, the study cohort was relevant and well-characterized as it included individuals who initiated ART during acute infection, 12 of whom were followed longitudinally for 3 years, providing unique insights into the beneficial effects of early treatment on both immune responses and the viral reservoir. The study uses advanced methodology. I enjoyed reading the paper. 

Weaknesses: 

All participants were male (acknowledged by the authors), potentially reducing the generalizability of the findings to broader populations. A control group receiving ART during chronic infection would have been an interesting comparison. 

We thank the reviewer for their appreciation of our study. Although we had indeed acknowledged the fact that all participants were male, we have clarified why this is a limitation of the study (Discussion, lines 296-298). The reviewer raises the point that it would be useful to compare our data to a control group. Unfortunately, these samples are not yet available, but our study protocol allows for a control group (chronic infection) to ensure we can include a control group in the future.

Reviewer #1 (Recommendations for the authors): 

Minor: 

On the introduction: 

(1) One large topic that is mostly missing completely is the emerging evidence of selection on HIV proviruses during ART from the groups of Xu Yu and Matthias Lichterfeld, and Ya Chi Ho, among others. 

Previously, it was only touched upon in the Discussion. Now we have also included this in the Introduction (lines 77-80).

(2) References 4 and 5 don't quite match with the statement here about reservoir seeding; we don't completely understand this process, and certainly, the tissue seeding aspect is not known. 

Line 61-62: references were changed and this paragraph was rewritten to clarify.

(3) Shelton et al. showed a strong relationship with HIV DNA size and timing of ART initiation across many studies. I believe Ananwaronich also has several key papers on this topic. 

References by Ananwaronich are included (lines 91-94).

(4) "the viral levels decline within weeks of AHI", this is imprecise, there is a peak and a decline, and an equilibrium. 

We agree and have rewritten the paragraph accordingly.

(5) The impact of CD8 cells on viral evolution during primary infection is complex and likely not relevant for this paper. 

We have left viral evolution out of the introduction in order to keep a focus on the current subject.

(6) The term "reservoir" is somewhat polarizing, so it might be worth mentioning somewhere exactly what you think the reservoir is, I think, as written, your definition is any HIV DNA in a person on ART? 

Indeed, we refer to the reservoir when we talk about the several aspects of the reservoir that we have quantified with our assays (total HIV DNA, unspliced RNA, intact and defective proviral DNA, and replication-competent virus). In most instances we try to specify which measurement we are referring to. We have added additional reservoir explanation to clarify our definition to the introduction (lines 55-58).

(7) I think US might be used before it is defined. 

We thank the reviewer for this notification, we have now also defined it in the Results section (line 131).

(8) In Figure 1 it's also not clear how statistics were done to deal with undetectable values, which can be tricky but important. 

We have now clarified this in the legend to Figure 2 (former Figure 1). Paired Wilcoxon tests were performed to test the significance of the differences between the time points. Pairs where both values were undetectable were always excluded from the analysis. Pairs where one value was undetectable and its detection limit was higher than the value of the detectable partner, were also excluded from the analysis. Pairs where one value was undetectable and its detection limit was lower than the value of the detectable partner, were retained in the analysis.

In the discussion: 

(1) "This confirms that the existence of a replication-competent viral reservoir is linked to the presence of intact HIV DNA." I think this statement is indicative of many of the overinterpretations without statistical justification. There are 4 of 12 individuals with QVOA+ detectable proviruses, which means there are 8 without. What are their intact HIV DNA levels? 

We thank the reviewer for the question that is raised here. We have now compared the intact DNA levels (measured by IPDA) between participants with positive vs. negative QVOA output, and observed a significant difference. We rephrased the wording as follows: “We compared the intact HIV DNA levels at the 24-week timepoint between the six participants, from whom we were able to isolate replicating virus, and the fourteen participants, from whom we could not. Participants with positive QVOA had significantly higher intact HIV DNA levels than those with negative QVOA (p=0.029, Mann-Whitney test; Suppl. Fig. 3). Five of six participants with positive QVOA had intact DNA levels above 100 copies/106 PBMC, while thirteen of fourteen participants with negative QVOA had intact HIV DNA below 100 copies/106 PBMC (p=0.0022, Fisher’s exact test). These findings indicate that recovery of replication-competent virus by QVOA is more likely in individuals with higher levels of intact HIV DNA in IPDA, reaffirming a link between the two measurements.”

(2) "To determine whether early HIV-specific CD8+ T-cell responses at 24 weeks were predictive for the change in reservoir size". This is a fundamental miss on correlation vs causation... it could be the inverse. 

We thank the reviewer for the remark. We have calculated the change in reservoir size (the difference between the reservoir size at 24 weeks and 156 weeks ART) and analyzed if the HIVspecific CD8+ T-cell response at 24 weeks ART are predictive for this change. We do not think it can be inverse, as we have a chronological relationship (CD8+ responses at week 24 predict the subsequent change in the reservoir).

(3) "This may suggest that active viral replication drives the CD8+ T-cell response." I think to be precise, you mean viral transcription drives CD8s, we don't know about the full replication cycle from these data. 

We agree with the reviewer and have changed “replication” to “transcription” (line 280).

(4) "Remarkably, we observed that the defective HIV DNA levels declined significantly between 24 weeks and 3 years on ART. This is in contrast to previous observations in chronic HIV infection (30)". I don't find this remarkable or in contrast: many studies have analyzed and/or modeled defective HIV DNA decay, most of which have shown some negative slope to defective HIV DNA, especially within the first year of ART. See White et al., Blankson et al., Golob et al., Besson et al., etc In addition, do you mean in long-term suppressed? 

The point we would like to make is that,  compared to other studies, we found a significant, prominent decrease in defective DNA (and not intact DNA) over the course of 3 years, which is in contrast to other studies (where usually the decrease in intact is significant and the decrease in defective less prominent). We have rephrased the wording (lines 227-230) as follows:

“We observed that the defective HIV DNA levels decreased significantly between 24 and 156 weeks of ART. This is different from studies in CHI, where no significant decrease during the first 7 years of ART (Peluso, Bacchetti et al. 2020, Gandhi, Cyktor et al. 2021), or only a significant decrease during the first 8 weeks on ART, but not in the 8 years thereafter, was observed (Nühn, Bosman et al. 2025).”

Reviewer #2 (Recommendations for the authors): 

(1) Page 4, paragraph 2 - will be informative to report the statistics here. 

(2) Page 4, paragraph 4 - "General phenotyping of CD4+ (Suppl. Fig. 3A) and CD8+ (Supplementary Figure 3B) T-cells showed no difference in frequencies of naïve, memory or effector CD8+ T-cells between 24 and 156 weeks." - What did the CD4+ phenotyping show? 

We thank the reviewer for the remark. Indeed, there were also no differences in frequencies of naïve, memory or effector CD4+ T-cells between 24 and 156 weeks. We have added this to the paragraph (now Suppl. Fig 4), lines 166-168.

(3) Page 5, paragraph 3 - "Similarly, a broad HIV-specific CD8+ T-cell proliferative response to at least three different viral proteins was observed in the majority of individuals at both time points" - should specify n=? for the majority of individuals. 

At time point 24 weeks, 6/11 individuals had a response to env, 10/11 to gag, 5/11 to nef, and 4/11 to pol. At 156 weeks, 8/11 to env, 10/11 to gag, 8/11 to nef and 9/11 to pol. We have added this to the text (lines 188-191).

(4) Seven of 22 participants had non-subtype B infection. Can the authors explain the use of the IPDA designed by Bruner et. al. for subtype B HIV, and how this may have affected the quantification in these participants? 

Intact HIV DNA was detectable in all 22 participants. We cannot completely exclude influence of primer/probe-template mismatches on the quantification results, however such mismatches could also have occurred in subtype B participants, and droplet digital PCR that IPDA is based on is generally much less sensitive to these mismatches than qPCR.

(5) Page 7, paragraph 2 - the authors report a difference in findings from a previous study ("a decline in CD8 T cell responses over 2 years" - reference 21), but only provide an explanation for this on page 9. The authors should consider moving the explanation to this paragraph for easier understanding. 

We agree with the reviewer that this causes confusion. Therefore, we have revised and changed the order in the Discussion.

(6) Page 7, paragraph 2 - Following from above, the previous study (21) reported this contradicting finding "a decline in CD8 T cell responses over 2 years" in a CHI (chronic HIV) treated cohort. The current study was in an acute HIV treated cohort. The authors should explain whether this may also have resulted in the different findings, in addition to the use of different readouts in each study.

We thank the reviewer for this attentiveness. Indeed, the study by Takata et al. investigates the reservoir and HIV-specific CD8+ T-cell responses in both the RV254/ SEARCH010 study who initiated ART during AHI and the RV304/ SEARCH013 who initiated ART during CHI. We had not realized that the findings of the decline in CD8 T cell responses were solely found in the RV304/ SEARCH013 (CHI cohort). It appears functional HIV specific immune responses were only measured in AHI at 96 weeks, so we have clarified this in the Discussion. 

Besson, G. J., C. M. Lalama, R. J. Bosch, R. T. Gandhi, M. A. Bedison, E. Aga, S. A. Riddler, D. K. McMahon, F. Hong and J. W. Mellors (2014). "HIV-1 DNA decay dynamics in blood during more than a decade of suppressive antiretroviral therapy." Clin Infect Dis 59(9): 1312-1321.

Gandhi, R. T., J. C. Cyktor, R. J. Bosch, H. Mar, G. M. Laird, A. Martin, A. C. Collier, S. A. Riddler, B. J. Macatangay, C. R. Rinaldo, J. J. Eron, J. D. Siliciano, D. K. McMahon and J. W. Mellors (2021). "Selective Decay of Intact HIV-1 Proviral DNA on Antiretroviral Therapy." J Infect Dis 223(2): 225-233.

Gandhi, R. T., D. K. McMahon, R. J. Bosch, C. M. Lalama, J. C. Cyktor, B. J. Macatangay, C. R. Rinaldo, S. A. Riddler, E. Hogg, C. Godfrey, A. C. Collier, J. J. Eron and J. W. Mellors (2017). "Levels of HIV-1 persistence on antiretroviral therapy are not associated with markers of inflammation or activation." PLoS Pathog 13(4): e1006285.

Nühn, M. M., K. Bosman, T. Huisman, W. H. A. Staring, L. Gharu, D. De Jong, T. M. De Kort, N. Buchholtz, K. Tesselaar, A. Pandit, J. Arends, S. A. Otto, E. Lucio De Esesarte, A. I. M. Hoepelman, R. J. De Boer, J. Symons, J. A. M. Borghans, A. M. J. Wensing and M. Nijhuis (2025). "Selective decline of intact HIV reservoirs during the first decade of ART followed by stabilization in memory T cell subsets." Aids 39(7): 798-811.

Peluso, M. J., P. Bacchetti, K. D. Ritter, S. Beg, J. Lai, J. N. Martin, P. W. Hunt, T. J. Henrich, J. D. Siliciano, R. F. Siliciano, G. M. Laird and S. G. Deeks (2020). "Differential decay of intact and defective proviral DNA in HIV-1-infected individuals on suppressive antiretroviral therapy." JCI Insight 5(4).

La Réflexion Dialogique : Synthèse des Idées de Steve Mann

Synthèse Exécutive

Le professeur Steve Mann (Université de Warwick), lors de sa résidence à l'Institut d'Études Avancées (IEA) de Paris, présente son projet de recherche sur la "réflexion dialogique".

Il la définit comme une forme de conversation collaborative et médiatisée, conçue pour examiner les expériences et les idées, contrastant fortement avec la vision traditionnelle de la réflexion en tant qu'exercice solitaire et individuel.

L'argument central de sa présentation est que les êtres humains possèdent un "moteur interactionnel" inné, une capacité fondamentale à l'empathie, à l'écoute et à l'interaction, rendant les pratiques dialogiques non pas artificielles, mais au contraire profondément ancrées dans notre nature.

Mann suggère que l'IEA, dont la mission est de favoriser le dialogue, pourrait systématiquement documenter et analyser ces interactions fertiles, voire positionner la réflexion dialogique comme une de ses méthodes de recherche.

Son propre plan de travail à l'institut consiste à réexaminer ses corpus de données à la recherche de marqueurs linguistiques de la réflexion dialogique, tout en explorant des domaines comme les études néonatales pour en consolider les fondements théoriques.

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1. Définition et Fondements de la Réflexion Dialogique

La réflexion dialogique est présentée comme un processus collaboratif qui vise à dépasser la pensée individuelle à travers une interaction dynamique et une multiplicité de perspectives.

• Définition : Il s'agit d'une forme d'enquête par la parole, souvent structurée, qui permet d'examiner les expériences, les idées et les présupposés.

Elle est fondamentalement médiatisée et collaborative.

• Origines du Concept : L'intérêt de Steve Mann pour ce sujet provient de plusieurs sources :

◦ "Cooperative Development" (Développement Coopératif) : Un modèle développé par son superviseur de thèse, Julian Edge, fortement influencé par les idées de Carl Rogers (respect, empathie, sincérité).

Ce modèle met l'accent sur l'écoute active et utilise des techniques linguistiques spécifiques comme le "reflet" (reflecting) et la "focalisation" (focusing) pour soutenir l'émergence des idées du locuteur.   

◦ Travaux antérieurs : Un chapitre co-écrit avec le professeur Steve Walsh sur la réflexion dialogique, que Mann a estimé n'avoir fait qu'effleurer le sujet.   

◦ Recherche sur la Réflexivité : Des travaux sur la réflexivité dans les entretiens de recherche qualitative, analysant comment les chercheurs réfléchissent à leur propre identité et méthodologie.

2. Contestation de la Vision Traditionnelle de la Réflexion

Mann remet en question la sémiotique dominante qui présente la réflexion comme une pratique purement individuelle et solitaire.

• L'image du "Penseur" : La sculpture "Le Penseur" de Rodin est citée comme l'archétype de cette vision de la pensée individuelle et isolée.

Mann note l'influence de Charles Baudelaire sur Rodin, soulignant le lien entre la forme physique et l'exploration des états émotionnels internes.

• La connotation négative : Cette vision individualiste a un "côté sombre", incarné par le mythe de Narcisse.

La pratique réflexive est ainsi souvent perçue de manière péjorative comme une forme d'introspection excessive ou de "nombrilisme" (navel-gazing).

• Le Contexte Éducatif : Le système éducatif est souvent décrit comme "monologique", dominé par la parole de l'enseignant qui fournit des réponses à des questions que les élèves n'ont pas posées.

Le travail de Mann vise à "perturber" ou "intervenir" dans ces normes d'interaction pour les rendre plus dialogiques.

3. Le Concept du "Moteur Interactionnel"

Pour contrer l'idée que le dialogue structuré est artificiel, Mann s'appuie sur des recherches en études néonatales, notamment celles de Stephen Levinson.

• Preuves chez les nouveau-nés : Des études montrent que les nouveau-nés interagissent avec leurs soignants quelques jours seulement après la naissance.

On observe des preuves de prise de tour (turn-taking) et d'organisation séquentielle dans leur regard et leurs interactions.

• Une Capacité Innée : Levinson propose l'existence d'un "moteur interactionnel" (interactional engine), une capacité humaine spéciale et innée pour l'interaction.

Cette capacité inclut des compétences cognitives comme l'attention conjointe, l'empathie et la recherche d'un terrain d'entente (common ground).

• Implications Fondamentales : Si l'empathie et l'écoute sont des aspects fondamentaux de l'expérience humaine dès le début de la vie, alors les pratiques qui les favorisent ne sont pas artificielles mais exploitent une disposition naturelle.

• Neurosciences et Interaction : Mann cite des études montrant que les processus cérébraux et cognitifs fonctionnent différemment lorsque les individus sont en interaction.

Par exemple, le cerveau d'un nourrisson réagit différemment à une écoute dirigée vers lui par rapport à une écoute périphérique.

De plus, les messages soutenus par des éléments multimodaux sont mieux assimilés par le cerveau.

4. Outils et Méthodes pour la Pratique Dialogique

Pour être efficace, la réflexion dialogique doit être médiatisée par des outils et un "étayage" (scaffolding) appropriés, au sens vygotskien du terme.

Outil / Approche

Description

Outils vidéo (Iris Connect, VEO)

Permettent aux praticiens (enseignants, médecins) d'analyser leurs propres interactions.

E-portfolios et Podcasts

Offrent des moyens multimodaux pour la création de sens et la réflexion.

Mentorat et Coaching

Projets qui structurent la pratique réflexive et l'intègrent dans le développement professionnel.

Recherche-Action

Approche visant à modifier les normes d'interaction au sein des séminaires ou des formations.

5. Perspectives pour l'Institut d'Études Avancées de Paris

Mann souligne l'alignement entre son projet et la mission de l'IEA, qui est de "promouvoir des discussions qui encouragent la réflexion".

• Témoignages de Résidents : Il cite le rapport annuel de l'institut, où des résidents témoignent de l'importance des conversations informelles et de la manière dont ces interactions ont fait évoluer de manière significative leur projet de recherche.

◦ _« Très enrichissant de discuter de manière informelle pendant le déjeuner et les apéritifs aussi.

Ces conversations m'ont aidé à la fois à voir mon propre projet d'un point de vue non spécialiste et à avoir une idée des développements importants dans d'autres domaines. »_   

◦ « Grâce à l'interaction à l'IEA, l'orientation initiale de ma recherche a considérablement évolué depuis sa création. Cela m'a amené à examiner les questions de pouvoir, les structures sociétales et leur impact sur l'atteinte des objectifs de durabilité. »

• Propositions pour l'Institut :

1. Documenter les processus : L'IEA pourrait-il systématiquement documenter et analyser les types d'interactions et de réflexions dialogiques qui s'y déroulent ?   

2. Une nouvelle méthode de recherche : L'institut pourrait-il positionner la réflexion dialogique comme l'une de ses nouvelles méthodes de recherche, valorisant ainsi les processus collaboratifs au même titre que les productions écrites ?

6. Plan de Recherche de Steve Mann

Durant sa résidence, Mann prévoit de se concentrer sur plusieurs axes :

• Analyse de Données Existantes : Réexaminer ses corpus de données (les siens et ceux de ses étudiants) pour identifier des exemples de réflexion dialogique.

• Identification de Marqueurs Linguistiques : Rechercher des preuves linguistiques spécifiques de la réflexion, telles que :

◦ La création de liens et de résonances.  

◦ L'utilisation de métaphores, de récits, d'anecdotes.    ◦ Les stratégies d'atténuation (hedging) et de spéculation.   

◦ La signalisation de "zones grises" ou de "tiers-espaces".    ◦ Les "moments eurêka" (light bulb moments).

• Influence de Bakhtine : Explorer la nature multimodale et intertextuelle de la réflexion dialogique, en s'appuyant sur le concept d'hétéroglossie de Bakhtine (les voix, concepts et cadres internalisés que nous mobilisons dans le dialogue).

• Tension Centripète/Centrifuge : Étudier comment l'esprit oscille entre un désir de focalisation (centripète) et une volonté d'élargir les perspectives (centrifuge).

7. Échanges avec les autres Chercheurs

La présentation a suscité des réactions et des connexions avec les travaux d'autres résidents.

• Dialogue avec Sadi :

◦ Sadi exprime son intérêt pour l'approche de Mann afin d'améliorer les "formats" de l'IEA et mentionne l'approche de l'enquête humble (humble inquiry) d'Edgar Schein.  

◦ Il partage une expérience utilisant des micro-caméras qui révèlent une synchronisation des regards entre des personnes résolvant un problème.

Cela illustre le "triangle psychosocial" : l'ego, l'alter et l'objet. 

◦ Il émet l'hypothèse que le succès de l'IEA réside dans l'absence de hiérarchie ou de compétition, ce qui permet aux chercheurs de se concentrer sur l'objet de la discussion plutôt que sur les relations interpersonnelles.

• Dialogue avec Eleanor :

◦ Eleanor établit un lien avec le concept de "co-construction" du sens (une poignée de main nécessite deux personnes).   

◦ Elle cite les travaux de Charles Goodwin ("Co-operation"), qui a analysé à un niveau micro-temporel comment la pensée se forme pendant que l'on parle.    ◦

Elle recommande deux chercheuses françaises travaillant sur ces sujets : Aude-Marie Morgenstern et Maya Gratier, qui étudient les interactions entre mères et nourrissons et leur dimension "musicale".

La Créativité : Perspectives Croisées des Neurosciences, de l'Art, de la Musique et de l'Intelligence Artificielle

Résumé

Ce document de synthèse analyse les thèmes et les arguments clés d'une table ronde sur la créativité, réunissant des experts en neurosciences, composition musicale, arts plastiques et intelligence artificielle.

La discussion s'articule autour d'un cadre conceptuel définissant la créativité humaine selon quatre dimensions : la nouveauté, l'adéquation, l'authenticité et l'agentivité.

Les intervenants explorent comment ces dimensions se manifestent dans leurs domaines respectifs.

En intelligence artificielle, la créativité émerge par des mécanismes de curiosité et des algorithmes évolutionnistes, permettant à des robots de découvrir de manière autonome des solutions nouvelles et efficaces à des problèmes complexes, comme le démontrent les exemples du jeu de Go ou de l'apprentissage moteur.

Dans le domaine artistique et musical, la créativité oscille entre la génération au sein de contraintes strictes (l'algorithme de composition de Mozart) et la transgression délibérée des conventions pour créer de l'inédit (l'hybridation chez Beethoven).

Les bases neuroscientifiques révèlent le rôle central du cortex préfrontal, qui agit comme un moniteur capable d'inhiber des stratégies inefficaces pour laisser émerger de nouvelles solutions issues de la mémoire.

Enfin, des exemples tirés du monde animal, notamment le poulpe et sa capacité de camouflage et de ruse ("métis"), suggèrent que la créativité est un phénomène plus large que l'activité purement humaine.

La discussion conclut sur les limites actuelles de l'IA, qui excelle à produire des surfaces cohérentes mais peine encore à générer des œuvres dotées de la profondeur structurelle et de l'authenticité caractéristiques de la création humaine.

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1. Un Cadre Théorique pour la Créativité

Étienne Koechlin, neuroscientifique, propose un modèle standard pour décomposer le concept de créativité en quatre dimensions fondamentales.

Ce cadre sert de référence tout au long de la discussion pour analyser les différentes manifestations de la créativité.

Dimension

Description

Concepts Clés

Cognitives

Nouveauté

La capacité à produire quelque chose qui n'existait pas auparavant. Cette possibilité est inhérente même aux systèmes formels les plus fermés, comme le démontre le théorème de Gödel.

Génération, innovation, possibilité de l'inédit.

Adéquation

La production nouvelle doit être pertinente par rapport à un contexte externe. Cela peut être la solution à un problème, ou une œuvre d'art qui résonne avec un public.

Évaluation, pertinence, contexte, originalité (articulation nouveauté/adéquation).

Conatives

Authenticité

L'acte créatif est l'expression d'un individu, souvent issue d'un déséquilibre interne (insatisfaction, état extatique).

Le créateur cherche à répondre à ce déséquilibre.

Expression individuelle, déséquilibre interne, énergie créatrice.

Agentivité

La créativité est une action visant à transformer ou influencer le monde. Il y a une volonté d'être effectif, d'avoir un impact.

Action, volonté, transformation du monde, effectivité.

Koechlin souligne que ces dimensions peuvent être présentes à des degrés divers selon l'activité (humaine, animale ou artificielle).

Par exemple, une IA comme AlphaGo fait preuve de nouveauté et d'adéquation (coups créatifs pour gagner), et d'une forme d'agentivité (interagir avec un joueur humain), mais son authenticité est considérée comme très réduite.

2. La Créativité dans les Systèmes Artificiels

Pierre-Yves Oudeyer, chercheur en IA, présente comment des machines peuvent générer des comportements et des connaissances à la fois nouveaux, pertinents et efficaces, remplissant ainsi plusieurs critères de la créativité.

2.1. La Curiosité comme Moteur de l'Exploration

Le travail de l'équipe de P-Y. Oudeyer se concentre sur la modélisation de la curiosité, comprise comme le mécanisme poussant un agent (enfant ou robot) à explorer spontanément son environnement.

• Apprentissage Autonome : Un robot quadrupède, initialement sans connaissance de son corps ou de l'environnement, apprend par expérimentation.

Guidé par des algorithmes de curiosité, il teste des actions (bouger ses membres, vocaliser) et observe les résultats.

• Découverte de Régularités : Le robot découvre progressivement des relations de cause à effet : pousser un objet avec son bras le fait bouger, vocaliser vers un autre robot provoque une imitation.

Cette exploration, motivée par la curiosité, le mène à découvrir les interactions sociales.

Étienne Koechlin relie cette approche à la recherche en neurosciences sur les moteurs de l'action.

Il oppose deux visions : l'action pour accumuler des ressources (récompenses) et l'action pour acquérir de l'information et améliorer ses modèles internes du monde.

La curiosité est au cœur de cette seconde vision : on agit là où l'on pense pouvoir apprendre le plus.

2.2. Algorithmes Évolutionnistes et Apprentissage par Renforcement

Des algorithmes inspirés de l'évolution biologique permettent de générer des solutions créatives que des ingénieurs n'auraient pas envisagées.

• Créatures Virtuelles : Dans une simulation, des "créatures" composées de cellules virtuelles (muscles, cellules rigides) sont générées aléatoirement.

Un critère de "fitness" (capacité à avancer vite) est défini.

Les créatures les plus performantes sont sélectionnées, leurs "gènes" sont mutés aléatoirement pour créer une nouvelle génération.

Au fil des générations, des formes de corps et des stratégies de locomotion efficaces et inattendues émergent.

• Robots Physiques : Un robot physique apprend à se déplacer par essais et erreurs (apprentissage par renforcement). Initialement, ses mouvements sont aléatoires et maladroits.

En quelques minutes, il découvre comment se retourner, puis se mettre sur ses pattes et marcher de manière robuste, capable de réagir aux perturbations.

La stratégie de mouvement finale n'a pas été programmée par un humain, mais découverte par le robot lui-même.

Ces mêmes méthodes sont à la base des succès d'AlphaGo, qui a produit des coups jugés "hautement créatifs" par les experts humains.

3. La Créativité dans la Pratique Artistique

Les intervenants issus des domaines de la musique et des arts plastiques illustrent la tension créative entre la contrainte et la liberté, et entre la tradition et l'innovation.

3.1. Musique : Algorithmes et Transgressions

Le compositeur Floris Guédy présente deux modèles de création musicale :

• Le Jeu de Dés de Mozart : Un système algorithmique pour composer des menuets.

En lançant des dés, on sélectionne des mesures pré-écrites dans une matrice.

Bien que basé sur le hasard, le système est ultra-contraint par des règles d'harmonie tonale (fonctions harmoniques : sujet, verbe, complément).

Le résultat est toujours cohérent et varié, générant des milliards de combinaisons possibles.

Ce système peut être généralisé pour simuler, avec le même modèle de base, les styles de compositeurs ultérieurs (Schumann, Debussy) en changeant simplement les paramètres.

• L'Hybridation chez Beethoven : L'analyse des brouillons de la 30ème sonate pour piano montre un processus créatif différent. Beethoven oppose deux éléments musicaux (A : monodique et piqué ; B : accords liés) et crée un troisième élément (C) en hybridant leurs caractéristiques.

Ses carnets révèlent un processus de recherche active, d'essais et d'erreurs pour trouver le contraste maximal rendant l'hybridation la plus audible possible.

Pour F. Guédy, ce type de créativité, qui consiste à "casser les conventions" d'une infinité de manières possibles, est difficilement simulable par une IA qui cherche plutôt à reproduire ce qui est statistiquement probable.

3.2. Arts et Artisanat : Co-création et Matière Active

Patricia Ribault, spécialiste en arts plastiques, met en lumière la créativité dans les processus de "faire" et les interactions.

• La Co-création à Murano : Lors d'un workshop, des étudiants en design présentent des dessins aux maîtres verriers de Murano.

Les artisans, confrontés à des formes qui dépassent leur savoir-faire traditionnel, doivent inventer de nouvelles techniques.

Ce moment de "cocréation" pousse les techniques traditionnelles au-delà de leurs limites.

• La Matière Active ("Active Matter") : Elle décrit son travail au sein du cluster d'excellence "Matters of Activity", où des chercheurs de toutes disciplines (scientifiques, ingénieurs, designers) étudient des pratiques comme le filtrage, le tissage ou la découpe sous l'angle de la matière elle-même comme agent actif.

• Visualisation de la Neuroplasticité : Elle présente le projet "Brain Roads", une collaboration entre artistes, designers et neurochirurgiens visant à visualiser la complexité de la plasticité cérébrale.

Face aux limites des imageries traditionnelles (tractographie), les artistes proposent de nouveaux modèles graphiques (inspirés des cartes de métro, des voxels) pour mieux guider le geste du chirurgien et représenter l'expérience des patients en chirurgie éveillée.

4. Les Bases Biologiques et Neuroscientifiques

La discussion explore les mécanismes cérébraux sous-jacents à la créativité humaine ainsi que ses manifestations dans le monde animal.

4.1. Le Rôle du Cortex Préfrontal

Étienne Koechlin explique que le cortex préfrontal est la région clé qui "autorise" la créativité chez l'homme.

• Le Mécanisme de Contrôle et d'Ouverture : Cette région du cerveau monitore en permanence nos comportements et stratégies mentales.

Lorsqu'une stratégie est jugée non pertinente ou inefficace, le cortex préfrontal l'inhibe.

Cette inhibition permet à de nouvelles options, issues d'un "remixage" contextualisé de la mémoire à long terme, d'émerger.

• Gestion de la Propre Limitation : Le système est conçu pour prendre en compte sa propre limitation. Il accepte de "perdre le contrôle" pour permettre l'émergence de la nouveauté.

Les nouvelles options sont ensuite évaluées : si elles sont probantes, elles sont confirmées et consolidées en mémoire, enrichissant le répertoire de l'individu pour de futures créations.

• L'Exemple du Test des 9 Points : Ce test classique illustre le processus.

Pour relier 9 points avec 4 segments de droite sans lever le crayon, il faut abandonner des modèles mentaux implicites (ne pas sortir du carré, ne pas repasser sur un trait).

La solution émerge lorsqu'on transgresse ces règles auto-imposées.

4.2. La Créativité Animale : Le Poulpe et la "Métis"

Patricia Ribault utilise l'exemple du poulpe pour illustrer une forme d'intelligence créative non-humaine, la "métis" (la ruse), théorisée par Marcel d'Étienne et Jean-Pierre Vernand.

• Un Être sans Structure Rigide : Le poulpe peut prendre et perdre forme, ce qui lui confère une plasticité exceptionnelle.

• Maître du Camouflage : Sa créativité s'exprime dans sa capacité à interagir avec la perception de l'autre.

Le camouflage n'est pas seulement se fondre, mais "tromper celui ou ceux qui vous regardent". Il peut être défensif ou offensif (hypnotiser une proie).

• Le "Mimic Octopus" : Cette espèce est capable non seulement de se camoufler mais de changer son comportement pour imiter d'autres animaux en fonction de la situation.

• La Métis comme Forme de Créativité : La métis est décrite comme une "intelligence à l'œuvre dans le devenir", utilisant "la prudence, la perspicacité, la promptitude", mais aussi "la ruse, voire le mensonge".

L'être "amétis", comme le poulpe, est "insaisissable" et capable de "retourner constamment des situations".

5. Thèmes Transversaux et Conclusion

La discussion finale aborde plusieurs questions clés sur la nature de la créativité et les distinctions entre l'humain et la machine.

• Authenticité et Subjectivité : La question de l'authenticité reste la plus difficile à attribuer aux IA.

L'authenticité humaine est liée à un déséquilibre interne et à une intention expressive.

Les IA peuvent simuler une forme de subjectivité primaire (en ayant des modèles de leurs propres connaissances), mais l'expressivité profonde reste un attribut humain.

• Hasard et Contrainte : Le hasard est une composante essentielle du fonctionnement cérébral, notamment via le "bruit neuronal" qui augmente lorsque les modèles du monde sont mis en défaut, ouvrant le "champ des possibles".

Cependant, comme le montre le jeu de Mozart, un hasard apparent peut opérer au sein de contraintes très fortes.

La créativité réside dans ce jeu entre ouverture (pensée divergente) et fermeture (pensée convergente).

• Les Limites Actuelles de l'IA : Une anecdote est partagée sur une IA chargée d'improviser dans le style de L'Art de la Fugue de Bach.

Le résultat était bluffant en surface ("la chair"), mais ignorait complètement la structure fondamentale de l'œuvre.

De même, un texte rédigé par une IA est décrit comme "très fluide", "cohérent en surface", mais sans "corps" ni profondeur sémantique.

• Sérendipité : Il est souligné que la créativité ne peut pas être planifiée.

Elle émerge souvent de la sérendipité : la découverte de quelque chose d'intéressant par hasard en cherchant autre chose.

Pour être efficace, la sérendipité nécessite cependant une capacité de reconnaissance de ce qui est intéressant, ce qui renvoie à la subjectivité et au modèle interne du créateur.

Reviewer #1 (Public review):

Summary:

The authors attempt to study how oocyte incomplete cytokinesis occurs in the mouse ovary.

Strengths:

The finding that UPR components are highly expressed during zygotene is an interesting result that has broad implications for how germ cells navigate meiosis. The findings that proteasome activity increases in germ cells compared to somatic cells suggest that the germline might have a quantitatively different response for protein clearance.

Weaknesses:

(1) The microscopy images look saturated, for example, Figure 1a, b, etc? Is this a normal way to present fluorescent microscopy?

(2) The authors should ensure that all claims regarding enrichment/lower vs lower values have indicated statistical tests.

(a) In Figure 2f, the authors should indicate which comparison is made for this test. Is it comparing 2 vs 6 cyst numbers?

(b) Figures 4d and 4e do not have a statistical test indicated.

(3) Because the system is developmentally dynamic, the major conclusions of the work are somewhat unclear. Could the authors be more explicit about these and enumerate them more clearly in the abstract?

(4) The references for specific prior literature are mostly missing (lines 184-195, for example).

(5) The authors should define all acronyms when they are first used in the text (UPR, EGAD, etc).

(6) The jumping between topics (EMA, into microtubule fragmentation, polarization proteins, UPR/ERAD/EGAD, GCNA, ER, balbiani body, etc) makes the narrative of the paper very difficult to follow.

(7) The heading title "Visham participates in organelle rejuvenation during meiosis" in line 241 is speculative and/or not supported. Drawing upon the extensive, highly rigorous Drosophila literature, it is safe to extrapolate, but the claim about regeneration is not adequately supported.

Reviewer #3 (Public review):

This manuscript provides evidence that mice have a fusome, a conserved structure most well studied in Drosophila that is important for oocyte specification. Overall, a myriad of evidence is presented demonstrating the existence of a mouse fusome that the authors term visham. This work is important as it addresses a long-standing question in the field of whether mice have fusomes and sheds light on how oocytes are specified in mammals. Concerns that need to be addressed revolve around several conclusions that are overstated or unclear and are listed below.

(1) Line 86 - the heading for this section is "PGCs contain a Golgi-rich structure known as the EMA granule" but there is nothing in this section that shows it is Golgi-rich. It does show that the structure is asymmetric and has branches.

(2) Line 105-106, how do we know if what's seen by EM corresponds to the EMA1 granule?

(3) Line 106-107-states "Visham co-stained with the Golgi protein Gm130 and the recycling endosomal protein Rab11a1". This is not convincing as there is only one example of each image, and both appear to be distorted.

(4) Line 132-133---while visham formation is disrupted when microtubules are disrupted, I am not convinced that visham moves on microtubules as stated in the heading of this section.

(5) Line 156 - the heading for this section states that Visham associates with polarity and microtubule genes, including pard3, but only evidence for pard3 is presented.

(6) Lines 196-210 - it's strange to say that UPR genes depend on DAZ, as they are upregulated in the mutants. I think there are important observations here, but it's unclear what is being concluded.

(7) Line 257-259---wave 1 and 2 follicles need to be explained in the introduction, and how this fits with the observations here clarified.

Author response:

Reviewer #1 (Public Review):

Summary

We thank the reviewer for the constructive and thoughtful evaluation of our work. We appreciate the recognition of the novelty and potential implications of our findings regarding UPR activation and proteasome activity in germ cells.

(1) The microscopy images look saturated, for example, Figure 1a, b, etc. Is this a normal way to present fluorescent microscopy?

The apparent saturation was not present in the original images, but likely arose from image compression during PDF generation. While the EMA granule was still apparent, in the revised submission, we will provide high-resolution TIFF files to ensure accurate representation of fluorescence intensity and will carefully optimize image display settings to avoid any saturation artifacts.

(2) The authors should ensure that all claims regarding enrichment/lower vs. lower values have indicated statistical tests.

We fully agree. In the revised version, we will correct any quantitative comparisons where statistical tests were not already indicated, with a clear statement of the statistical tests used, including p-values in figure legends and text.

(a) In Figure 2f, the authors should indicate which comparison is made for this test. Is it comparing 2 vs. 6 cyst numbers?

We acknowledge that the description was not sufficiently detailed. Indeed, the test was not between 2 vs 6 cyst numbers, but between all possible ways 8-cell cysts or the larger cysts studied could fragment randomly into two pieces, and produce by chance 6-cell cysts in 13 of 15 observed examples. We will expand the legend and main text to clarify that a binomial test was used to determine that the proportion of cysts producing 6-cell fragments differed very significantly from chance.

Revised text:

“A binomial test was used to assess whether the observed frequency of 6-cell cyst products differed from random cyst breakage. Production of 6-cell cysts was strongly preferred (13/15 cysts; ****p < 0.0001).”

(b) Figures 4d and 4e do not have a statistical test indicated.

We will include the specific statistical test used and report the corresponding p-values directly in the figure legends.

(3) Because the system is developmentally dynamic, the major conclusions of the work are somewhat unclear. Could the authors be more explicit about these and enumerate them more clearly in the abstract?

We will revise the abstract to better clarify the findings of this study. We will also replace the term Visham with mouse fusome to reflect its functional and structural analogy to the Drosophila and Xenopus fusomes, making the narrative more coherent and conclusive.

(4) The references for specific prior literature are mostly missing (lines 184-195, for example).

We appreciate this observation of a problem that occurred inadvertently when shortening an earlier version.  We will add 3–4 relevant references to appropriately support this section.

(5) The authors should define all acronyms when they are first used in the text (UPR, EGAD, etc).

We will ensure that all acronyms are spelled out at first mention (e.g., Unfolded Protein Response (UPR), Endosome and Golgi-Associated Degradation (EGAD)).

(6)  The jumping between topics (EMA, into microtubule fragmentation, polarization proteins, UPR/ERAD/EGAD, GCNA, ER, balbiani body, etc) makes the narrative of the paper very difficult to follow.

We are not jumping between topics, but following a narrative relevant to the central question of whether female mouse germ cells develop using a fusome.  EMA, microtubule fragmentation, polarization proteins, ER, and balbiani body are all topics with a known connection to fusomes. This is explained in the general introduction and in relevant subsections. We appreciate this feedback that further explanations of these connections would be helpful. In the revised manuscript, use of the unified term mouse fusome will also help connect the narrative across sections.  UPR/ERAD/EGAD are processes that have been studied in repair and maintenance of somatic cells and in yeast meiosis.  We show that the major regulator XbpI is found in the fusome, and that the fusome and these rejuvenation pathway genes are expressed and maintained throughout oogenesis, rather than only during limited late stages as suggested in previous literature.

(7) The heading title "Visham participates in organelle rejuvenation during meiosis" in line 241 is speculative and/or not supported. Drawing upon the extensive, highly rigorous Drosophila literature, it is safe to extrapolate, but the claim about regeneration is not adequately supported.

We believe this statement is accurate given the broad scope of the term "participates." It is supported by localization of the UPR regulator XbpI to the fusome. XbpI is the ortholog of HacI a key gene mediating UPR-mediated rejuvenation during yeast meiosis.  We also showed that rejuvenation pathway genes are expressed throughout most of meiosis (not previously known) and expanded cytological evidence of stage-specific organelle rejuvenation later in meiosis, such as mitochondrial-ER docking, in regions enriched in fusome antigens. However, we recognize the current limitations of this evidence in the mouse, and want to appropriately convey this, without going to what we believe would be an unjustified extreme of saying there is no evidence. 

Reviewer #2 (Public Review):

We thank the reviewer for the comprehensive summary and for highlighting both the technical achievement and biological relevance of our study. We greatly appreciate the thoughtful suggestions that have helped us refine our presentation and terminology.

(1) Some titles contain strong terms that do not fully match the conclusions of the corresponding sections.

(1a) Article title “Mouse germline cysts contain a fusome-like structure that mediates oocyte development”

We will change the statement to: “Mouse germline cysts contain a fusome that supports germline cyst polarity and rejuvenation.”

(1b) Result title “Visham overlaps centrosomes and moves on microtubules” We acknowledge that “moves” implies dynamics. We will include additional supplementary images showing small vesicular components of the mouse fusome on spindle-derived microtubule tracks.

(1c) Result title “Visham associates with Golgi genes involved in UPR beginning at the onset of cyst formation”

We will revise this title to: “The mouse fusome associates with the UPR regulatory protein Xbp1 beginning at the onset of cyst formation” to reflect the specific UPR protein that was immunolocalized. 

(1d) Result title “Visham participates in organelle rejuvenation during meiosis”

We will revise this to: “The mouse fusome persists during organelle rejuvenation in meiosis.”

(2) The authors aim to demonstrate that Visham is a fusome-like structure. I would suggest simply referring to it as a "fusome-like structure" rather than introducing a new term, which may confuse readers and does not necessarily help the authors' goal of showing the conservation of this structure in Drosophila and Xenopus germ cells. Interestingly, in a preprint from the same laboratory describing a similar structure in Xenopus germ cells, the authors refer to it as a "fusome-like structure (FLS)" (Davidian and Spradling, BioRxiv, 2025).

We appreciate the reviewer’s insightful comment. To maintain conceptual clarity and align with existing literature, we will refer to the structure as the mouse fusome throughout the manuscript, avoiding introduction of a new term.

Reviewer #3 (Public Review):

We thank the reviewer for emphasizing the importance of our study and for providing constructive feedback that will help us clarify and strengthen our conclusions.

(1) Line 86 - the heading for this section is "PGCs contain a Golgi-rich structure known as the EMA granule" 

We agree that the enrichment of Golgi within the EMA PGCs was not shown until the next section. We will revise this heading to:

“PGCs contain an asymmetric EMA granule.”

(2)  Line 105-106, how do we know if what's seen by EM corresponds to the EMA1 granule?

We will clarify that this identification is based on co-localization with Golgi markers (GM130 and GS28) and response to Brefeldin A treatment, which will be included as supplementary data. These findings support that the mouse fusome is Golgi-derived and can therefore be visualized by EM. The Golgi regions in E13.5 cyst cells move close together and associate with ring canals as visualized by EM (Figure 1E), the same as the mouse fusomes identified by EMA.

(3) Line 106-107-states "Visham co-stained with the Golgi protein Gm130 and the recycling endosomal protein Rab11a1". This is not convincing as there is only one example of each image, and both appear to be distorted.

Space is at a premium in these figures, but we have no limitation on data documenting this absolutely clear co-localization. We will replace the existing images with high-resolution, non-compressed versions for the final figures to clearly illustrate the co-staining patterns for GM130 and Rab11a1.

(4) Line 132-133---while visham formation is disrupted when microtubules are disrupted, I am not convinced that visham moves on microtubules as stated in the heading of this section.

We will include additional supplementary data showing small mouse fusome vesicles aligned along microtubules.

(5) Line 156 - the heading for this section states that Visham associates with polarity and microtubule genes, including pard3, but only evidence for pard3 is presented.

We agree and will revise the heading to: “Mouse fusome associates with the polarity protein Pard3.” We are adding data showing association of small fusome vesicles on microtubules.  

(6)  Lines 196-210 - it's strange to say that UPR genes depend on DAZ, as they are upregulated in the mutants. I think there are important observations here, but it's unclear what is being concluded.

UPR genes are not upregulated in DAZ in the sense we have never documented them increasing. We show that UPR genes during this time behave like pleuripotency genes and normally decline, but in DAZ mutants their decline is slowed.  We will rephrase the paragraph to clarify that Dazl mutation partially decouples developmental processes that are normally linked, which alters UPR gene expression relative to cyst development.

(7) Line 257-259-wave 1 and 2 follicles need to be explained in the introduction, and how these fits with the observations here clarified.

Follicle waves are too small a focus of the current study to explain in the introduction, but we will request readers to refer to the cited relevant literature (Yin and Spradling, 2025) for further details.

We sincerely thank all reviewers for their insightful and constructive feedback. We believe that the planned revisions—particularly the refined terminology, improved image quality, clarified statistics, and restructured abstract—will substantially strengthen the manuscript and enhance clarity for readers.

Reviewer #1 (Public review):

Summary:

In this paper, the authors conduct both experiments and modeling of human cytomegalovirus (HCMV) infection in vitro to study how the infectivity of the virus (measured by cell infection) scales with the viral concentration in the inoculum. A naïve thought would be that this is linear in the sense that doubling the virus concentration (and thus the total virus) in the inoculum would lead to doubling the fraction of infected cells. However, the authors show convincingly that this is not the case for HCMV, using multiple strains, two different target cells, and repeated experiments. In fact, they find that for some regimens (inoculum concentration), infected cells increase faster than the concentration of the inoculum, which they term "apparent cooperativity". The authors then provided possible explanations for this phenomenon and constructed mathematical models and simulations to implement these explanations. They show that these ideas do help explain the cooperativity, but they can't be conclusive as to what the correct explanation is. In any case, this advances our knowledge of the system, and it is very important when quantitative experiments involving MOI are performed.

Strengths:

Careful experiments using state-of-the-art methodologies and advancing multiple competing models to explain the data.

Weaknesses:

There are minor weaknesses in explaining the implementation of the model. However, some specific assumptions, which to this reviewer were unclear, could have a substantial impact on the results. For example, whether cell infection is independent or not. This is expanded below.

Suggestions to clarify the study:

(1) Mathematically, it is clear what "increase linearly" or "increase faster than linearly" (e.g., line 94) means. However, it may be confusing for some readers to then look at plots such as in Figure 2, which appear linear (but on the log-log scale) and about which the authors also say (line 326) "data best matching the linear relationship on a log-log scale".

(2) One of the main issues that is unclear to me is whether the authors assume that cell infection is independent of other cells. This could be a very important issue affecting their results, both when analyzing the experimental data and running the simulations. One possible outcome of infection could be the generation of innate mediators that could protect (alter the resistance) of nearby cells. I can imagine two opposite results of this: i) one possibility is that resistance would lead to lower infection frequencies and this would result in apparent sub-linear infection (contrary to the observations); or ii) inoculums with more virus lead to faster infection, which doesn't allow enough time for the "resistance" (innate effect) to spread (potentially leading to results similar to the observations, supra-linear infection).

(3) Another unclear aspect of cell infection is whether each cell only has one chance to be infected or multiple chances, i.e., do the authors run the simulation once over all the cells or more times?

(4) On the other hand, the authors address the complementary issue of the virus acting independently or not, with their clumping model (which includes nice experimental measurements). However, it was unclear to me what the assumption of the simulation is in this case. In the case of infection by a clump of virus or "viral compensation", when infection is successful (the cell becomes infected), how many viruses "disappear" and what happens to the rest? For example, one of the viruses of the clump is removed by infection, but the others are free to participate in another clump, or they also disappear. The only thing I found about this is the caption of Figure S10, and it seems to indicate that only the infected virus is removed. However, a typical assumption, I think, is that viruses aggregate to improve infection, but then the whole aggregate participates in infection of a single cell, and those viruses in the clump can't participate in other infections. Viral cooperativity with higher inocula in this case would be, perhaps, the result of larger numbers of clumps for higher inocula. This seems in agreement with Figure S8, but was a little unclear in the interpretation provided.

(5) In algorithm 1, how does P_i, as defined, relate to equation 1?

(6) In line 228, and several other places (e.g., caption of Table S2), the authors refer to the probability of a single genome infecting a cell p(1)=exp(-lambda), but shouldn't it be p(1)=1-exp(-lambda) according to equation 1?

(7) In line 304, the accrued damage hypothesis is defined, but it is stated as a triggering of an antiviral response; one would assume that exposure to a virion should increase the resistance to infection. Otherwise, the authors are saying that evolution has come up with intracellular viral resistance mechanisms that are detrimental to the cell. As I mentioned above, this could also be a mechanism for non-independent cell infection. For example, infected cells signal to neighboring cells to "become resistance" to infection. This would also provide a mechanism for saturation at high levels.

(8) In Figure 3, and likely other places, t-tests are used for comparisons, but with only an n=5 (experiments). Many would prefer a non-parametric test.

Reviewer #3 (Public review):

Summary:

The authors dilute fluorescent HCMV stocks in small steps (df ≈ 1.3-1.5) across 23 points, quantify infections by flow cytometry at 3 dpi, and fit a power-law model to estimate a cooperativity parameter n (n > 1 indicates apparent cooperativity). They compare fibroblasts vs epithelial cells and multiple strains/reporters, and explore alternative mechanisms (clumping, accrued damage, viral compensation) via analytical modeling and stochastic simulations. They discuss implications for titer/MOI estimation and suggest a method for detecting "apparent cooperativity," noting that for viruses showing this behavior, MOI estimation may be biased.

Strengths:

(1) High-resolution titration & rigor: The small-step dilution design (23 serial dilutions; tailored df) improves dose-response resolution beyond conventional 10× series.

(2) Clear quantitative signal: Multiple strain-cell pairs show n > 1, with appropriate model fitting and visualization of the linear regime on log-log axes.

(3) Mechanistic exploration: Side-by-side modeling of clumping vs accrued damage vs compensation frames testable hypotheses for cooperativity.

Weaknesses:

(1) Secondary infection control: The authors argue that 3 dpi largely avoids progeny-mediated secondary infection; this claim should be strengthened (e.g., entry inhibitors/control infections) or add sensitivity checks showing results are robust to a small secondary-infection contribution.

(2) Discriminating mechanisms: At present, simulations cannot distinguish between accrued damage and viral compensation. The authors should propose or add a decisive experiment (e.g., dual-color coinfection to quantify true coinfection rates versus "priming" without coinfection; timed sequential inocula) and outline expected signatures for each mechanism.

(3) Decline at high genomes/cell: Several datasets show a downturn at high input. Hypotheses should be provided (cytotoxicity, receptor depletion, and measurement ceiling) and any supportive controls.

(4) Include experimental data: In Figure 6, please include the experimentally measured titers (IU/mL), if available.

(5) MOI guidance: The practical guidance is important; please add a short "best-practice box" (how to determine titer at multiple genomes/cell and cell densities; when single-hit assumptions fail) for end-users.

Author response:

Reviewer #1 (Public review):

Summary:

In this paper, the authors conduct both experiments and modeling of human cytomegalovirus (HCMV) infection in vitro to study how the infectivity of the virus (measured by cell infection) scales with the viral concentration in the inoculum. A naïve thought would be that this is linear in the sense that doubling the virus concentration (and thus the total virus) in the inoculum would lead to doubling the fraction of infected cells. However, the authors show convincingly that this is not the case for HCMV, using multiple strains, two different target cells, and repeated experiments. In fact, they find that for some regimens (inoculum concentration), infected cells increase faster than the concentration of the inoculum, which they term "apparent cooperativity". The authors then provided possible explanations for this phenomenon and constructed mathematical models and simulations to implement these explanations. They show that these ideas do help explain the cooperativity, but they can't be conclusive as to what the correct explanation is. In any case, this advances our knowledge of the system, and it is very important when quantitative experiments involving MOI are performed.

Strengths:

Careful experiments using state-of-the-art methodologies and advancing multiple competing models to explain the data.

Weaknesses:

There are minor weaknesses in explaining the implementation of the model. However, some specific assumptions, which to this reviewer were unclear, could have a substantial impact on the results. For example, whether cell infection is independent or not. This is expanded below.

Suggestions to clarify the study:

(1) Mathematically, it is clear what "increase linearly" or "increase faster than linearly" (e.g., line 94) means. However, it may be confusing for some readers to then look at plots such as in Figure 2, which appear linear (but on the log-log scale) and about which the authors also say (line 326) "data best matching the linear relationship on a log-log scale". 

This is a good point. In our revision, we will include a clarification to indicate that linear on the log-log scale relationship does not imply linear relationship on the linear-linear scale.

(2) One of the main issues that is unclear to me is whether the authors assume that cell infection is independent of other cells. This could be a very important issue affecting their results, both when analyzing the experimental data and running the simulations. One possible outcome of infection could be the generation of innate mediators that could protect (alter the resistance) of nearby cells. I can imagine two opposite results of this: i) one possibility is that resistance would lead to lower infection frequencies and this would result in apparent sub-linear infection (contrary to the observations); or ii) inoculums with more virus lead to faster infection, which doesn't allow enough time for the "resistance" (innate effect) to spread (potentially leading to results similar to the observations, supra-linear infection). 

In our models we assumed cells to be independent of each other (see also responses to other similar points). Because we measure infection in individual cells, assuming cells are independent is a reasonable first approximation. However, the reviewer makes an excellent point that there may be some between-cell signaling happening in the culture that “alerts” or “conditions” cells to change their “resistance”. It is also possible that at higher genome/cell numbers, exposure of cells to virions or virion debris may change the state of cells in the culture, and more cells become “susceptible” to infection. This is a good point that we will list in Limitations subsection of Discussion; it is a good hypothesis to test in our future experiments.

(3) Another unclear aspect of cell infection is whether each cell only has one chance to be infected or multiple chances, i.e., do the authors run the simulation once over all the cells or more times? 

Each cell has only one chance to be infected. Algorithm 1 clearly states that; we will add an extra sentence in “Agent-based simulations” to indicate this point.

(4) On the other hand, the authors address the complementary issue of the virus acting independently or not, with their clumping model (which includes nice experimental measurements). However, it was unclear to me what the assumption of the simulation is in this case. In the case of infection by a clump of virus or "viral compensation", when infection is successful (the cell becomes infected), how many viruses "disappear" and what happens to the rest? For example, one of the viruses of the clump is removed by infection, but the others are free to participate in another clump, or they also disappear. The only thing I found about this is the caption of Figure S10, and it seems to indicate that only the infected virus is removed. However, a typical assumption, I think, is that viruses aggregate to improve infection, but then the whole aggregate participates in infection of a single cell, and those viruses in the clump can't participate in other infections. Viral cooperativity with higher inocula in this case would be, perhaps, the result of larger numbers of clumps for higher inocula. This seems in agreement with Figure S8, but was a little unclear in the interpretation provided. 

This is a good point. We did not remove the clump if one of the virions in the clump manages to infect a cell, and indeed, this could be the reason why in some simulations we observe apparent cooperativity when modeling viral clumping. This is something we will explore in our revision.

(5) In algorithm 1, how does P_i, as defined, relate to equation 1? 

These are unrelated because eqn.(1) is a phenomenological model that links infection per cell to genomes per cell. P_i in algorithm 1 is “physics-inspired” potential barrier.

(6) In line 228, and several other places (e.g., caption of Table S2), the authors refer to the probability of a single genome infecting a cell p(1)=exp(-lambda), but shouldn't it be p(1)=1-exp(-lambda) according to equation 1?

Indeed, it was a typo, p(1)=1-exp(-lambda) per eqn 1. Thank you, it will be corrected in the revised paper.

(7) In line 304, the accrued damage hypothesis is defined, but it is stated as a triggering of an antiviral response; one would assume that exposure to a virion should increase the resistance to infection. Otherwise, the authors are saying that evolution has come up with intracellular viral resistance mechanisms that are detrimental to the cell. As I mentioned above, this could also be a mechanism for non-independent cell infection. For example, infected cells signal to neighboring cells to "become resistance" to infection. This would also provide a mechanism for saturation at high levels. 

We do not know how exposure of a cell to one virion would change its “antiviral state”, i.e., to become more or less resistant to the next infection. If a cell becomes more resistant, there is no possibility to observe apparent cooperativity in infection of cells, so this hypothesis cannot explain our observations with n>1. Whether this mechanism plays a role in saturation of cell infection rate at lower than 1 value when genome/cell is large is unclear but is a possibility. We will add this point to Discussion in revision.

(8) In Figure 3, and likely other places, t-tests are used for comparisons, but with only an n=5 (experiments). Many would prefer a non-parametric test. 

We repeated the analyses in Fig 3 with Mann-Whitney test, results were the same, so we would like to keep results from the t-test in the paper.

Reviewer #2 (Public review):

In their article, Peterson et al. wanted to show to what extent the classical "single hit" model of virion infection, where one virion is required to infect a cell, does not match empirical observations based on human cytomegalovirus in vitro infection model, and how this would have practical impacts in experimental protocols.

They first used a very simple experimental assay, where they infected cells with serially diluted virions and measured the proportion of infected cells with flow cytometry. From this, they could elegantly show how the proportion of infected cells differed from a "single hit" model, which they simulated using a simple mathematical model ("powerlaw model"), and better fit a model where virions need to cooperate to infect cells. They then explore which mechanism could explain this apparent cooperation:

(1) Stochasticity alone cannot explain the results, although I am unsure how generalizable the results are, because the mathematical model chosen cannot, by design, explain such observations only by stochasticity. 

Our null model simulations are not just about stochasticity; they also include variability in virion infectivity and cell resistance to infection. We agree that simulations cannot truly prove that such variability cannot result in apparent cooperativity; however, we also provide a mathematical proof that increase in frequency of infected cells should be linear with virion concentration at small genome/cell numbers.

(2) Virion clumping seemed not to be enough either to generally explain such a pattern. For that, they first use a mathematical model showing that the apparent cooperation would be small. However, I am unsure how extreme the scenario of simulated virion clumping is. They then used dynamic light scattering to measure the distribution of the sizes of clumps. From these estimates, they show that virion clumps cannot reproduce the observed virion cooperation in serial dilution assays. However, the authors remain unprecise on how the uncertainty of these clumps' size distribution would impact the results, as most clumps have a size smaller than a single virion, leaving therefore a limited number of clumps truly containing virions. 

As we stated in the paper, clumping may explain apparent cooperativity in simulations depending on how stock dilution impacts distribution of virions/clump. This could be explored further, however, better experimental measurements of virions/clump would be highly informative (but we do not have resources to do these experiments at present). Our point is that the degree of apparent cooperativity is dependent on the target cell used (n is smaller on epithelial cells than on fibroblasts) that is difficult to explain by clumping which is a virion property. Per comment by reviewer 1, we will do some more analyses of the clumping model to investigate importance of clump removal per successful infection on the detected degree of apparent cooperativity.

The two models remain unidentifiable from each other but could explain the apparent virion cooperativity: either due to an increase in susceptibility of the cell each time a virion tries to infect it, or due to viral compensation, where lesser fit viruses are able to infect cells in co-infection with a better fit virion. Unfortunately, the authors here do not attempt to fit their mathematical model to the experimental data but only show that theoretical models and experimental data generate similar patterns regarding virion apparent cooperation. 

In the revision we will provide examples of simulations that “match” experimental data with a relatively high degree of apparent cooperativity; we have done those before but excluded them from the current version since they are a bit messy. Fitting simulations to data may be an overkill.

Finally, the authors show that this virions cooperation could make the relationship between the estimated multiplicity of infection and viruses/cell deviate from the 1:1 relationship. Consequently, the dilution of a virion stock would lead to an even stronger decrease in infectivity, as more diluted virions can cooperate less for infection.

Overall, this work is very valuable as it raises the general question of how the estimate of infectivity can be biased if extrapolated from a single virus titer assay. The observation that HCMV virions often cooperate and that this cooperation varies between contexts seems robust. The putative biological explanations would require further exploration.

This topic is very well known in the case of segmented viruses and the semi-infectious particles, leading to the idea of studying "sociovirology", but to my knowledge, this is the first time that it was explored for a nonsegmented virus, and in the context of MOI estimation. 

Thank you.

Reviewer #3 (Public review): 

Summary:

The authors dilute fluorescent HCMV stocks in small steps (df ≈ 1.3-1.5) across 23 points, quantify infections by flow cytometry at 3 dpi, and fit a power-law model to estimate a cooperativity parameter n (n > 1 indicates apparent cooperativity). They compare fibroblasts vs epithelial cells and multiple strains/reporters, and explore alternative mechanisms (clumping, accrued damage, viral compensation) via analytical modeling and stochastic simulations. They discuss implications for titer/MOI estimation and suggest a method for detecting "apparent cooperativity," noting that for viruses showing this behavior, MOI estimation may be biased.

Strengths:

(1) High-resolution titration & rigor: The small-step dilution design (23 serial dilutions; tailored df) improves dose-response resolution beyond conventional 10× series.

(2) Clear quantitative signal: Multiple strain-cell pairs show n > 1, with appropriate model fitting and visualization of the linear regime on log-log axes.

(3) Mechanistic exploration: Side-by-side modeling of clumping vs accrued damage vs compensation frames testable hypotheses for cooperativity. 

Thank you.

Weaknesses:

(1) Secondary infection control: The authors argue that 3 dpi largely avoids progeny-mediated secondary infection; this claim should be strengthened (e.g., entry inhibitors/control infections) or add sensitivity checks showing results are robust to a small secondary-infection contribution. 

This is an important point. We do believe that the current knowledge about HCMV virion production time – it takes 3-4 days to make virions per multiple papers (see Fig 7 in Vonka and Benyesh-Melnick JB 1966; Fig 3B in Stanton et al JCI 2010; and Fig 1A in Li et al. PNAS 2015) – is sufficient to justify our experimental design but we do agree that an additional control to block novel infections with would be useful. We had previously performed experiments with a HCMV TB-gL-KO that cannot make infectious virions (but the stock virions can be made from complemented target cells). We will investigate if our titration experiments with this virus strain have sufficient resolution to detect apparent cooperativity. However, at present we do not have the resources to perform novel experiments.  

(2) Discriminating mechanisms: At present, simulations cannot distinguish between accrued damage and viral compensation. The authors should propose or add a decisive experiment (e.g., dual-color coinfection to quantify true coinfection rates versus "priming" without coinfection; timed sequential inocula) and outline expected signatures for each mechanism. 

Excellent suggestion. Because infection of a cell is a result of the joint viral infectivity and cell resistance, it may be hard to discriminate between these alternatives unless we specify them as particular molecular mechanisms. But we will try our best and list potential future experiments in the revised version of the paper.

(3) Decline at high genomes/cell: Several datasets show a downturn at high input. Hypotheses should be provided (cytotoxicity, receptor depletion, and measurement ceiling) and any supportive controls. 

Another good point. We do not have a good explanation, but we do not believe this is because of saturation of available target cells.  It seemed to only happen (or was most pronounced) with the ME stocks, which are typically lower in titer and so the higher MOI were nearly undiluted stock. It may be the effect of the conditioned medium.  Or perhaps there are non-infectious particles like dense bodies (enveloped particles that lack a capsid and genome) and non-infectious, enveloped particles (NIEPs) that compete for receptors or otherwise damage cells and these don’t get diluted out at the higher doses.  We plan to include these points in Discussion of the revised version of the paper.

(4) Include experimental data: In Figure 6, please include the experimentally measured titers (IU/mL), if available. 

This is a model-simulated scenario, and as such, there is no measured titers.

(5) MOI guidance: The practical guidance is important; please add a short "best-practice box" (how to determine titer at multiple genomes/cell and cell densities; when single-hit assumptions fail) for end-users. 

Good suggestion. We will include best-practice box using guidelines developed in Ryckman lab over the years in the revised version of the paper.

Overall note to all reviews: We have deposited our codes and the data on github; yet, none of the reviewers commented on it.

Reviewer #1 (Public review):

Summary:

The authors use high-resolution ribosome profiling (Ezra-seq) and eRF1 pulldown-based ribosome profiling (eRF1-seq) developed in their lab to identify a GA rich sequence motif located upstream of the stop codon responsible for translation termination pausing. They then perform a massively parallel assay with randomly generated sequences to further characterize this motif. Using mouse tissues, they show that termination pausing signatures can be tissue-specific. They use a series of published ribosome structures and 18S rRNA mutants, and eS26 knockdown experiments to propose that the GA rich sequence interacts with the 3′-end of the 18S rRNA.

Strengths:

(1) Robust ribosome profiling data and clear analyses clarify the subtle behavior of terminating ribosomes near the stop codon.

(2) Novel termination or "false termination" sites revealed by eRF1-seq in the 5′-UTR, 3′-UTR, and CDS highlight a previously underappreciated facet of translation dynamics.

Weakness:

(1) Modest effects seen in ABCE1 knockdown do not seem to add up to the level of regulation. The authors state "ABCE1 regulates terminating ribosomes independent of the sequence context" on pg 9, and "ABCE1 modulates termination pausing independent of the mRNA sequence context" in the figure caption for Figure S4. Given the modest effect of the knockdown, such phrasing is most likely not supported. Further clarification of "ABCE1 plays a generic role in translation termination" is necessary.

(2) The authors propose that the GA rich sequence element upstream of the stop codon on the mRNA could potentially base pair with the 3′-end of the 18S rRNA. In the PDBs the authors reference in their paper and also in 3JAG, 3JAH, 3JAI (structures of terminating ribosomes with the stop codon in the A-site and eRF1), the mRNA exiting the ribosome and the 3′-end of the 18S rRNA are about 25-30 A apart. In addition, a segment of eS26 is wedged in between these two RNA segments. This reviewer noted this arrangement in a random sampling of 5 other PDBs of mammalian and human ribosome 80S structures. How do the authors anticipate the base pairing they have proposed to occur in light of these steric hindrances? RpsS26 is known to be released by Tsr2 in yeast during very specific stresses. Is it their expectation that termination pausing in human/mammalian cells happens during stressful conditions only?

(3) The authors say, "It is thus likely that mRNA undergoes post-decoding scanning by 18S rRNA." (pg. 10). It is unclear what the authors mean by "scanning." Do they mean that the mRNA gets scanned in a manner similar to scanning during initiation? There is no evidence presented to support that particular conclusion.

(4) Role of termination pausing in the testis is highly speculative. The authors state: "It is thus conceivable that the wide range of ribosome density at stop codons in testis facilitates functional division of ribosome occupancy beyond the coding region." It is unclear what type of functional division they are referring to.

Reviewer #4 (Public review):

Summary:

This manuscript by Qian and colleagues utilizes ribosome profiling, and reporter assays to dissect translation termination. Unfortunately, the data do not support the conclusions of the paper, controls are missing and several assays are not well validated and do not reproduce previous findings from others.

Specific comments:

• Translation termination has been studied in several organisms including mammalian cells and yeast. In those cases what is analyzed is not the peak height at the stop codon, but rather the difference in the ribosome density before and after the stop. Thus, analyzing peak height is not validated. I understand that this is relevant only for the ribosome profiling experiments (and Ezra-seq) not the RF1 profiling. But much of the data was acquired that way.

• Moreover, the data do not reproduce previous findings and no effort is made to connect them to previous data. Previous data has shown that stop codon efficacy varies. This is not reproduced (S1C). Similarly, an effect from the +1 residue is not reproduced. The data isn't even stratified by different stop codons as previous work has shown that different surrounding residues have different effects in the context of different stop codons. Thus, none of the sequencing data is validated or trusted and does not reproduce previous findings.

• The GA-rich sequence identified by Ezra-Seq and RF1 seq is not the same and it differs from previous sequences (Wangen &Green).

• The authors claim that the majority of Rf1 peaks is at stop codons, but that is not true. It is only about 30% of the peaks. Also, not all mRNAs have peaks at the stop codons. That is at best problematic. Finally, there are mRNAs that are known to "suffer" from NMD, what do these look like in the Ezra-Seq and RF1-Seq? How about mRNAs that have programmed frameshifts? This raises questions on the validity of the eRF1 data.

• Figure 4: First, instead of M/P ratio, one should analyze M/M+P, to normalize out differences in the loading and effects from collisions, which are guaranteed to occur here, but not considered or analyzed. Second, the data are analyzed as if what matters are codons in the P and E site (and beyond, where there are definitely NOT recognized codons). While there is evidence for some interactions, one would think that an additional analysis based on sequence would be helpful. Also, the supplemental data indicates that very rarely are there reciprocal changes (as should be the case), and as seen for stop codons.

• Regarding the HiBit reporter assay: The two sequecnes clearly have effects on translation without considering stop codon context (Figure 4C), which need to be taken into account. Also, the effect from the sequences varies in the context of the assay in 4C and 4D (2-fold vs .5 fold), further questioning the assay. Moreover, the authors claim that re-initiation cannot account for Hibit levels, but that is clearly incorrect. The western in Figure 4E does not reproduce the data in 4D. While Hibit goes up (as in 4D, the putative GFP-fusion goes down. Finally, while the second reading frame should be more efficient is not explained and further argues for an artifact. Previous work (and work herein) suggests that read-through occurs equally in each reading frame. No controls for these assays are presented: e.g. stimulation by antibiotics, ABCE1 depletion, etc.

• Figure 5 has similar problems. I don't understand how the Figure in 5A is made, but when you overlay the cited structures on Rps26, the molecules are identical. I guess the authors used some fantasy to build non-existing sequences differently into the structure. There is no basis for that. In panel C and the same in Figure 7, the number of analyzed mRNAs varies. This could influence the outcome and the EXACT same set of mRNAs should be analyzed. But the main problem here is that the authors need to analyze readthrough and not peak height as detailed above. Essential controls are missing that show what fraction of the 18S rRNA is mutated. Previous work has shown that 2 nt truncated 18S rRNA is actively degraded. It is hard to believe how 15% of altered ribosomes can abolish 100% of the effect from the C-rich sequences. Important validation is missing: the authors should analyze rRNA sequences in their ribo-seq dataset to demonstrate that they have the mutated rRNAs, and that these enrich and de-enrich as predicted.

• In Figure 5-7 the authors develop a model that the sequence selectivity arises from base pairing between 18S rRNA and the mRNA. If so, then they should really stratify the data by number of WC pairs that can be formed. And only WC pairs, as GU pairs have a totally different geometry that will likely be discriminated against in this context. Also, the mutation is in a part of the helix that has no effect (Figure S3G). Thus, the data within the manuscript are inconsistent.

• Figure 6 does not agree with published data (Li et al., Nature 2022). Previous work did not show testis-depletion of Rps26 in purified ribosomes. This is the critical difference as the authors here did not purify ribosomes. Also, another Rps is an essential control, even if purified ribosomes are used. The validity of this dataset is thus questionable . Depletion from polysomes is hard to believe, as overall there is less signal in the polysomes.

• Figure 7 has similar problems as figure 5. Different pools of mRNAs are analyzed; peak height is not validated. Overexpression of Rps26 is not shown, as only Myc is shown, not Rps26. Beyond that, increased occupancy in ribosomes needs to be shown for the effect to come from ribosomes. Given how sick the cells are it is most likely that all effects are secondary and arise from whatever else is going on in the overexpression or depletion of Rps26. No controls are presented to show specific effects from Rps26.

• The authors need to check Rli1/ABCE levels in their cells. Their data have features that are indicative of low ABCE1 levels. These include a very small effect from ABCE1 depletion. These could be responsible for some of the effects they observe.

Author response:

We thank the editor and reviewers for their thoughtful feedback. We agree with eLife’s overall assessment that, while profiling terminating ribosomes is informative in revealing termination dynamics, the underlying mechanisms require more evidence. Our revision will focus on three conceptual points.

(1) We will tone down the statement that putative mRNA:rRNA interaction contributes to sequence-specific termination pausing.

(2) We will clarify the potential role of Rps26 in regulating translation termination.

(3) We will expand the discussion of tissue-specific termination pausing.

Reviewer #1 (Public Review):

(1) We admit that the modest effects of ABCE1 were partly due to the incomplete ABCE1 knockdown in HEK293 cells. Since the elevated ribosome density occurred at all stop codons, we argue that the action of ABCE1 is likely independent of the sequence context. We will rephrase relevant statements in the revised manuscript.

(2) In terms of Rps26 structures, we agree the structural rearrangement in the absence of Rps26 is highly speculative. However, we do not believe the Rps26 stoichiometry is solely dependent on stress. We will clarify this important point in the revised manuscript.

(3) We apologize for the confusion about 18S rRNA “scanning” and will revise the sentence in the main text.

(4) We agree that functional significance of testis-specific termination dynamics is unclear. Since other reviewers raised similar concern, we will expand the discussion of tissue-specific termination pausing in the revised manuscript.

Reviewer #2 (Public Review):

We appreciate the Reviewer’s time and efforts in reviewing our manuscript. We are grateful for the insightful comments and many recommendations made by the reviewer to improve our manuscript. We feel that the reviewer may have some misunderstanding in terms of the sequence motif associated with the termination pausing, partly because of the lack of clarity in our original description of the results from MPRA and reporter assays. We will ensure that the reviewer’s points are fully addressed in the revised manuscript.

Reviewer #3 (Public Review):

We thank the reviewer’s positive comment on our manuscript. We agree that the tissue-specific termination differences were poorly described in the main text. Notably, other reviewers raised similar concerns. We will expand the relevant discussion in the revised manuscript, outlining this as a limitation and a future direction.

Reviewer #4 (Public Review):

We believe the reviewer mixed xthe public view with recommendation comments. The reviewer appears to be preoccupied by previous studies and questioned some inconsistency in our results. With the development of new technology such as eRF1-seq, we are encouraged to present “new” and “different” findings. All other reviewers appreciate the development of eRF1-seq to profile terminating ribosomes. In fact, we do not believe our data is fundamentally different from the established principles. Rather, our data provides new perspectives to further our understanding of ribosome dynamics at stop codons. We thank the reviewer for understanding.

The reviewer is quite confused by our sequencing analysis based on peak height, or read density, which is commonly used to infer ribosome dynamics such as pausing. Regarding the sequencing analysis and reporter assays in cells expressing 18S mutant (Figure 5) and Rps26 (Figure 7), we feel that the reviewer has some misunderstanding. In the revised manuscript, we will do our best to clarify those relevant issues. Finally, the reviewer’s comment on base pairing is well-received and we will thoroughly revise the main text and discussion in the revised manuscript.

Reviewer #1 (Public review):

Microglia are mononuclear phagocytes in the CNS and play essential roles in physiology and pathology. In some conditions, circulating monocytes may infiltrate in the CNS and differentiated into microglia or microglia-like cells. However, the specific mechanism is large unknown. In this study, the authors explored the epigenetic regulation of this process. The quality of this study will be significantly improved if a few questions are addressed.

(1) The capacity of circulating myeloid cell-derived microglia are controversial. In this study, the authors utilized CX3CR1-GFP/CCR2-DsRed (hetero) mice as a lineage tracing line. However, this animal line is not an appropriate approach for this purpose. For example, when the CX3CR1-GFP/CCR2-DsRed as the undifferentiated donor cell, they are GFP+ and DsRed+. When the cell fate has been changed to microglia, they will change into GFP+ and DsRed- cells. However, this process is mediated with busulfan and artificially introduced bone marrow cells in the circulating cell, which is not existed in physiological and pathological conditions. These artifacts will potentially bring in artifacts and confound the conclusion, as the classical wrong text book knowledge of the bone marrow derived microglia theory and subsequently corrected by Fabio Rossi lab1,2. This is the most risk for drawing this conclusion. The top evidence is from the parabiosis animal model. Therefore, A parabiosis study before making this conclusion, combining a CX3CR1-GFP (hetero) mouse with a WT mouse without busulfan conditioning and looking at whether there are GFP+ microglia in the GFP- WT mouse brain. If there are no GFP+ microglia, the author should clarify this is not a physiological or pathological condition, but a defined artificial host condition, as previously study did3.

(2) In some conditions, peripheral myeloid cells can infiltrate and replace the brain microglia4,5. Discuss it would be helpful to better understand the mechanism of microglia replacement.

References:

(1) Ajami, B., Bennett, J.L., Krieger, C., Tetzlaff, W., and Rossi, F.M. (2007). Local self-renewal can sustain CNS microglia maintenance and function throughout adult life. Nature neuroscience 10, 1538-1543. 10.1038/nn2014.

(2) Ajami, B., Bennett, J.L., Krieger, C., McNagny, K.M., and Rossi, F.M.V. (2011). Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool. Nature neuroscience 14, 1142-1149. http://www.nature.com/neuro/journal/v14/n9/abs/nn.2887.html#supplementary-information.

(3) Mildner, A., Schmidt, H., Nitsche, M., Merkler, D., Hanisch, U.K., Mack, M., Heikenwalder, M., Bruck, W., Priller, J., and Prinz, M. (2007). Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions. Nature neuroscience 10, 1544-1553. 10.1038/nn2015.

(4) Wu, J., Wang, Y., Li, X., Ouyang, P., Cai, Y., He, Y., Zhang, M., Luan, X., Jin, Y., Wang, J., et al. (2025). Microglia replacement halts the progression of microgliopathy in mice and humans. Science 389, eadr1015. 10.1126/science.adr1015.

(5) Xu, Z., Rao, Y., Huang, Y., Zhou, T., Feng, R., Xiong, S., Yuan, T.F., Qin, S., Lu, Y., Zhou, X., et al. (2020). Efficient strategies for microglia replacement in the central nervous system. Cell reports 32, 108041. 10.1016/j.celrep.2020.108041.

'Écoute dans le Développement Humain : Une Analyse de la Perspective de la Professeure Elinor Ochs

Résumé Analytique

Ce document de synthèse analyse les arguments principaux de la professeure Elinor Ochs concernant le rôle sous-estimé de l'écoute dans le développement de l'enfant.

La thèse centrale est que les études développementales dominantes, principalement menées dans les sociétés occidentales post-industrielles, se sont concentrées de manière excessive sur la production de la parole par l'enfant dans des contextes dyadiques (parent-enfant), tout en négligeant la compétence cruciale de l'écoute, en particulier l'écoute incidente ("overhearing") au sein d'interactions multipartites.

En s'appuyant sur des décennies de recherche ethnographique, notamment son travail fondateur au Samoa, Ochs démontre que dans de nombreuses sociétés, les enfants sont socialisés dès leur plus jeune âge pour devenir des auditeurs compétents au sein de conversations de groupe.

Cette "formation" à l'écoute est facilitée par des "affordances" culturelles spécifiques, telles que l'architecture ouverte des habitations, les postures corporelles qui orientent l'enfant vers l'espace public, et une économie domestique qui valorise la continuité générationnelle et les ressources partagées.

En contraste, le modèle occidental, avec ses espaces privés et son accent sur l'individualisme économique, favorise des interactions dyadiques centrées sur l'enfant, amplifiant son rôle de locuteur plutôt que d'auditeur.

En conclusion, la professeure Ochs soutient que les interactions multipartites offrent des avantages développementaux uniques, exposant les enfants à une plus grande diversité de locuteurs, de perspectives et de variétés linguistiques.

Ses recherches remettent en question l'universalité des modèles actuels d'acquisition du langage et appellent à une réévaluation du rôle de l'écoute comme une compétence socio-culturellement construite, essentielle à l'apprentissage, à la coopération et à l'intégration sociale.

Introduction : La Perspective d'une Anthropologue Linguistique

La professeure Elinor Ochs, de l'UCLA, est une anthropologue linguistique qui combine les disciplines de la linguistique et de l'anthropologie.

Sa méthodologie principale est le travail de terrain ethnographique, utilisant des enregistrements audio et vidéo pour documenter de manière détaillée comment la communication façonne les situations sociales, les relations et les modes de pensée.

• Domaine de spécialisation : Elle a co-créé le sous-domaine de la "socialisation langagière", qui postule qu'en apprenant une langue, les enfants acquièrent simultanément une compétence socioculturelle pour devenir une "personne" au sein de leur communauté.

• Expérience de recherche :

◦ Samoa (1978-1988) : Étude longitudinale sur l'acquisition du langage chez de jeunes enfants dans un village rural.  

◦ États-Unis (années 80 et 2000) : Recherches sur les différences de classe sociale dans le discours de résolution de problèmes et une étude interdisciplinaire à grande échelle documentant la vie de 32 familles de la classe moyenne.   

◦ Autisme (depuis 1997) : Étude des pratiques communicatives des enfants sur le spectre autistique à la maison et à l'école.

Le Paradigme Dominant dans les Études Développementales : La Primauté de la Parole sur l'Écoute

La professeure Ochs commence par un constat : bien que la parole et l'écoute soient deux pratiques communicatives universelles, la parole reste de loin l'objet d'intérêt principal dans tous les domaines qui étudient le langage. L'accent est mis sur la production du langage, et non sur le processus qui distingue l'audition de l'écoute.

Les Limites des Études Quantitatives

Les études quantitatives sur le développement du langage chez l'enfant se concentrent sur la langue produite par l'enfant, souvent réduite au nombre de mots.

Une préoccupation majeure du public, notamment concernant les différences socio-économiques ("word gap"), est née de ces études.

• Le Modèle Dyadique : La généralisation dominante est que "plus un enfant entend de mots qui lui sont directement adressés, plus son vocabulaire sera étendu".

• Conditions Idéales Supposées : Ce modèle repose sur des conditions très spécifiques :

1. L'enfant est l'allocutaire principal dans une conversation dyadique (un locuteur, un auditeur).  

2. L'interaction est en face à face.  

3. Le langage utilisé est simplifié et affectif (langage adressé à l'enfant ou "parler bébé").

• La Négation de l'Écoute Incidente : Dans ce cadre, l'écoute de conversations d'autres personnes ("overhearing") est considérée comme ayant "peu ou pas de bénéfice développemental".

• Biais Culturel : Ces études sont principalement situées dans des sociétés occidentales post-industrielles, avec très peu de recherches menées dans des sociétés aux économies sociopolitiques différentes.

Un Modèle Alternatif : L'Apprentissage par l'Écoute en Contexte Multipartite

La thèse centrale de la professeure Ochs, étayée par des recherches ethnographiques, est qu'un autre modèle d'apprentissage existe et est courant dans de nombreuses sociétés.

Arguments Clés

Argument

Description

Argument 1

Les études développementales valorisent les conversations dyadiques fréquentes où le jeune enfant est locuteur ou allocutaire principal, motivant des interventions éducatives dans le monde entier.

Argument 2

Des études ethnographiques montrent que dans certaines sociétés, les nourrissons et les tout-petits participent régulièrement à des conversations multipartites en tant qu'auditeurs incidents légitimes ("legitimate overhearers") ou participants secondaires.

Argument 3

Qu'ils soient immergés dans des contextes multipartites ou dyadiques, les enfants neurotypiques acquièrent le langage avec succès dans différents contextes socioculturels.

Argument 4

Les interactions multipartites possèdent leurs propres affordances développementales, exposant les enfants à une diversité de locuteurs, de perspectives et de variétés linguistiques, et leur apprenant à adapter leur discours à différents interlocuteurs ("recipient design").

Argument 5

Les compétences d'écoute sont renforcées dès la petite enfance par des alignements corporels multipartites tournés vers l'extérieur et par des environnements construits ouverts qui offrent un accès auditif et visuel aux espaces publics.

Étude de Cas Ethnographique : Le Village Samoan

Le travail de terrain de la professeure Ochs au Samoa, il y a près de 50 ans, constitue la principale source de données pour son argumentaire.

Contexte Linguistique et Social

• Langue Complexe : La langue samoane est ergative, avec des ordres de mots multiples, deux registres phonologiques, et un vocabulaire de respect complexe.

• Société Hiérarchique : La société est structurée avec des personnes titrées (grands chefs, orateurs) et non titrées.

• Absence de "Parler Bébé" : Les soignants n'utilisent généralement pas de langage simplifié ou de "parler bébé" avec les nourrissons. Ils n'étiquettent pas les objets et posent rarement des questions dont ils connaissent la réponse.

• Apprentissage Immersif : Les enfants acquièrent le samoan parlé en étant au milieu d'interactions multipartites.

Les Affordances Environnementales et Corporelles pour l'Écoute

Ochs identifie deux types principaux d'affordances qui favorisent une culture de l'écoute.

1. Environnements Construits Ouverts :

◦ Les maisons traditionnelles samoanes n'ont ni murs extérieurs ni murs intérieurs. L'espace est ouvert, avec des nattes en feuilles de cocotier pour l'ombre.   

◦ Les maisons sont regroupées en concessions familiales ouvertes et proches de la route principale, donnant accès aux conversations publiques.  

◦ Les interactions simultanées à l'intérieur et à l'extérieur de la maison sont courantes, et les habitants sont habitués à écouter plusieurs conversations à la fois.  

◦ En revanche, les maisons de style européen (coloniales), bien que prestigieuses, sont murées, rectangulaires et moins appréciées car elles limitent l'accès auditif et sont très chaudes.

2. Alignements Corporels Orientés vers l'Extérieur :

◦ Nourrissons : Ils sont souvent "nichés" dans les bras d'un soignant (adulte ou aîné) de manière à faire face à l'extérieur, vers l'espace public et la communauté. Ils sont portés sur le dos, sur la hanche, ou assis devant le soignant, regardant dans la même direction que les autres participants.  

◦ Enfants plus âgés : Ils doivent s'asseoir en tailleur (ne pas montrer la plante des pieds) et observer activement les personnes à l'intérieur de la maison ainsi que celles sur la route depuis le bord de la maison. Leurs tâches (messagers, service, etc.) les rendent mobiles et actifs dans la communauté.  

◦ Le mot samoan pour "respect" (fa'aaloalo) est composé du préfixe fa'a et de alo, qui signifie "visage", impliquant l'idée de "se tourner vers l'autre".

Hypothèses Socio-Économiques et Questions Ouvertes

La professeure Ochs relie ces différents modes d'interaction à la structure économique de la famille.

• Le Modèle de la Continuité Familiale (ex: Samoa) :

◦ Les enfants sont élevés pour soutenir les ressources économiques partagées de la famille et assurer la continuité générationnelle des biens.  

◦ Dans ce contexte, "la famille a un investissement pour que l'enfant écoute". L'écoute est une compétence essentielle pour apprendre les dynamiques sociales et économiques du groupe.  

◦ Ce modèle favorise la participation de l'enfant en tant qu'auditeur dans des conversations multipartites.

• Le Modèle de l'Indépendance Individuelle (ex: familles néolibérales américaines) :

◦ Les enfants sont élevés pour devenir des individus économiquement indépendants, un héritage culturel où les droits de succession ont été abolis bien avant la révolution industrielle.    ◦ L'accent est mis sur le développement rapide de l'enfant en tant qu'individu, ce qui favorise les interactions dyadiques intenses et centrées sur l'enfant.

Questions Centrales pour la Recherche Future

La présentation se termine par une série de questions fondamentales :

1. Les habitats (ouverts ou murés) et les orientations corporelles peuvent-ils influencer la phénoménologie de l'écoute dans la petite enfance ?

2. Ces facteurs socioculturels agissent-ils comme des "amplificateurs culturels" ?

Un habitat privé et clos amplifie-t-il l'écoute en tant qu'allocutaire dyadique, tandis qu'un habitat ouvert amplifie l'écoute en tant que participant secondaire ?

3. Les études développementales actuelles n'examinent-elles qu'une "fraction des possibilités" en matière d'environnements et d'affordances pour l'écoute ?

Crise, Inégalités et Précarité : Synthèse des Analyses d'Esther Duflo, Claire Hédon et Frédéric Worms

Résumé

Ce document de synthèse analyse les interventions d'Esther Duflo, Claire Hédon et Frédéric Worms sur l'impact de la crise du coronavirus sur les inégalités et la précarité. Les conclusions clés sont les suivantes :

• Aggravation des Inégalités : La crise a un effet immédiat et délétère, exacerbant les inégalités existantes tant au sein des pays qu'entre eux.

Les populations les plus pauvres et les plus vulnérables subissent de manière disproportionnée les chocs sanitaires et économiques.

Aux États-Unis, par exemple, la probabilité de décès du coronavirus pour une personne noire est quatre fois supérieure à celle d'une personne blanche, à âge égal.

• Disparité des Réponses Économiques : Les pays riches ont pu mobiliser 20% de leur PIB pour soutenir leurs économies, contre 6% pour les pays émergents et seulement 2% pour les pays pauvres, ce qui laisse présager un enlisement de la pauvreté dans ces derniers.

• Révélation des Failles Systémiques : La crise a mis en lumière des problèmes structurels profonds :

• une méfiance institutionnalisée envers les pauvres qui rend les systèmes de protection sociale punitifs,
• un recul des services publics qui complique l'accès aux droits (notamment à cause de la dématérialisation), et
• une incapacité de la communauté internationale à organiser une solidarité efficace.

• Opportunités de Changement : Malgré ses effets négatifs, la crise offre des opportunités.

Elle a démontré que le gouvernement est une solution essentielle pour gérer les crises, et non le problème.

L'expérience massive du chômage partiel pourrait également changer la perception de la redistribution, en montrant que chacun peut avoir besoin d'aide, et potentiellement ouvrir la voie à des systèmes plus respectueux de la dignité.

• Approche Structurelle : Le traitement des inégalités n'est pas seulement une conséquence à gérer, mais une condition préalable à la gestion efficace des crises futures, qu'elles soient sanitaires, climatiques ou démocratiques.

La confiance dans un système de redistribution juste est indispensable pour obtenir l'adhésion collective aux efforts nécessaires.

• Enjeux de l'Accès au Droit : La crise a aggravé le phénomène de "non-recours" aux droits, où les personnes les plus précaires, confrontées à la fermeture des services physiques et à la barrière numérique, ne parviennent pas à obtenir les aides auxquelles elles ont droit.

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1. L'Impact Immédiat et Disproportionné de la Crise

La crise du coronavirus, loin d'être un "grand égaliseur", a frappé de manière asymétrique, aggravant les vulnérabilités existantes.

1.1. Inégalités au sein des Pays Riches

• Sur le plan sanitaire : Esther Duflo souligne que les populations les plus pauvres et minoritaires ont été les plus touchées.

Aux États-Unis, en ajustant pour l'âge, une personne noire a quatre fois plus de chances de mourir du coronavirus qu'une personne blanche.

Une étude de l'INSEE en France, citée par Claire Hédon, montre également une corrélation entre le niveau de vie de la commune et la mortalité.

• Sur le plan économique :

◦ La reprise est inégale. Aux États-Unis, le quart le plus riche de la population a retrouvé ses niveaux d'emploi et de salaire d'avant-crise, tandis que les plus pauvres, notamment dans le secteur des services, s'installent dans une crise durable.  

◦ Les dispositifs de solidarité, comme le chômage partiel en Europe, se sont principalement basés sur l'existence d'un emploi préalable, laissant de côté les personnes déjà en grande précarité.   

◦ Claire Hédon rapporte que les personnes aux minima sociaux ont vu leur situation se dégrader (courses plus chères dans les commerces de proximité, enfants non scolarisés à la cantine à 1€) sans bénéficier d'aides supplémentaires significatives.

1.2. Inégalités entre les Pays

Esther Duflo met en évidence un fossé immense dans la capacité de réponse économique à la crise.

Catégorie de pays

Dépenses de soutien fiscal (en % du PIB)

Pays riches

20 %

Pays émergents

6 %

Pays pauvres

2 % (d'un PIB déjà beaucoup plus petit)

Cette disparité a des conséquences majeures :

• Les pays riches ont pu emprunter massivement pour protéger leurs populations, une option inaccessible aux pays pauvres.

• Alors qu'une reprise économique rapide est attendue dans les pays riches grâce à la vaccination, les pays pauvres risquent un "enlisement de la crise" et un renfermement de la pauvreté sur elle-même.

2. Les Failles Systémiques Révélées et Exacerbées

La crise a agi comme un révélateur de dysfonctionnements structurels profonds dans nos sociétés et nos institutions.

2.1. La Méfiance envers les Pauvres et le Carcan Punitif de la Redistribution

Esther Duflo affirme que nos systèmes de protection sociale sont qualitativement faibles et "punitifs à leur cœur" en raison d'une méfiance profonde envers les pauvres, perçus comme "paresseux".

Cette vision, qualifiée de "victorienne", érige des barrières pour éviter que les bénéficiaires "ne se vautrent pas dans la complaisance".

Claire Hédon confirme ce constat avec des exemples concrets :

• Le soupçon de fraude permanent : Elle cite le cas d'un homme ayant mis 15 mois à obtenir le RSA, ou ceux de personnes accusées de fraude pour avoir vendu leurs vêtements ou leur voiture pour survivre.

• Un regard culpabilisateur : "J'ai le sentiment qui est ancré dans la société un regard très culpabilisateur qui est aussi qu'est-ce que vous avez raté dans votre vie pour vous retrouver dans cette situation là."

Elle soutient que c'est la société qui a échoué envers ces personnes, et non l'inverse.

2.2. Le Recul des Services Publics et le Non-Recours aux Droits

Claire Hédon, en tant que Défenseure des droits, alerte sur un "recul de la présence de l'État" qui a été aggravé par la crise.

• La dématérialisation comme barrière : La fermeture des services physiques (CAF, postes) a rendu l'accès aux droits quasi impossible pour les personnes sans connexion internet, sans matériel adéquat ou sans compétences numériques.

Pour les plus précaires, la dématérialisation aboutit à un "non accès au droit".

• Le phénomène du non-recours : Beaucoup de personnes éligibles n'arrivent pas à faire valoir leurs droits. La lutte contre la fraude, en complexifiant les démarches, génère de fait du non-recours.

• Qualité de l'accueil : Même l'accès physique est semé d'embûches, comme l'illustre l'exemple d'un homme devant parcourir 30 km pour se rendre à la CAF, se voir refuser l'entrée faute de rendez-vous pris sur internet, puis être jugé "pas motivé" par les agents d'accueil.

2.3. L'Échec de la Solidarité Internationale

Esther Duflo déplore que les pays riches, qui ont dépensé des "trillions de dollars" pour leurs propres économies, aient été "aux grands abonnés absents" pour aider les pays pauvres.

L'appel à un "plan Marshall pour les pays pauvres" qu'elle a lancé au début de la crise n'a pas été entendu.

Cette incapacité à agir collectivement en temps de crise est un signal inquiétant pour les défis à venir, notamment le changement climatique.

3. Les Crises comme Catalyseurs de Changements Potentiels

Malgré le constat sombre, les intervenants identifient des lueurs d'espoir et des opportunités de repenser certains paradigmes.

3.1. Le Rôle Essentiel de l'État

Pour Esther Duflo, la crise a apporté une leçon majeure : "le gouvernement n'est pas le problème, le gouvernement est la solution."

Seul l'État a la capacité :

• D'imposer des mesures de santé publique (port du masque).

• D'investir massivement dans la recherche et l'achat de vaccins.

• D'emprunter au nom de la population pour la protéger des chocs économiques.

Cette prise de conscience pourrait mener à un "regain d'appréciation pour l'importance du rôle du gouvernement".

3.2. Vers une Nouvelle Perception de la Redistribution

L'expérience massive et souple du chômage partiel en Europe a montré que "tout le monde peut avoir besoin d'aide".

Des personnes "tout à fait vertueuses" se sont retrouvées dépendantes d'un soutien public.

• Espoir d'un changement de mentalité : Esther Duflo espère que cette expérience pourra "nous libérer un peu de ce carcan victorien" et permettre une redistribution "plus fluide, plus respectueuse, mettant la dignité des individus au cœur".

• Débat sur le revenu des jeunes : Claire Hédon note que la crise a rendu moins tabou le débat sur un revenu d'existence pour les 18-25 ans (via le RSA ou la généralisation de la Garantie Jeune).

4. Une Approche Structurelle : Traiter les Inégalités pour Prévenir les Crises

Frédéric Worms propose une analyse en trois niveaux de la réponse à la crise et plaide pour une vision structurelle à long terme.

4.1. Trois Types de Réponses à la Crise

1. La réponse "hypocrite" : Consiste à dire que, puisque les mesures sanitaires aggravent les inégalités, il ne fallait pas y répondre (ou pas autant).

Frédéric Worms et Esther Duflo réfutent cet argument en soulignant qu'il n'y a pas d'arbitrage entre le sanitaire et l'économique : les pays qui ont mal géré la crise sanitaire ont aussi les pires résultats économiques.

2. La réponse "honnête" (démocratie sociale) : Consiste à répondre aux deux dangers simultanément, en conjuguant les impératifs sanitaires, économiques et sociaux.

3. La réponse "structurelle" (la plus forte) : Consiste à affirmer que le traitement des inégalités est la condition même de la réponse aux dangers sanitaires du 21e siècle. Les inégalités ne sont pas un effet secondaire, mais une cause première des crises.

4.2. La Confiance comme Prérequis à l'Action Collective

Cette approche structurelle est essentielle car, comme le souligne Esther Duflo, on ne peut pas gérer une crise (COVID, climatique) qui implique des sacrifices sans la confiance des citoyens.

• Confiance et redistribution : Les gens n'accepteront des mesures difficiles (ex: taxe carbone) que s'ils ont confiance dans le fait qu'ils seront justement compensés.

Cette confiance est impossible sans un système de redistribution perçu comme "efficace, généreux et qui respecte les gens".

• Le cercle vicieux de la défiance : Frédéric Worms pointe une "défiance mutuelle" :

celle des citoyens envers le gouvernement, mais aussi celle du gouvernement envers les citoyens (soupçon de fraude).

Briser ce cercle nécessite de s'appuyer sur le savoir, la science, et des "institutions du désaccord" solides.

5. Pistes d'Action et Solutions

La discussion a également abordé des solutions concrètes pour lutter contre la pauvreté et les inégalités.

• Revenu Minimum Garanti vs. Revenu Universel :

◦ Pour les pays pauvres, Esther Duflo préconise un revenu universel très faible, accessible sur simple demande.

L'enjeu principal y est la perte de dignité, et même un revenu modeste peut suffire à "mettre de quoi manger à vos enfants trois fois par jour".   

◦ Pour les pays riches, elle privilégie un revenu minimum garanti (sur le principe du RSA), qui concentre les ressources sur ceux qui en ont le plus besoin, car les informations pour les cibler existent.

Elle insiste sur le fait que la dignité y est aussi liée au travail, qui nécessite plus que de l'argent (logement, garde d'enfants, etc.).

Ce doit être un droit, non une charité.

• Le Droit au Travail : Claire Hédon et Esther Duflo s'accordent sur l'importance du droit au travail.

Les personnes en situation de précarité souhaitent travailler, car c'est un "moyen d'être inséré dans la société".

• L'Approche Expérimentale : Esther Duflo plaide pour l'importation d'une attitude apprise dans son travail dans les pays pauvres :

l'humilité de reconnaître qu'on ne sait pas toujours ce qui marche et la nécessité de tester rigoureusement les politiques publiques avant de les généraliser.

Des études ont par exemple montré que la sécurité financière encourage l'initiative plutôt qu'elle ne la limite.

• Droit à l'accès au numérique : Face à la dématérialisation généralisée, Claire Hédon estime qu'il faut désormais réfléchir à un "droit à l'accès au numérique".

Author response:

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

Reviewer #1 (Public Review):

Summary:

In this manuscript, Bisht et al address the hypothesis that protein folding chaperones may be implicated in aggregopathies and in particular Tau aggregation, as a means to identify novel therapeutic routes for these largely neurodegenerative conditions.

The authors conducted a genetic screen in the Drosophila eye, which facilitates the identification of mutations that either enhance or suppress a visible disturbance in the nearly crystalline organization of the compound eye. They screened by RNA interference all 64 known Drosophila chaperones and revealed that mutations in 20 of them exaggerate the Tau-dependent phenotype, while 15 ameliorated it. The enhancer of the degeneration group included 2 subunits of the typically heterohexameric prefoldin complex and other co-translational chaperones.

The authors characterized in depth one of the prefoldin subunits, Pfdn5, and convincingly demonstrated that this protein functions in the regulation of microtubule organization, likely due to its regulation of proper folding of tubulin monomers. They demonstrate convincingly using both immunohistochemistry in larval motor neurons and microtubule binding assays that Pfdn5 is a bona fide microtubule-associated protein contributing to the stability of the axonal microtubule cytoskeleton, which is significantly disrupted in the mutants.

Similar phenotypes were observed in larvae expressing Frontotemporal dementia with Parkinsonism on chromosome 17-associated mutations of the human Tau gene V377M and R406W. On the strength of the phenotypic evidence and the enhancement of the TauV377Minduced eye degeneration, they demonstrate that loss of Pfdn5 exaggerates the synaptic deficits upon expression of the Tau mutants. Conversely, the overexpression of Pfdn5 or Pfdn6 ameliorates the synaptic phenotypes in the larvae, the vacuolization phenotypes in the adult, and even memory defects upon TauV377M expression.

Strengths

The phenotypic analyses of the mutant and its interactions with TauV377M at the cell biological, histological, and behavioral levels are precise, extensive, and convincing and achieve the aims of characterization of a novel function of Pfdn5. 

Regarding this memory defect upon V377M tau expression. Kosmidis et al (2010), PMID: 20071510, demonstrated that pan-neuronal expression of Tau^V377M disrupts the organization of the mushroom bodies, the seat of long-term memory in odor/shock and odor/reward conditioning. If the novel memory assay the authors use depends on the adult brain structures, then the memory deficit can be explained in this manner. 

(1) If the mushroom bodies are defective upon Tau^V377M. expression, does overexpression of Pfdn5 or 6 reverse this deficit? This would argue strongly in favor of the microtubule stabilization explanation.

We thank the reviewer for this insightful comment. Consistent with Kosmidis et al. (2010), we confirm that expression of hTau^V377M disrupts the architecture of mushroom bodies.   In addition, we find, as suggested by the reviewer, that coexpression of either Pfdn5 or Pfdn6 with hTau^V377M significantly restores the organization of the mushroom bodies. These new findings strongly support the hypothesis that Pfdn5 or Pfdn6 mitigate hTau^V377M -induced memory deficits by preserving the structure of the mushroom body, likely through stabilizing the microtubule network. This data has now been included in the revised manuscript (Figure 7H-O).

(2) The discovery that Pfdn5 (and 6 most likely) affects tauV377M toxicity is indeed a novel and important discovery for the Tauopathies field. It is important to determine whether this interaction affects only the FTDP-17-linked mutations or also WT Tau isoforms, which are linked to the rest of the Tauopathies. Also, insights on the mode(s) that Pfdn5/6 affect Tau toxicity, such as some of the suggestions above, are aiming at will likely be helpful towards therapeutic interventions.

We agree that determining whether prefoldin modulates the toxicity of both mutant and wildtype Tau is critical for understanding its broader relevance to Tauopathies. We have now performed additional experiments required to address this issue. These new data show that loss of Pfdn5 also exacerbates toxicity associated with wildype Tau (hTau^WT), in a manner similar to that observed with hTau^V337M or hTau^R406W. Specifically, overexpression of hTau^WT in a Pfdn5 mutant background leads to Tau aggregate formation (Figure S7G-I), and coexpression of Pfdn5 with hTau^WT reduces the associated synaptic defects (Figure S11F-L). These findings underscore a general role for Pfdn5 in modulating diverse Tauopathy-associated phenotypes and suggest that it could be a broadly relevant therapeutic target. 

Weakness

(3) What is unclear, however, is how Pfdn5 loss or even overexpression affects the pathological Tau phenotypes. Does Pfdn5 (or 6) interact directly with TauV377M? Colocalization within tissues is a start, but immunoprecipitations would provide additional independent evidence that this is so.

We appreciate this important suggestion. To investigate a potential direct interaction between Pfdn5 and Tau^V377M, we performed co-immunoprecipitation experiments using lysates from adult fly brain expressing hTau^V337M. Under the conditions tested, we did not detect a direct physical interaction. While this does not support a direct interaction, it does not strongly refute it either. We note that Pfdn5 and Tau are colocalized within axons (Figure S13J-K). At this stage, we are unable to resolve the issue of direct vs indirect association. If indirect, then Tau and Pfdn5 act within the same subcellular compartments (axon); if direct, then either only a small fraction of the total cellular proteins is in the Tau-Pfdn5 complex and therefore difficult to detect in bulk protein westerns, or the interactions are dynamic or occur in conditions that we have not been able to mimic in vitro. 

(4) Does Pfdn5 loss exacerbate Tau^V377M phenotypes because it destabilizes microtubules, which are already at least partially destabilized by Tau expression? Rescue of the phenotypes by overexpression of Pfdn5 agrees with this notion. 

However, Cowan et al (2010) pmid: 20617325 demonstrated that wildtype Tau accumulation in larval motor neurons indeed destabilizes microtubules in a Tau phosphorylation-dependent manner. So, is Tau^V377M hyperphosphorylated in the larvae?? What happens to Tau^V377M phosphorylation when Pfdn5 is missing and presumably more Tau is soluble and subject to hyperphosphorylation as predicted by the above?

We completely agree that it is important to link Tau-induced phenotypes with the microtubule destabilization and phosphorylation state of Tau.   We performed immunostaining using futsch antibody to check the microtubule organization at the NMJ and observed a severe reduction in futsch intensity when Tau^V337M was expressed in the Pfdn5 mutant (ElavGal4>Tau^V337M; DPfdn5^15/40), suggesting that Pfdn5 absence exacerbates the hTau^V337M defects due to more microtubule destabilization (Figure S6F-J). 

We have performed additional experiments to examine the phosphorylation state of hTau in Drosophila larval axons. Immunocytochemistry indicated that only a subset of hTau aggregates in Pfdn5 mutants (Elav-Gal4>Tau^V337M; DPfdn5^15/40) are recognized by phospho-hTau antibodies.   For instance, the AT8 antibody (targeting pSer202/pThr205) (Goedert et al., 1995) labelled only a subset of aggregates identified by the total hTau antibody (D5D8N) (Figure S9AE). Moreover, feeding these larvae (Elav-Gal4>Tau<sup>V337M</sup; DPfdn5^15/40) with LiCl, which blocks GSK3b, still showed robust Tau aggregation (Figure S9F-J). 

These results imply that: a) soluble phospho-hTau levels in Pfdn5 mutants are low and not reliably detected with a single phospholylation-specific antibody; b) Loss of Pfdn5 results in Tau aggregation in a hyperphosphorylation-independent manner similar to what has been reported earlier (LI et al. 2022); and c) the destabilization of microtubules in Elav-Gal4>Tau^V337M; DPfdn5^15/40 results in Tau dissociation and aggregate formation. These data and conclusions have been incorporated into the revised manuscript.

(5) Expression of WT human Tau (which is associated with most common Tauopathies other than FTDP-17) as Cowan et al suggest has significant effects on microtubule stability, but such Tauexpressing larvae are largely viable. Will one mutant copy of the Pfdn5 knockout enhance the phenotype of these larvae?? Will it result in lethality? Such data will serve to generalize the effects of Pfdn5 beyond the two FDTP-17 mutations utilized.

We have now examined whether heterozygous loss of Pfdn5 (∆Pfdn5/+) enhances the effect of Tau expression. While each genotype (hTau^V337M, hTau^WT or ∆Pfdn5/+) alone is viable, Elav-Gal4 driven expression of hTau^V337M or hTau^WT in Pfdn5 heterozygous background does not cause lethality. 

(6) Does the loss of Pfdn5 affect TauV377M (and WTTau) levels?? Could the loss of Pfdn5 simply result in increased Tau levels? And conversely, does overexpression of Pfdn5 or 6 reduce Tau levels?? This would explain the enhancement and suppression of Tau^V377M (and possibly WT Tau) phenotypes. It is an easily addressed, trivial explanation at the observational level, which, if true, begs for a distinct mechanistic approach.

To test whether Pfdn5 modulates Tau phenotypes by altering Tau protein levels, we performed western blot analysis under Pfdn5 or Pfdn6 overexpression conditions and observed no change in hTau^V337M levels (Figure 6O). However, in the absence of Pfdn5, both hTau^V337M and hTau^WT form large, insoluble aggregates that are not detected in soluble lysates by standard western blotting but are visualized by immunocytochemistry (Figure S7G-I). Thus, the apparent reduction in Tau levels on western blots reflects a solubility shift, not an actual decrease in Tau expression. These findings argue against a simple model in which Pfdn5 regulates Tau abundance and instead support a mechanism in which Pfdn5 loss leads to change in Tau conformation, leading to its sequesteration away for already destabilized microtubules.  

(7) Finally, the authors argue that Tau^V377M forms aggregates in the larval brain based on large puncta observed especially upon loss of Pfdn5. This may be so, but protocols are available to validate this molecularly the presence of insoluble Tau aggregates (for example, pmid: 36868851) or soluble Tau oligomers, as these apparently differentially affect Tau toxicity. Does Pfdn5 loss exaggerate the toxic oligomers, and overexpression promote the more benign large aggregates??

We have performed additional experiments to analyze the nature of these aggregates using 1,6-HD. The 1,6-hexanediol can dissolve the Tau aggregate seeds formed by Tau droplets, but cannot dissolve the stable Tau aggregates (WEGMANN et al. 2018). We observed that 5% 1,6hexanediol failed to dissolve these Tau aggregates (Figure S8), demonstrating the formation of stable filamentous flame-shaped NFT-like aggregates in the absence of Pfdn5 (Figure 5D and Figure S9).

Reviewer #2 (Public review):

Bisht et al detail a novel interaction between the chaperone, Prefoldin 5, microtubules, and taumediated neurodegeneration, with potential relevance for Alzheimer's disease and other tauopathies. Using Drosophila, the study shows that Pfdn5 is a microtubule-associated protein, which regulates tubulin monomer levels and can stabilize microtubule filaments in the axons of peripheral nerves. The work further suggests that Pfdn5/6 may antagonize Tau aggregation and neurotoxicity. While the overall findings may be of interest to those investigating the axonal and synaptic cytoskeleton, the detailed mechanisms for the observed phenotypes remain unresolved and the translational relevance for tauopathy pathogenesis is yet to be established. Further, a number of key controls and important experiments are missing that are needed to fully interpret the findings.

The strength of this study is the data showing that Pfdn5 localizes to axonal microtubules and the loss-of-function phenotypic analysis revealing disrupted synaptic bouton morphology. The major weakness relates to the experiments and claims of interactions with Tau-mediated neurodegeneration. 

In particular, it is unclear whether knockdown of Pfdn5 may cause eye phenotypes independent of Tau. 

Our new experiments confirm that knockdown of Pfdn5 alone does not cause eye phenotypes.

Further, the GMR>tau phenotype appears to have been incorrectly utilized to examine agedependent, neurodegeneration.

In response, we have modulated and explained our conclusions in this regard as described later in our “rebuttal.”

This manuscript argues that its findings may be relevant to thinking about mechanisms and therapies applicable to tauopathies; however, this is premature given that many questions remain about the interactions from Drosophila, the detailed mechanisms remain unresolved, and absent evidence that Tau and Pfdn may similarly interact in the mammalian neuronal context. Therefore, this work would be strongly enhanced by experiments in human or murine neuronal culture or supportive evidence from analyses of human data.

The reviewer is correct that the impact would be greater if Pfdn5-Tau interactions were also examined in human tissue.   While we have not attempted these experiments ourselves, we hope that our observations will stimulate others to test the conservation of phenomena we describe. There are, however, several lines of circumstantial evidence from human Alzheimer’s disease datasets that implicate PFDN5 in disease pathology. For example, recent compilations and analyses of proteomic data show reductions of CCT components, TBCE, as well as Prefoldin subunits, including PFDN5, in AD tissue (HSIEH et al. 2019; TAO et al. 2020; JI et al. 2022; ASKENAZI et al. 2023; LEITNER et al. 2024; SUN et al. 2024). Furthermore, whole blood mRNA expression data from Alzheimer's patients revealed downregulation of PFDN5 transcript (JI et al. 2022). Together, these findings from human data are consistent with the roles of PFDN5 in suppressing diverse neurodegenerative processes. We have incorporated these points into the discussion section of the revised manuscript.

Reviewer #1 (Recommendations for the authors):

See public review for experimental recommendations focusing on the Tau Pfdn interactions.  I would refrain from using the word aggregates, I would call them puncta, unless there is molecular or visual (ie AFM) evidence that they are indeed insoluble aggregates.  Finally, although including the full genotypes written out below the axis in the bar graphs is appreciated, it nevertheless makes them difficult to read due to crowding in most cases and somewhat distracting from the figure. 

In my opinion, a more reader-friendly manner of reporting the phenotypes will be highly helpful. For example, listing each component of the genotype on the left of each bar graph and adding a cross or a filled circle under the bar to inform of the full genotype of the animals used.

As described in the response to the previous comment, we now have strong direct evidences to support our view that the observed puncta are stable Tau aggregates. Thus, we feel justified to use the term Tau-aggregates in preference to Tau puncta. 

We have tried to write the genotypes to make them more reader-friendly.

Reviewer #2 (Recommendations for the authors):

(1) Lines 119-121: 35 modifiers from 64 seem like an unusually high hit rate. Are these individual genes or lines? Were all modifiers supported by at least 2 independent RNAi strains targeting non-overlapping sequences? A supplemental table should be included detailing all genes and specific strains tested, with corresponding results.

We agree with the reviewer that 35 modifiers from 64 genes may be too high. However, since the genes knocked down in the study are chaperones, crucial for maintaining proteostasis, we may have got unusually high hits. The information related to individual genes and lines is provided in Supplemental Table 1. We have now included an additional Supplemental Table 3, which lists the genes and the RNAi lines used in Figure 1, detailing the sequence target information. The table also specifies the number of independent RNAi strains used and the corresponding results. 

(2) Figure 1: The authors quantify the areas of ommatidial fusion and necrosis as degeneration, but it is difficult to appreciate the aberrations in the photos provided. Was any consideration given to also quantifying eye size?

We have processed the images to enhance their contrast and make the aberrations clearer. The percentage of degenerated eye area (Figure 1M) was normalized with total eye area. The method for quantifying degenerated area has been explained in the materials and methods section.

(3) Figure 1: a) Only enhancers of rough eyes are shown but no controls are included to evaluate whether knockdown of these genes causes eye toxicity in the absence of Tau. These are important missing controls. All putative Tau enhancers, including Pdn5/6, need to be tested with GMR-GAL4 independently of Tau to determine whether they cause a rough eye. In a previous publication from some of the same investigators (Raut et al 2017), knockdown of Pfdn using eyGAL4 was shown to induce severe eye morphology defects - this raises questions about the results shown here. 

We agree that assessing the effects of HSP knockdown independent of Tau is essential to confirm modifier specificity. We have now performed these knockdowns, and the data are reported in Supplemental Table 1. For RNAi lines represented in Figure 1, which enhanced Tau-induced degeneration/eye developmental defect, except for one of the RNAi lines against Pfdn6 (GD34204), no detectable eye defects were observed when knocked down with GMR-Gal4 at 25°C, suggesting that enhancement is specific to the Tau background. 

Use of a more eye-specific GMR-Gal4 driver at 25°C versus broader expressing ey-Gal4 at 29°C in prior work (Raut et al. 2017) likely reflects the differences in the eye morphological defects.

(b) Besides RNAi, do the classical Pdn5 deletion alleles included in this work also enhance the tau rough eye when heterozygous? Please also consider moving the Pfdn5/6 overexpression studies to evaluate possible suppression of the Tau rough eye to Figure 1, as it would enhance the interpretation of these data (but see also below).

GMR-Gal4 driven expression of hTau^V337M or hTau^WT in Pfdn5 heterozygous background does not enhance rough eye phenotype. 

(4) For genes of special interest, such as Pdn5, and other genes mentioned in the results, the main figure, or discussion, it is also important to perform quantitative PCR to confirm that the RNAi lines used actually knock down mRNA expression and by how much. These studies will establish specificity.

We agree that confirming RNAi efficiency via quantitative PCR (qPCR) is essential for validating the knockdown efficiency. We have now included qPCR data, especially for key modifiers, confirming effective knockdown (Figure S2).

(5) Lines 235-238: how do you conclude whether the tau phenotype is "enhanced" when Pfdn5 causes a similar phenotype on its own? Could the combination simply be additive? Did overexpression of Pdn5 suppress the UAS-hTau NMJ bouton phenotype (see below)? 

Although Pfdn5 mutants and hTau expression individually increase satellite boutons, their combination leads to a significantly more severe and additional phenotype, such as significantly decreased bouton size and increased bouton number, indicating an enhancing rather than purely additive interaction (Figure 4 and Figure S6C). Moreover, we now show that overexpression of Pfdn5 significantly suppressed the hTau^V337M-induced NMJ phenotypes. This new data has been incorporated as Figure S11F-L in the revised manuscript. 

Alternatively, did the authors consider reducing fly tau in the Pdn5 mutant background?

In new additional experiments, we observe that double mutants for Drosophila Tau (dTau) and Pfdn5 also exhibit severe NMJ defects, suggesting genetic interactions between dTau and Pfdn5. This data is shown below for the reviewer.

Author response image 1.

A double mutant combination of dTau and Pfdn5 aggravates the synaptic defects at the Drosophila NMJ. (A-D') Confocal images of NMJ synapses at muscle 4 of A2 hemisegment showing synaptic morphology in (A-A') control, (B-B') ΔPfdn5<SUP>15/40</SUP>, (C-C') dTauKO/dTauKO (Drosophila Tau mutant), (D-D') dTauKO/dTauKO; ∆Pfdn5<SUP>15/40</SUP> double immunolabeled for HRP (green), and CSP (magenta). The scale bar in D for (A-D') represents 10 µm. 

(6) It may be important to further extend the investigation to the actin cytoskeleton. It is noted that Pfdn5 also stabilizes actin. Importantly, tau-mediated neurodegeneration in Drosophila also disrupts the actin cytoskeleton, and many other regulators of actin modify tau phenotypes.

We appreciate the suggestion to examine the actin cytoskeleton. While prior studies indicate that Pfdn5 might regulate the actin cytoskeleton and that Tau^V377M hyperstabilizes the actin cytoskeleton, we did not observe altered actin levels in Pfdn5 mutants (Figure 2G). However, actin dynamics may represent an additional mechanism through which Pfdn5 might temporally influence Tauopathy. Future work will address potential actin-related mechanisms in Tauopathy.

(7) Figure 2: in the provided images, it is difficult to appreciate the futsch loops. Please include an image with increased magnification. It appears that fly strains harboring a genomic rescue BAC construct are available for Pfdn-this would be a complementary reagent to test besides Pfdn overexpression.

We have updated Figure 2 to include high magnification NMJ images as insets, clearly showing the Futsch loops. While we have not yet tested a genomic rescue BAC construct for Pfdn5, we plan to use the fly line harboring this construct in future work.

(8) Figure 3: Some of the data is not adequately explained. The use of Ran as a loading control seems rather unusual. What is the justification? Pfdn appears to only partially co-localize with a-tubulin in the axon; can the authors discuss or explain this? Further, in Pfdn5 mutants, there appears to be a loss of a-tubulin staining (3b'); this should also be discussed.

We appreciate the reviewer's concern regarding the choice of loading control for our Western blot analysis. Importantly, since Tubulin levels and related pathways were the focus of our analysis, traditional loading controls such as α- or β-tubulin or actin were deemed unsuitable due to potential co-regulation. Ran, a nuclear GTPase involved in nucleocytoplasmic transport, is not known to be transcriptionally or post-translationally regulated by Tubulin-associated signaling pathways. To ensure its reliability as a loading control, we confirmed by densitometric analysis that Ran expression showed minimal variability across all samples. Hence, we used Ran for accurate normalization in the Western blot data represented in this manuscript. We have also used GAPDH as a loading control and found no difference with respect to Ran as a loading control across samples.

We appreciate the reviewer's comment regarding the interpretation of our Pearson's correlation coefficient (PCC) results. While the mean colocalization value of 0.6 represents a moderate positive correlation (MUKAKA 2012), which may not reach the conventional threshold for "high positive" colocalization (usually considered 0.7-0.9), it nonetheless indicates substantial spatial overlap between the proteins of interest. Importantly, colocalization analysis provides supportive but indirect evidence for molecular proximity.  To further validate the interaction, we performed a microtubule binding assay, which directly demonstrates the binding of Pfdn5 to stabilized microtubules.

In accordance with the western blot analysis shown in Figure 2G-I, the levels of Tubulin are reduced in the Pfdn5 mutants (Figure 3B''). We have incorporated and discussed this in the revised manuscript.

(9) Figure 4: Overexpression of Pfdn appears to rescue the supernumerary satellite bouton numbers induced by human Tau; however, interpretation of this experiment is somewhat complicated as it is performed in Pfdn mutant genetic background. Can overexpression of Pfdn on its own rescue the Tau bouton defect in an otherwise wildtype background?

We have now coexpressed Pfdn5 and hTau<SUP>V337M</SUP> in an otherwise wild-type background. As shown in Figure S11F-L, Pfdn5 overexpression suppresses Tau-induced bouton defects. We have incorporated the data in the Results section to support the role of Pfdn5 as a modifier of Tau toxicity.

(10) Lines 256-263 / Figure 5: (a) What exactly are these tau-positive structures (punctae) being stained in larval brains in Fig 5C-E? Most prior work on tau aggregation using Drosophila models has been done in the adult brain, and human wildtype or mutant Tau is not known to form significant numbers of aggregates in neurons (although aggregates have been described following glia tau expression). 

Therefore, the results need to be further clarified. Besides the provided schematic, a zoomed-out image showing the whole larval brain is needed here for orientation. Have these aggregates been previously characterized in the literature? 

We agree with the reviewer that the expression of the wildtype or mutant form of human Tau in Drosophila is not known to form aggregates in the larval brain, in contrast to the adult brain (JACKSON et al. 2002; OKENVE-RAMOS et al. 2024). Consistent with previous reports, we also observed that Tau expression on its own does not form aggregates in the Drosophila larval brain.

However, in the absence of Pfdn5, microtubule disruption is severe, leading to reduced Taumicrotubule binding and formation of globular/round or flame-shaped tangles like aggregates in the larval brain. Previous studies have reported that 1,6-hexanediol can dissolve the Tau aggregate seeds formed by Tau droplets, but cannot dissolve the stable Tau aggregates (WEGMANN et al. 2018). We observed that 5% 1,6-Hexanediol failed to dissolve these Tau puncta, demonstrating the formation of stable aggregates in the absence of Pfdn5. Additionally, we now performed a Tau solubility assay and show that in the absence of Pfdn5, a significant amount of Tau goes in the pellet fraction, which could not be detected by phospho-specific AT8 Tau antibody (targeting pSer202/pThr205) but was detected by total hTau antibody (D5D8N) on the western blots (Figure S8). These data further reinforce our conclusion that  Pfdn5 prevents the transition of hTau from soluble and/or microtubule-associated state to an aggregated, insoluble, and pathogenic state. These new data have been incorporated into the revised manuscript.

(b) Can additional markers (nuclei, cell membrane, etc.) be used to highlight whether the taupositive structures are present in the cell body or at synapses?

We performed the co-staining of Tau and Elav to assess the aggregated Tau localization. We found that in the presence of Pfdn5, Tau is predominantly cytoplasmic and localised to the cell body and axons. In the absence of Pfdn5, Tau forms aggregates but is still localized to the cell body or axons. However, some of the aggregates are very large, and the subcellular localization could not be determined (Figure S8M-N'). These might represent brain regions of possible nuclear breakdown and cell death (JACKSON et al. 2002).

(c) It would also be helpful to perform western blots from larval (and adult) brains examining tau protein levels, phospho-tau species, possible higher-molecular weight oligomeric forms, and insoluble vs. soluble species. These studies would be especially important to help interpret the potential mechanisms of observed interactions.

Western blot analysis revealed that overexpression of Pfdn5 does not alter total Tau levels (Figure 6O). In Pfdn5 mutants, however, hTau^V337M levels were reduced in the supernatant fraction and increased in the pellet fraction, indicating a shift from soluble monomeric Tau to aggregated Tau.

(d) Does overexpression of Pdn5 (UAS-Pdn5) suppress the formation of tau aggregates? I would therefore recommend that additional experiments be performed looking at adult flies (perhaps in Pfdn5 heterozygotes or using RNAi due to the larval lethality of Pdn5 null animals).

Overexpression of Pfdn5 significantly reduced Tau-aggregates (Elav-Gal4/UASTau^V337M; UAS-Pfdn5; DPfdn5^15/40) observed in Pfdn5 mutants (Figure 5E). Coexpression of Pfdn5 and hTau^V337M suppresses the Tau aggregates/puncta in 30-day adult brain. Since heterozygous DPfdn¹⁵/+ did not show a reduction in Pfdn5 levels, we did not test the suppression of Tau aggregates in  DPfdn¹⁵/+; Elav>UAS-Pfdn5, UAS-Tau^V337M.

(11) Figure 6, panels A-N: The GMR>Tau rough eye is not a "neurodegenerative" but rather a predominantly developmental phenotype. It results from aberrant retinal developmental patterning and the subsequent secretion/formation of the overlying eye cuticle (lenslets). I am confused by the data shown suggesting a "shrinking eye size" and increasing roughened surface over time (a GMR>tau eye similar to that shown in panel B cannot change to appear like the one in panel H with aging). The rough eye can be quite variable among a population of animals, but it is usually fixed at the time the adult fly ecloses from the pupal case, and quite stable over time in an individual animal. Therefore, any suppression of the Tau rough eye seen at 30 days should be appreciable as soon as the animals eclose. These results need to be clarified. If indeed there is robust suppression of Tau rough eye, it may be more intuitive and clearer to include these data with Figure 1, when first showing the loss-of-function enhancement of the Tau rough eye. Also, why is Pfdn6 included in these experiments but not in the studies shown in Figures 2-5?

We thank the reviewer for their careful and knowledgeable assessment of the GMR>Tau rough eye model. We appreciate the clarification that the rough eye phenotype could be “developmental” rather than neurodegenerative.”  Our initial observations regarding "shrinking eye size" and "increased surface roughness" clearly show age-related progression of structural change.   Such progression has been observed and reported by others (IIJIMA-ANDO et al. 2012; PASSARELLA AND GOEDERT 2018).   We observed an age-dependent increase in the number of fused ommatidia in GMR-Gal4 >Tau, which were rescued by Pfdn5 or Pfdn6 expression. We noted that adult-specific induction of hTau^V337M adult flies using the Gal80^ts and GMR-GeneSwitch (GMR-GS) systems was not sufficient to induce a significant eye phenotype; thus, early expression of Tau in the developing eye imaginal disc appears to be required for the adult progressive phenotype that we observe. We feel that it is inadequate to refer to this adult progressive phenotype as “developmental,” and while admittedly arguable whether this can be termed “degenerative.”   

To address neurodegeneration more directly, we focused on 30-day-old adult fly brains and demonstrated that Pfdn5 overexpression suppresses age-dependent Tau-induced neurodegeneration in the central nervous system (Figure 6H-N and Figure S12). This supports our central conclusion regarding the neuroprotective role of Pfdn5 in age-associated Tau pathology. Since we found an enhancement in the Tau-induced synaptic and eye phenotypes by Pfdn6 knockdown, we also generated CRISPR/Cas9-mediated loss-of-function mutants for Pfdn6. However, loss of Pfdn6 resulted in embryonic/early first instar lethality, which precluded its detailed analysis at the larval stages.

(12) Figure 6, panels O-T: the elav>tau image appears to show a different frontal section plane compared to the other panels. It is advisable to show images at a similar level in all panels since vacuolar pathology can vary by region. It is also useful to be able to see the entire brain at a lower power, but the higher power inset view is obscuring these images. I would recommend creating separate panels rather than showing them as insets.

In the revised figure, we now display the low- and high-magnification images as separate, clearly labeled panels instead of using insets. This improves visibility of the brain morphology while providing detailed views of the vacuolar pathology (Figure 6H-L).

(13) Figure 6/7: For the experiments in which Pfdn5/6 is overexpressed and possibly suppresses tau phenotypes (brain vacuoles and memory), it is important to use controls that normalize the number of UAS binding sites, since increased UAS sites may dilute GAL4 and reduced Tau expression levels/toxicity. Therefore, it would be advisable to compare with Elav>Tau flies that also include a chromosome with an empty UAS site or other transgenes, such as UAS-GFP or UAS-lacZ.

We thank the reviewer for the suggestion. Now we have incorporated proper controls in the brain vacuolization, the mushroom body, and ommatidial fusion rescue experiments. Also, we have independently verified whether Gal4 dilution has any effect on the Tau phenotypes (Figure 6H-L, Figure 7, and Figure S11A-B).

(14) Lines 311-312: the authors say vacuolization occurs in human neurodegenerative disease, which is not really true to my knowledge and definitely not stated in the citation they use. Please re-phrase.

Now we have made the appropriate changes in the revised manuscript.

(15) Figure 7: The authors claim that Pfdn5/6 expression does not impact memory behavior, but there in fact appears to be a decrease in preference index (panel D vs panel B). Does this result complicate the interpretation of the potential interaction with Tau (panel F). Are data from wildtype control flies available?

In our memory assay, a decrease in performance index (PI) of the trained flies compared to the naïve flies indicates memory formation (normal memory in control flies, Figure 7B). In contrast, a lack of significant difference in PI indicates a memory defect (Figure 7C: hTau^V337M overexpressed flies). "Decrease in preference index (panel D vs panel B)" is not a sign of memory defect; it may be interpreted as a better memory instead. Hence, neuronal overexpression of Pfdn5 (Figure 7D) or Pfdn6 (Figure 7E) in wildtype neurons does not cause memory deficits. In addition, coexpression of Pfdn5/6 and hTau^V337M successfully rescues the Tau-induced memory defect (significant drop in PI compared to the PI of naïve flies in Figure 7F-G). Moreover, almost complete rescue of the Tau-induced mushroom body defect on Pfdn5 or Pfdn6 expression further establishes potential interaction between Pfdn5/6 and Tau. This data has been incorporated into the revised manuscript.

The memory assay itself with extensive data on wildtype flies and various other genotype will shortly be submitted for publication in another manuscript (Majumder et al, manuscript under preparation); However, we can confirm for the reviewer that wildtype flies, trained and assayed by the protocol described, show a significant decrease in performance index compared to the naïve flies, indicative of strong learning and memory performance, very similar to the control genotype data shown in Figure 7B. 

Additional minor considerations

(16) Lines 50-52: there are many therapeutic interventions for treating tauopathies, but not curative or particularly effective ones.

Now we have made the appropriate changes in the revised manuscript.

(17) Lines 87-106 seem like a duplication of the abstract. Consider deleting or condensing.

We have made the appropriate changes in the revised manuscript.

(18) Where is pfdn5 expressed? Development v. adult? Neuron v. glia? Conservation?

Prefoldin5 is expressed throughout development but strongly localized to the larval trachea and neuronal axons. Drosophila Pfdn5 shows 35% overall identity with human PFDN5. 

(19) Liine 187: is pfdn5 truly "novel"?

The role of Pfdn5 as microtubule-binding and stabilizing is a new finding and has not been predicted or described before. Hence, it is a novel neuronal microtubule-associated protein.  

(20) Figure 5, panel F, genotype labels on the x-axis are confusing; consider simplifying to Control, DPfdn, and Rescue.

We have made appropriate changes in the figure for better readability.

(21) Figures 5/8: it might be preferable to use consistent colors for Tau/HRP--Tau is labeled green in Figure 5 and then purple in Figure 8.

We have made these changes where possible. 

(22) Lines 311-312: Vacuolar neuropathology is NOT typically observed in human Tauopathy.

We thank the reviewer for pointing this out. We have made the appropriate changes in the revised manuscript.

(23) Lines 328-349: The explanation could be made more clear. Naïve flies should not necessarily be called controls. Also, a more detailed explanation of how the preference index is computed would be helpful. Why are some datapoints negative values?

(a) We have rewritten this paragraph to make the description and explanation clearer. The detailed method and formula to calculate the Preference index have been incorporated in the Materials and Methods section.

(b) We have replaced the term Control with Naïve. 

(c) Datapoints with negative values appeared in some of the 'Trained' group flies. It indicates that post-CuSO₄ training, some groups showed repulsion towards the otherwise attractive odor 2,3B. As 2,3B is an attractive odorant, naïve or control flies show attraction towards it compared to air, which is evident from a higher number of flies in the Odor arm (O) compared to that of the Air arm (A) of the Y-maze; thus, the PI [(O-A/O+A)*100] is positive in case of naïve fly groups. Training of the flies led to an association of the attractive odorant with bitter food, leading to a decrease of attraction, and even repulsion towards the odorant in a few instances, resulting in less fly count in the odor arm compared to the air arm. Hence, the PI becomes negative as (O-A) is negative in such instances. Thus, it is not an anomaly but indicates strong learning. 

(24) Line 403: misspelling "Pdfn"

We have corrected this.

(25) Lines 423-425: recommend re-phrasing, since tauopathies are human diseases. Mice and other animal models may be susceptible to tau-mediated neuronal dysfunction but not Tauopathy, per see.

We have made the appropriate changes in the revised manuscript.

(26) Lines 468-469: "tau neuropathology" rather than "tau associated neuropathies".

We have made the appropriate changes in the revised manuscript. 

References

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Hsieh, Y. C., C. Guo, H. K. Yalamanchili, M. Abreha, R. Al-Ouran et al., 2019 Tau-Mediated Disruption of the Spliceosome Triggers Cryptic RNA Splicing and Neurodegeneration in Alzheimer's Disease. Cell Rep 29: 301-316 e310.

Iijima-Ando, K., M. Sekiya, A. Maruko-Otake, Y. Ohtake, E. Suzuki et al., 2012 Loss of axonal mitochondria promotes tau-mediated neurodegeneration and Alzheimer's disease-related tau phosphorylation via PAR-1. PLoS Genet 8: e1002918.

Jackson, G. R., M. Wiedau-Pazos, T. K. Sang, N. Wagle, C. A. Brown et al., 2002 Human wildtype tau interacts with wingless pathway components and produces neurofibrillary pathology in Drosophila. Neuron 34: 509-519.

Ji, W., K. An, C. Wang and S. Wang, 2022 Bioinformatics analysis of diagnostic biomarkers for Alzheimer's disease in peripheral blood based on sex differences and support vector machine algorithm. Hereditas 159: 38.

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Mershin, A., E. Pavlopoulos, O. Fitch, B. C. Braden, D. V. Nanopoulos et al., 2004 Learning and memory deficits upon TAU accumulation in Drosophila mushroom body neurons. Learn Mem 11: 277-287.

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board game: mid-weight, beginner-friendly

Synthèse du projet Sympa

Résumé Exécutif

Sympa est un gestionnaire de listes de diffusion open-source (GPLv2), développé en Perl depuis 17 ans.

Initialement conçu au sein de l'université Comète-Résu, il est aujourd'hui hébergé par Renater, le réseau national de télécommunications pour la technologie, l'enseignement et la recherche en France.

Bien qu'il assure les fonctions de base d'un gestionnaire de listes, Sympa se distingue par des fonctionnalités avancées qui en font un outil puissant pour les grandes organisations.

Ses principaux atouts sont sa capacité d'intégration profonde avec les systèmes d'information existants (bases de données, annuaires LDAP, systèmes d'authentification), ses mécanismes d'industrialisation pour la création et la gestion de milliers de listes, et un système d'autorisation par scénarios extrêmement flexible et expressif.

Le projet, bien que mature et utilisé par des institutions prestigieuses (90% des universités françaises, ministères, entreprises comme Orange et Atos), fait face aux défis d'un code historique de 17 ans.

Pour y répondre, l'équipe de développement a entamé une refonte majeure du code pour la future version 7.0.

Cette version introduira une architecture modernisée, des tests unitaires, une nouvelle interface web et une migration vers Git pour faciliter les contributions externes.

La vision à long terme inclut le déploiement en mode SaaS, la diffusion de messages multi-supports (SMS, web) et un système de plugins.

Le projet lance un appel actif à la communauté pour contribuer au développement, à la documentation, au support et à la gestion du projet, offrant même un service d'hébergement gratuit pour la communauté Perl afin de promouvoir l'utilisation d'outils libres.

1. Introduction à Sympa

Définition et Origine

• Nom : Sympa est l'acronyme de "Système de Multi-postage Automatique".

• Âge : Il s'agit d'un logiciel mature, dont la première version a été publiée le 1er avril 1997, soit il y a 17 ans au moment de la présentation.

• Fonction de base : Comme Mailman ou PHPList, Sympa permet d'envoyer un seul e-mail à un serveur qui se charge de le distribuer à un grand nombre d'abonnés.

• Hébergement et Licence : Le projet est hébergé par Renater, l'équivalent français du réseau national pour la recherche et l'éducation. C'est un logiciel libre sous licence GPLv2.

• Philosophie Perl : L'équipe revendique fièrement l'utilisation de Perl, affirmant que malgré les questions sur l'utilisation d'un langage "plus moderne", Sympa reste l'un des meilleurs gestionnaires de listes de diffusion et "il fonctionne".

Statistiques et Utilisateurs Clés

Sympa est utilisé par une base d'utilisateurs majoritairement internationale, malgré son origine française.

Métrique

Chiffre Record

Contexte

Plus grande liste

1,6 million d'abonnés

Plus grand nombre d'hôtes virtuels

30 000

Sur un seul serveur, par l'hébergeur Infomaniac

Plus grand nombre de listes

32 000

Sur un seul serveur

Plus grand nombre d'abonnés

3 millions

Sur un seul serveur

Principaux utilisateurs :

• Recherche et Éducation : 90% des universités et centres de recherche en France.

• Secteur Public : Plusieurs ministères français.

• Entreprises privées : Orange, Atos.

• Hébergeurs : Infomaniac, Switch (fourni par défaut à leurs clients).

• Organisations non gouvernementales : riseup.net, NAA, UNESCO, CGT.

2. Fonctionnalités Principales et Différenciatrices

Au-delà de l'envoi d'e-mails, Sympa se distingue par des capacités avancées conçues pour les environnements complexes.

Gestion Avancée des E-mails

• Envoi en masse optimisé : Sympa permet de regrouper les e-mails par domaine et de personnaliser la fréquence d'envoi pour éviter d'être identifié comme un spammeur tout en assurant une distribution rapide.

• Support des standards (RFC) : Il prend en charge S/MIME (signature et chiffrement), DKIM et offre une protection contre DMARC, ce qui a été crucial lorsque Yahoo a modifié sa politique en avril, cassant de nombreux systèmes de listes de diffusion.

• Gestion des erreurs : La gestion des bounces est automatique et gérée par Sympa, non par l'expéditeur original. Le support de VERP (Variable Envelope Return Path) permet de traiter automatiquement les erreurs pour les adresses e-mail transférées.

• Suivi des e-mails : Un suivi respectueux de la vie privée (sans "spy pixels") permet de savoir ce qui est arrivé à un e-mail pour chaque utilisateur, en se basant sur les RFC.

• Personnalisation (Mail Merging) : Il est possible de fusionner des données utilisateur dans un e-mail pour envoyer des messages personnalisés.

• Archives Web : Sympa dispose d'archives web avec un contrôle d'accès fin.

Intégration aux Systèmes d'Information (SI)

Sympa est conçu pour s'intégrer nativement avec les briques logicielles d'un système d'information d'entreprise ou d'université.

Composant

Technologies Supportées

Serveur de messagerie (MTA)

Sendmail, Postfix, Exim

Base de données (SGBDR)

MySQL, PostgreSQL, Oracle, SQLite, Sybase ("sans espoir")

Serveur Web

Apache, lighttpd, Nginx

Sources de données (Référentiels)

Bases de données relationnelles, LDAP, fichiers plats, services web (texte brut)

Systèmes d'authentification

Natif (email/mot de passe), CAS, Shibboleth, LDAP

Industrialisation de la Gestion des Listes

Pour les environnements nécessitant la création de centaines ou de milliers de listes (par exemple, chaque année dans une université), Sympa offre des mécanismes d'automatisation.

1. Création Manuelle : Un simple formulaire web où l'utilisateur remplit les informations de base (nom, objet, propriétaire).

Les valeurs par défaut sont fournies par la configuration globale et un modèle de liste (Template Toolkit - tt2).

2. Familles de Listes : Un mécanisme pour créer des listes en masse.

Il utilise un modèle tt2 commun et un fichier XML qui définit les paramètres spécifiques de chaque liste à créer.

Une seule commande permet de générer ou de mettre à jour toutes les listes de la famille.

3. Listes Automatiques : Conçues pour les cas où il existe un très grand nombre de listes potentielles mais où seulement une fraction sera utilisée.

◦ Le nom de la liste contient lui-même les paramètres (ex: prefix-field1_value1-field2_value2).  

◦ La liste n'est créée dynamiquement que lors du premier envoi d'un message à cette adresse.   

◦ Une interface web a été développée pour simplifier la composition de ces adresses complexes.

4. Familles de Familles : Il est possible de créer des familles de listes automatiques, permettant une industrialisation à plusieurs niveaux.

Mécanisme d'Autorisation par Scénarios

C'est l'une des fonctionnalités les plus originales et puissantes de Sympa.

• Principe : Les autorisations pour chaque action (envoyer un message, consulter les archives, etc.) sont définies dans des fichiers appelés "scénarios" (ex: send.scenario).

• Structure d'un scénario : C'est une séquence de règles évaluées de haut en bas.

Chaque règle a la forme : test(arguments) 'auth_method' -> decision.

• Évaluation : Le traitement s'arrête à la première règle dont le test est vrai.

• Tests : De nombreux tests sont disponibles (is_subscriber, is_list_owner, etc.).

Il est possible d'ajouter des tests personnalisés via des modules Perl (custom_condition).

• Méthodes d'authentification : Permettent d'appliquer des règles différentes selon la robustesse de l'authentification (ex: smime, smtp pour le champ From:, md5 pour un utilisateur authentifié sur le web).

• Décisions : Vont au-delà du simple "oui/non". Les décisions possibles incluent do_it (accepter), reject (rejeter), owner (modération par le propriétaire), etc.

Ce système offre une grande expressivité pour définir des politiques d'accès très fines.

Capacités de Gestion de Groupes

Sympa peut être utilisé comme un gestionnaire de groupes pour des applications tierces.

• Interface SOAP (et REST en développement) : Une interface SOAP permet à d'autres applications d'interroger les données internes de Sympa (créer une liste, abonner un utilisateur, etc.).

• Intégration : Des plugins pour des applications comme DokuWiki ou LimeSurvey permettent d'interroger Sympa pour savoir à quelles listes (donc à quels groupes) un utilisateur appartient.

L'application tierce peut alors accorder des privilèges en fonction de cette appartenance.

• Hiérarchie de groupes : Sympa permet d'inclure des listes dans d'autres listes, créant ainsi des groupes plus larges.

Personnalisation Poussée

Presque tous les aspects de Sympa sont personnalisables à différents niveaux (serveur global, hôte virtuel, liste individuelle) selon un principe de cascade.

• Interface Web : Entièrement basée sur des modèles Template Toolkit.

• Messages de service : Les messages envoyés aux utilisateurs (bienvenue, etc.) peuvent être modifiés.

• Modèles de création de liste.

• Scénarios d'autorisation.

• Paramètres de liste : Il est possible de créer ses propres paramètres en plus de la centaine existante.

• Attributs utilisateur : Possibilité d'ajouter des champs personnalisés pour les utilisateurs, qui pourront être synchronisés avec LDAP ou une base de données dans une future version.

3. Architecture et Fonctionnement Technique

Le flux de traitement d'un e-mail illustre l'architecture modulaire de Sympa :

1. Réception : Un e-mail est envoyé à une liste et arrive sur le MTA entrant.

2. Traitement Initial : Le MTA transmet l'e-mail au démon sympa.pl, qui évalue les autorisations, personnalise le message, etc.

3. Stockage : Si le message est autorisé, il est stocké dans une base de données relationnelle (SGBDR). L'utilisation d'une base de données permet un accès concurrentiel sécurisé.

4. Distribution : Un démon dédié, bulk.pl, se charge exclusivement de l'envoi des e-mails.

Il lit les messages dans la base de données et ouvre de multiples sessions SMTP pour une distribution rapide et parallélisable sur plusieurs serveurs.

5. Archivage : Simultanément, une copie du message est traitée par le démon archived.pl pour être ajoutée aux archives web.

4. Le Projet Sympa : Développement et Communauté

Gouvernance et Équipe

• Développeurs principaux : Le projet est passé de 2 développeurs historiques à une équipe élargie de 5 personnes, dont 3 externes à Renater.

◦ Mark (Strasbourg) : Gourou Perl.   

◦ Guillaume : Responsable sécurité, expert en bonnes pratiques.    ◦ Soji (Tokyo) : Spécialiste des e-mails et des problèmes d'encodage (a mené la migration vers UTF-8).   

◦ Etienne : Développeur polyglotte.  

◦ David Verdin (le présentateur) : "Homme à tout faire" (documentation, gestion de communauté, présentations).

• Contributions : Le projet bénéficie de nombreuses contributions de la communauté Perl.

Défis d'un Logiciel Ancien

Avec 17 ans d'histoire, le code de Sympa est devenu très hétérogène, avec des styles de codage variés issus de nombreux contributeurs.

• Base installée : L'importante base d'utilisateurs en production impose une grande prudence lors des modifications du code.

• Dépendances : L'ajout de nouveaux modules CPAN est compliqué car les utilisateurs en production préfèrent installer via des paquets de distribution, qui doivent donc exister pour ces modules.

• Absence de tests : Historiquement, le logiciel n'avait pas de tests unitaires ; les tests étaient effectués "en direct" sur les serveurs de production.

5. L'Avenir de Sympa : Feuille de Route et Vision

Versions à Venir (6.2, 7.0, 7.1)

• Version 6.2 : Presque finalisée, elle subit actuellement des tests manuels intensifs avant une sortie en bêta.

• Version 7.0 : Il s'agit d'une refonte majeure.

◦ Nouveau code : Réécriture complète menée par Guillaume pour moderniser l'architecture. 

◦ Tests unitaires : Implémentation systématique de tests.    ◦ Nouvelle interface web : Plus simple, plus moderne et ergonomique, développée par un contributeur de Nouvelle-Zélande.  

◦ Migration vers Git : Pour faciliter le fork et les contributions externes (par exemple sur GitHub).

• Version 7.1 et au-delà :

◦ Mode SaaS (Software as a Service).  

◦ Diffusion multi-supports : Envoi de messages via SMS ou mise à jour de services web.  

◦ Système de plugins : Pour permettre l'ajout de petites fonctionnalités sans attendre une intégration au cœur du logiciel.  

◦ Support des adresses e-mail internationalisées.

Orientations Stratégiques

Un objectif clé est de maintenir la double capacité de Sympa :

1. Grandes installations : Capable de tourner sur des clusters en mode SaaS.

2. Petites installations : Rester simple à installer et à faire fonctionner sur un petit serveur autonome.

6. Appel à la Participation et Offres à la Communauté

Opportunités de Contribution

Le projet recherche activement de l'aide, y compris non technique :

• Développement : Correction de bugs, ajout de fonctionnalités.

• Documentation : La documentation est un wiki modifiable par tout utilisateur abonné à la liste sympa-users.

• Support : Aider les autres utilisateurs sur les listes de diffusion.

• Packaging : Créer des paquets pour différentes distributions Linux.

• Gestion de projet : Partage d'expérience sur la gestion d'un projet logiciel en pleine croissance.

Offre d'Hébergement Gratuit

Pour contrer l'utilisation de services comme Google Groups par les communautés du logiciel libre, l'équipe Sympa propose de fournir un service d'hébergement de listes de diffusion gratuit pour la communauté Perl mondiale.

L'infrastructure de Renater permet de déployer un nouvel hôte virtuel en 30 minutes.

7. Questions et Réponses Clés

• Nouvelle interface web (v7.0) : Elle sera plus simple, avec moins d'options par défaut pour ne pas submerger les nouveaux utilisateurs.

L'ergonomie sera plus moderne et proche de ce que l'on trouve sur les réseaux sociaux.

• Interface REST : Une interface REST existe déjà pour la gestion de groupes (basée sur OAuth), mais la refonte du code vise à rendre toutes les fonctionnalités de Sympa accessibles via toutes ses interfaces (ligne de commande, SOAP, REST, web et e-mail).

• Stockage des e-mails et des pièces jointes : Les e-mails des archives sont stockés de façon permanente.

L'anonymisation est un défi juridique et technique complexe.

Les pièces jointes sont stockées et accessibles via un lien.

Pour les listes qui le souhaitent, les pièces jointes volumineuses peuvent être automatiquement détachées et remplacées par un lien pour alléger les e-mails.

• Support des bases de données : MySQL est celle qui reçoit le plus d'attention car c'est la plus utilisée par l'équipe.

PostgreSQL et SQLite sont également très bien maintenus et leurs schémas sont mis à jour automatiquement.

Le support d'Oracle est plus difficile.

Reviewer #2 (Public review):

Summary:

The role of PRC2 in post neural crest induction was not well understood. This work developed an elegant mouse genetic system to conditionally deplete EED upon SOX10 activation. Substantial developmental defects were identified for craniofacial and bone development. The authors also performed extensive single-cell RNA sequencing to analyze differentiation gene expression changes upon conditional EED disruption.

Strengths:

(1) Elegant genetic system to ablate EED post neural crest induction.

(2) Single-cell RNA-seq analysis is extremely suitable for studying the cell type specific gene expression changes in developmental systems.

Original Weaknesses:

(1) Although this study is well designed and contains state-of-art single cell RNA-seq analysis, it lacks the mechanistic depth in the EED/PRC2-mediated epigenetic repression. This is largely because no epigenomic data was shown.

(2) The mouse model of conditional loss of EZH2 in neural crest has been previously reported, as the authors pointed out in the discussion. What is novelty in this study to disrupt EED? Perhaps a more detailed comparison of the two mouse models would be beneficial.

(3) The presentation of the single-cell RNA-seq data may need improvement. The complexity of the many cell types blurs the importance of which cell types are affected the most by EED disruption.

(4) While it's easy to identify PRC2/EED target genes using published epigenomic data, it would be nice to tease out the direct versus indirect effects in the gene expression changes (e.g Fig. 4e)

Comments on latest version:

The authors have addressed weaknesses 2 and 3 of my previous comment very well. For weaknesses 1 and 4, the authors have added a main Fig 5 and its associated supplemental materials, which definitely strengthen the mechanistic depth of the story. However, I think the audience would appreciate if the following questions/points could be further addressed regarding the Cut&Tag data (mostly related to main Figure 5):

(1) The authors described that Sox10-Cre would be expressed at E8.75, and in theory, EED-FL would be ablated soon after that. Why would E16.5 exhibit a much smaller loss in H3K27me3 compared to E12.5? Shouldn't a prolong loss of EED lead to even worse consequence?

(2) The gene expression change at E12.5 upon loss of EED (shown in Fig. 4h) seems to be massive, including many PRC2-target genes. However, the H3K27me3 alteration seems to be mild even at E12.5. Does this infer a PRC2 or H3K27 methylation - independent role of EED? To address this, I suggest the authors re-consider addressing my previously commented weakness #4 regarding the RNA-seq versus Cut&Tag change correlation. For example, a gene scatter plot with X-axis of RNA-seq changes versus Y-axis of H3K27me3 level changes.

(3) The CUT&Tag experiments seem to contain replicates according to the figure legend, but no statistical analysis was presented including the new supplemental tables. Also, for Fig. 5c-d, instead of showing the MRR in individual conditions, I think the audience would really want to know the differential MRR between Fl/WT and Fl/Fl. In other words, how many genes/ MRR have statistically lower H3K27me3 level upon EED loss.

Author response:

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

Reviewer #1 (Public review):

Epigenetic regulation complex (PRC2) is essential for neural crest specification, and its misregulation has been shown to cause severe craniofacial defects. This study shows that Eed, a core PRC2 component, is critical for craniofacial osteoblast differentiation and mesenchymal proliferation after neural crest induction. Using mouse genetics and single-cell RNA sequencing, the researcher found that conditional knockout of Eed leads to significant craniofacial hypoplasia, impaired osteogenesis, and reduced proliferation of mesenchymal cells in post-migratory neural crest populations.

Overall, the study is superficial and descriptive. No in-depth mechanism was analyzed and the phenotype analysis is not comprehensive.

We thank the reviewer for sharing their expertise and for taking the time to provide helpful suggestions to improve our study. We are gratified that the striking phenotypes we report from Eed loss in post-migratory neural crest craniofacial tissues were appreciated. The breadth and depth of our phenotyping techniques, including skeletal staining, micro-CT, echocardiogram, immunofluorescence, histology, and primary craniofacial cell culture provide comprehensive data in support our hypothesis that PRC2 is required for epigenetic control of craniofacial osteoblast differentiation. To provide mechanistic data in support of this hypothesis, we have now performed CUT&Tag H3K27me3 chromatin profiling on nuclei harvested from E12.5 or E16.5 Sox10-Cre Eed^Fl/WT and Sox10-Cre Eed^Fl/Fl craniofacial tissue. These new data, which are presented in Fig. 5, Supplementary Fig. 9, and Supplementary Tables 7-10 of our revised manuscript, validate our hypothesis that epigenetic regulation of chromatin architecture downstream of PRC2 activity underlies craniofacial osteoblast differentiation. In particular, we now show that Eed-dependent H3K27me3 methylation is associated with correct temporal expression of transcription factors that are necessary for craniofacial differentiation and patterning, such as including Msx1, Pitx1, Pax7, which were initially nominated by single-cell RNA sequencing of E12.5 Sox10-Cre Eed^Fl/WT and Sox10-Cre Eed^Fl/Fl craniofacial tissues in Fig. 4, Supplementary Fig. 5-7, and Supplementary Tables 1-6.

Reviewer #2 (Public review):

Summary:

The role of PRC2 in post-neural crest induction was not well understood. This work developed an elegant mouse genetic system to conditionally deplete EED upon SOX10 activation. Substantial developmental defects were identified for craniofacial and bone development. The authors also performed extensive single-cell RNA sequencing to analyze differentiation gene expression changes upon conditional EED disruption.

Strengths:

(1) Elegant genetic system to ablate EED post neural crest induction.

(2) Single-cell RNA-seq analysis is extremely suitable for studying the cell type-specific gene expression changes in developmental systems.

We thank the reviewer for their generous and helpful comments on our study. We are happy that our mouse genetic and single-cell RNA sequencing approaches were appropriate in pairing the craniofacial phenotypes we report with distinct gene expression changes in post-migratory neural crest tissues upon Eed deletion.

Weaknesses:

(1) Although this study is well designed and contains state-of-the-art single-cell RNA-seq analysis, it lacks the mechanistic depth in the EED/PRC2-mediated epigenetic repression. This is largely because no epigenomic data was shown.

Thank you for this suggestion. As described in response to Reviewer #1, we have now performed CUT&Tag H3K27me3 chromatin profiling on nuclei harvested from E12.5 or E16.5 Sox10-Cre Eed^Fl/WT and Sox10-Cre Eed^Fl/Fl craniofacial tissues to provide mechanistic epigenomic data in support of our hypothesis that hat PRC2 is required for craniofacial osteoblast differentiation. These new data, which are presented in Fig. 5, Supplementary Fig. 9, and Supplementary Tables 7-10 of our revised manuscript, integrate genome-wide and targeted metaplot visualizations across genotypes with in-depth analyses of methylation rich regions and genes associated with methylation rich loci. Broadly, these new data reveal that changes in H3K27me3 occupancy correlate with gene expression changes from single-cell RNA sequencing of E12.5 Sox10-Cre Eed^Fl/WT and Sox10-Cre Eed^Fl/Fl craniofacial tissues in Fig. 4, Supplementary Fig. 5-7, and Supplementary Tables 1-6.

(2) The mouse model of conditional loss of EZH2 in neural crest has been previously reported, as the authors pointed out in the discussion. What is novel in this study to disrupt EED? Perhaps a more detailed comparison of the two mouse models would be beneficial.

We acknowledge and cite the study the reviewer has indicated (Schwarz et al. Development 2014) in our initial and revised manuscripts. This elegant investigation uses Wnt1-Cre to delete Ezh2 and reports a phenotype similar to the one we observed with Sox10-Cre deletion of Eed, but our study adds depth to the understanding of PRC2’s vital role in neural crest development by ablating Eed, which has a unique function in the PRC2 complex by binding to H3K27me3 and allosterically activating Ezh2. In this sense, our study sheds light on whether phenotypes arising from deletion of Eed, the PRC2 “reader”, differ from phenotypes arising from deletion of Ezh2, the PRC2 “writer”, in neural crest derived tissues. Moreover, we provide the first single-cell RNA sequencing and epigenomic investigations of craniofacial phenotypes arising from PRC2 activity in the developing neural crest. Due to limitations associated with the Wnt1-Cre transgene (Lewis et al. Developmental Biology 2013), which targets pre-migratory neural crest cells, our investigations used Sox10Cre, which targets the migratory neural crest and is completely recombined by E10.5. We have included a detailed comparison of these mouse models in the Discussion section of our revised manuscript, and we thank the reviewer for this thoughtful suggestion. 

(3) The presentation of the single-cell RNA-seq data may need improvement. The complexity of the many cell types blurs the importance of which cell types are affected the most by EED disruption.

We thank the reviewer for the opportunity to improve the presentation of our single-cell RNA sequencing data. In response, we have added Supplementary Fig. 8 to our revised manuscript, which shows the cell clusters most affected by EED disruption in UMAP space across genotypes. Because we wanted to capture the fill diversity of cell types underlying the phenotypes we report, we did not sort Sox10+ cells (via FACS, for example) from craniofacial tissues before single-cell RNA sequencing. Our resulting single-cell RNA sequencing data are therefore inclusive of a diversity of cell types in UMAP space, and the prevalence of many of these cell types was unaffected by epigenetic disruption of neural crest derived tissues. The prevalence of the cell clusters that are most affected across genotypes and which are most relevant to our analyses of the developing neural crest are shown in Fig. 4c (and now also in Supplementary Fig. 8), including C0 (differentiating osteoblasts), C4 (mesenchymal stem cells), C5 (mesenchymal stem cells), and C7 (proliferating mesenchymal stem cells). Marker genes and pseudobulked differential expression analyses across these clusters are shown in Fig. 4d and Fig. 4e-h, respectively. 

(4) While it's easy to identify PRC2/EED target genes using published epigenomic data, it would be nice to tease out the direct versus indirect effects in the gene expression changes (e.g Figure 4e).

We agree with the reviewer that the single-cell RNA sequencing data in our initial submission do not provide insight into direct versus indirect changes in gene expression downstream of PRC2. In contrast, the CUT&Tag chromatin profiling data that we have generated for this revision provides mechanistic insight into H3K27me3 occupancy and direct effects on gene expression resulting from PRC2 inactivation in our mouse models.

REVIEWING EDITOR COMMENTS

The following are recommended as essential revisions

(1) The study is overall superficial and primarily descriptive, lacking in-depth mechanistic analysis and comprehensive phenotype evaluation.

Please see responses to Reviewer #1 and Reviewer #2 (weaknesses 1 and 4) above. 

(2) The authors did not investigate the temporal and spatial expression of Eed during cranial neural crest development, which is crucial for explaining the observed phenotypes.

The temporal and spatial expression of Eed during embryogenesis is well studied. Eed is ubiquitously expressed starting at E5.5, peaks at E9.5, and is downregulated but maintained at a high basal expression level through E18.5 (Schumacher et al. Nature 1996). Although comprehensive analysis of Eed expression in neural crest tissues has not been reported (to our knowledge), Eed physically and functionally interacts with Ezh2 (Sewalt et al. Mol Cell Biol 1998), which is enriched at a diversity of timepoints throughout all developing craniofacial tissues (Schwarz et al. Development 2014). In our study, we confirmed enrichment of Eed expression in craniofacial tissues throughout development using QPCR, and have provided a more detailed description of these published and new findings in the Discussion section of our revised manuscript. 

(3) There is no apoptosis analysis provided for any of the samples.

We evaluated the presence of apoptotic cells in E12.5 craniofacial sections using immunofluorescence for Cleaved Caspase 3 in Supplementary Fig. 3d. Although we found a modest increase in the labeling index of apoptotic cells, there was insufficient evidence to conclude that apoptosis is a substantial factor in craniofacial hypoplasia resulting from Eed loss in post-migratory neural crest craniofacial tissues. We have clarified these findings in the Results and Discussion sections of our revised manuscript. 

(4) As Eed is a core component of the PRC2 complex, were any other components altered in the Eed cKO mutant? How does Eed regulation influence osteogenic differentiation and proliferation through known pathways?

We thank the editors for this thoughtful inquiry. Although we did not specifically investigate expression or stability of other PRC2 components in Eed conditional mutants, and little is known about how Eed regulates osteogenic differentiation or proliferation through any pathway, our single-cell RNA sequencing data presented in Fig. 4, Supplementary Fig. 5-7, and Supplementary Tables 1-6 provide a significant conceptual advance with mechanistic implications for understanding bone development downstream of Eed and do not reveal any alterations in the expression of other PRC2 components across genotypes. We have clarified these important details in the Discussion section of our revised manuscript. 

(5) The authors may compare the Eed cKO phenotype with that of the previous EZH2 cKO mouse model since both Eed and EZH2 are essential subunits of PRC2.

Please see responses to editorial comment 2 above and the last paragraph of the Discussion section of our revised manuscript for comparisons between Eed and Ezh2 knockout phenotypes.

Author response:

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

Reviewer #1 (Public review):

Summary:

The authors validate the contribution of RAP2A to GB progression. RAp2A participates in asymmetric cell division, and the localization of several cell polarity markers, including cno and Numb.

Strengths:

The use of human data, Drosophila models, and cell culture or neurospheres is a good scenario to validate the hypothesis using complementary systems.

Moreover, the mechanisms that determine GB progression, and in particular glioma stem cells biology, are relevant for the knowledge on glioblastoma and opens new possibilities to future clinical strategies.

Weaknesses:

While the manuscript presents a well-supported investigation into RAP2A's role in GBM, several methodological aspects require further validation. The major concern is the reliance on a single GB cell line (GB5), which limits the generalizability of the findings. Including multiple GBM lines, particularly primary patient-derived 3D cultures with known stem-like properties, would significantly enhance the study's relevance.

Additionally, key mechanistic aspects remain underexplored. Further investigation into the conservation of the Rap2l-Cno/aPKC pathway in human cells through rescue experiments or protein interaction assays would be beneficial. Similarly, live imaging or lineage tracing would provide more direct evidence of ACD frequency, complementing the current indirect metrics (odd/even cell clusters, Numb asymmetry).

Several specific points require attention:

(1) The specificity of Rap2l RNAi needs further confirmation. Is Rap2l expressed in neuroblasts or intermediate neural progenitors? Can alternative validation methods be employed?

There are no available antibodies/tools to determine whether Rap2l is expressed in NB lineages, and we have not been able either to develop any. However, to further prove the specificity of the Rap2l phenotype, we have now analyzed two additional and independent RNAi lines of Rap2l along with the original RNAi line analyzed. We have validated the results observed with this line and found a similar phenotype in the two additional RNAi lines now analyzed. These results have been added to the text ("Results section", page 6, lines 142-148) and are shown in Supplementary Figure 3.

(2) Quantification of phenotypic penetrance and survival rates in Rap2l mutants would help determine the consistency of ACD defects.

In the experiment previously mentioned (repetition of the original Rap2l RNAi line analysis along with two additional Rap2l RNAi lines) we have substantially increased the number of samples analyzed (both the number of NB lineages and the number of different brains analyzed). With that, we have been able to determine that the penetrance of the phenotype was 100% or almost 100% in the 3 different RNAi lines analyzed (n>14 different brains/larvae analyzed in all cases). Details are shown in the text (page 6, lines 142-148), in Supplementary Figure 3 and in the corresponding figure legend.

(3) The observations on neurosphere size and Ki-67 expression require normalization (e.g., Ki-67+ cells per total cell number or per neurosphere size). Additionally, apoptosis should be assessed using Annexin V or TUNEL assays.

The experiment of Ki-67+ cells was done considering the % of Ki-67+ cells respect the total cell number in each neurosphere. In the "Materials and methods" section it is well indicated: "The number of Ki67+ cells with respect to the total number of nuclei labelled with DAPI within a given neurosphere were counted to calculate the Proliferative Index (PI), which was expressed as the % of Ki67+ cells over total DAPI+ cells"

Perhaps it was not clearly showed in the graph of Figure 5A. We have now changed it indicating: "% of Ki67+ cells/ neurosphere" in the "Y axis". 

Unfortunately, we currently cannot carry out neurosphere cultures to address the apoptosis experiments. 

(4) The discrepancy in Figures 6A and 6B requires further discussion.

We agree that those pictures can lead to confusion. In the analysis of the "% of neurospheres with even or odd number of cells", we included the neurospheres with 2 cells both in the control and in the experimental condition (RAP2A). The number of this "2 cell-neurospheres" was very similar in both conditions (27,7 % and 27 % of the total neurospheres analyzed in each condition), and they can be the result of a previous symmetric or asymmetric division, we cannot distinguish that (only when they are stained with Numb, for example, as shown in Figure 6B). As a consequence, in both the control and in the experimental condition, these 2-cell neurospheres included in the group of "even" (Figure 6A) can represent symmetric or asymmetric divisions. However, in the experiment shown in Figure 6B, it is shown that in these 2 cellneurospheres there are more cases of asymmetric divisions in the experimental condition (RAP2A) than in the control.

Nevertheless, to make more accurate and clearer the conclusions, we have reanalyzed the data taking into account only the neurospheres with 3-5-7 (as odd) or 4-6-8 (as even) cells. Likewise, we have now added further clarifications regarding the way the experiment has been analyzed in the methods.

(5) Live imaging of ACD events would provide more direct evidence.

We agree that live imaging would provide further evidence. Unfortunately, we currently cannot carry out neurosphere cultures to approach those experiments.

(6) Clarification of terminology and statistical markers (e.g., p-values) in Figure 1A would improve clarity.

We thank the reviewer for pointing out this issue. To improve clarity, we have now included a Supplementary Figure (Fig. S1) with the statistical parameters used. Additionally, we have performed a hierarchical clustering of genes showing significant or not-significant changes in their expression levels.

(7) Given the group's expertise, an alternative to mouse xenografts could be a Drosophila genetic model of glioblastoma, which would provide an in vivo validation system aligned with their research approach.

The established Drosophila genetic model of glioblastoma is an excellent model system to get deep insight into different aspects of human GBM. However, the main aim of our study was to determine whether an imbalance in the mode of stem cell division, favoring symmetric divisions, could contribute to the expansion of the tumor. We chose human GBM cell lines-derived neurospheres because in human GBM it has been demonstrated the existence of cancer stem cells (glioblastoma or glioma stem cells -GSCs--). And these GSCs, as all stem cells, can divide symmetric or asymmetrically. In the case of the Drosophila model of GBM, the neoplastic transformation observed after overexpressing the EGF receptor and PI3K signaling is due to the activation of downstream genes that promote cell cycle progression and inhibit cell cycle exit. It has also been suggested that the neoplastic cells in this model come from committed glial progenitors, not from stem-like cells.

With all, it would be difficult to conclude the causes of the potential effects of manipulating the Rap2l levels in this Drosophila system of GBM. We do not discard this analysis in the future (we have all the "set up" in the lab). However, this would probably imply a new project to comprehensively analyze and understand the mechanism by which Rap2l (and other ACD regulators) might be acting in this context, if it is having any effect. 

However, as we mentioned in the Discussion, we agree that the results we have obtained in this study must be definitely validated in vivo in the future using xenografts with 3D-primary patient-derived cell lines.

Reviewer #2 (Public review):

This study investigates the role of RAP2A in regulating asymmetric cell division (ACD) in glioblastoma stem cells (GSCs), bridging insights from Drosophila ACD mechanisms to human tumor biology. They focus on RAP2A, a human homolog of Drosophila Rap2l, as a novel ACD regulator in GBM is innovative, given its underexplored role in cancer stem cells (CSCs). The hypothesis that ACD imbalance (favoring symmetric divisions) drives GSC expansion and tumor progression introduces a fresh perspective on differentiation therapy. However, the dual role of ACD in tumor heterogeneity (potentially aiding therapy resistance) requires deeper discussion to clarify the study's unique contributions against existing controversies. Some limitations and questions need to be addressed.

(1) Validation of RAP2A's prognostic relevance using TCGA and Gravendeel cohorts strengthens clinical relevance. However, differential expression analysis across GBM subtypes (e.g., MES, DNA-methylation subtypes ) should be included to confirm specificity.

We have now included a Supplementary figure (Supplementary Figure 2), in which we show the analysis of RAP2A levels in the different GBM subtypes (proneural, mesenchymal and classical) and their prognostic relevance (i.e. the proneural subtype that presents RAP2A levels significantly higher than the others is the subtype that also shows better prognostic).

(2) Rap2l knockdown-induced ACD defects (e.g., mislocalization of Cno/Numb) are well-designed. However, phenotypic penetrance and survival rates of Rap2l mutants should be quantified to confirm consistency.

We have now analyzed two additional and independent RNAi lines of Rap2l along with the original RNAi line. We have validated the results observed with this line and found a similar phenotype in the two additional RNAi lines now analyzed. To determine the phenotypic penetrance, we have substantially increased the number of samples analyzed (both the number of NB lineages and the number of different brains analyzed). With that, we have been able to determine that the penetrance of the phenotype was 100% or almost 100% in the 3 different Rap2l RNAi lines analyzed (n>14 different brains/larvae analyzed in all cases). These results have been added to the text ("Results section", page 6, lines 142-148) and are shown in Supplementary Figure 3 and in the corresponding figure legend. 

(3) While GB5 cells were effectively used, justification for selecting this line (e.g., representativeness of GBM heterogeneity) is needed. Experiments in additional GBM lines (especially the addition of 3D primary patient-derived cell lines with known stem cell phenotype) would enhance generalizability.

We tried to explain this point in the paper (Results). As we mentioned, we tested six different GBM cell lines finding similar mRNA levels of RAP2A in all of them, and significantly lower levels than in control Astros (Fig. 3A). We decided to focus on the GBM cell line called GB5 as it grew well (better than the others) in neurosphere cell culture conditions, for further analyses. We agree that the addition of at least some of the analyses performed with the GB5 line using other lines (ideally in primary patientderive cell lines, as the reviewer mentions) would reinforce the results. Unfortunately, we cannot perform experiments in cell lines in the lab currently. We will consider all of this for future experiments.

(4) Indirect metrics (odd/even cell clusters, NUMB asymmetry) are suggestive but insufficient. Live imaging or lineage tracing would directly validate ACD frequency.

We agree that live imaging would provide further evidence. Unfortunately, we cannot approach those experiments in the lab currently.

(5) The initial microarray (n=7 GBM patients) is underpowered. While TCGA data mitigate this, the limitations of small cohorts should be explicitly addressed and need to be discussed.

We completely agree with this comment. We had available the microarray, so we used it as a first approach, just out of curiosity of knowing whether (and how) the levels of expression of those human homologs of Drosophila ACD regulators were affected in this small sample, just as starting point of the study. We were conscious of the limitations of this analysis and that is why we followed up the analysis in the datasets, on a bigger scale. We already mentioned the limitations of the array in the Discussion:

"The microarray we interrogated with GBM patient samples had some limitations. For example, not all the human genes homologs of the Drosophila ACD regulators were present (i.e. the human homologs of the determinant Numb). Likewise, we only tested seven different GBM patient samples. Nevertheless, the output from this analysis was enough to determine that most of the human genes tested in the array presented altered levels of expression"[....] In silico analyses, taking advantage of the existence of established datasets, such as the TCGA, can help to more robustly assess, in a bigger sample size, the relevance of those human genes expression levels in GBM progression, as we observed for the gene RAP2A."

(6) Conclusions rely heavily on neurosphere models. Xenograft experiments or patient-derived orthotopic models are critical to support translational relevance, and such basic research work needs to be included in journals.

We completely agree. As we already mentioned in the Discussion, the results we have obtained in this study must be definitely validated in vivo in the future using xenografts with 3D-primary patient-derived cell lines.

(7) How does RAP2A regulate NUMB asymmetry? Is the Drosophila Rap2l-Cno/aPKC pathway conserved? Rescue experiments (e.g., Cno/aPKC knockdown with RAP2A overexpression) or interaction assays (e.g., Co-IP) are needed to establish molecular mechanisms.

The mechanism by which RAP2A is regulating ACD is beyond the scope of this paper. We do not even know how Rap2l is acting in Drosophila to regulate ACD. In past years, we did analyze the function of another Drosophila small GTPase, Rap1 (homolog to human RAP1A) in ACD, and we determined the mechanism by which Rap1 was regulating ACD (including the localization of Numb): interacting physically with Cno and other small GTPases, such as Ral proteins, and in a complex with additional ACD regulators of the "apical complex" (aPKC and Par-6). Rap2l could be also interacting physically with the "Ras-association" domain of Cno (domain that binds small GTPases, such as Ras and Rap1). We have added some speculations regarding this subject in the Discussion:

"It would be of great interest in the future to determine the specific mechanism by which Rap2l/RAP2A is regulating this process. One possibility is that, as it occurs in the case of the Drosophila ACD regulator Rap1, Rap2l/RAP2A is physically interacting or in a complex with other relevant ACD modulators."

(8) Reduced stemness markers (CD133/SOX2/NESTIN) and proliferation (Ki-67) align with increased ACD. However, alternative explanations (e.g., differentiation or apoptosis) must be ruled out via GFAP/Tuj1 staining or Annexin V assays.

We agree with these possibilities.  Regarding differentiation, the potential presence of increased differentiation markers would be in fact a logic consequence of an increase in ACD divisions/reduced stemness markers. Unfortunately, we cannot approach those experiments in the lab currently.

(9) The link between low RAP2A and poor prognosis should be validated in multivariate analyses to exclude confounding factors (e.g., age, treatment history).

We have now added this information in the "Results section" (page 5, lines 114-123).

(10) The broader ACD regulatory network in GBM (e.g., roles of other homologs like NUMB) and potential synergies/independence from known suppressors (e.g., TRIM3) warrant exploration.

The present study was designed as a "proof-of-concept" study to start analyzing the hypothesis that the expression levels of human homologs of known Drosophila ACD regulators might be relevant in human cancers that contain cancer stem cells, if those human homologs were also involved in modulating the mode of (cancer) stem cell division. 

To extend the findings of this work to the whole ACD regulatory network would be the logic and ideal path to follow in the future.

We already mentioned this point in the Discussion:

"....it would be interesting to analyze in the future the potential consequences that altered levels of expression of the other human homologs in the array can have in the behavior of the GSCs. In silico analyses, taking advantage of the existence of established datasets, such as the TCGA, can help to more robustly assess, in a bigger sample size, the relevance of those human genes expression levels in GBM progression, as we observed for the gene RAP2A."

(11) The figures should be improved. Statistical significance markers (e.g., p-values) should be added to Figure 1A; timepoints/culture conditions should be clarified for Figure 6A.

Regarding the statistical significance markers, we have now included a Supplementary Figure (Fig. S1) with the statistical parameters used. Additionally, we have performed a hierarchical clustering of genes showing significant or notsignificant changes in their expression levels. 

Regarding the experimental conditions corresponding to Figure 6A, those have now been added in more detail in "Materials and Methods" ("Pair assay and Numb segregation analysis" paragraph).

(12) Redundant Drosophila background in the Discussion should be condensed; terminology should be unified (e.g., "neurosphere" vs. "cell cluster").

As we did not mention much about Drosophila ACD and NBs in the "Introduction", we needed to explain in the "Discussion" at least some very basic concepts and information about this, especially for "non-drosophilists". We have reviewed the Discussion to maintain this information to the minimum necessary.

We have also reviewed the terminology that the Reviewer mentions and have unified it.

Reviewer #1 (Recommendations for the authors):

To improve the manuscript's impact and quality, I would recommend:

(1) Expand Cell Line Validation: Include additional GBM cell lines, particularly primary patient-derived 3D cultures, to increase the robustness of the findings.

(2) Mechanistic Exploration: Further examine the conservation of the Rap2lCno/aPKC pathway in human cells using rescue experiments or protein interaction assays.

(3) Direct Evidence of ACD: Implement live imaging or lineage tracing approaches to strengthen conclusions on ACD frequency.

(4) RNAi Specificity Validation: Clarify Rap2l RNAi specificity and its expression in neuroblasts or intermediate neural progenitors.

(5) Quantitative Analysis: Improve quantification of neurosphere size, Ki-67 expression, and apoptosis to normalize findings.

(6) Figure Clarifications: Address inconsistencies in Figures 6A and 6B and refine statistical markers in Figure 1A.

(7) Alternative In Vivo Model: Consider leveraging a Drosophila glioblastoma model as a complementary in vivo validation approach.

Addressing these points will significantly enhance the manuscript's translational relevance and overall contribution to the field.

We have been able to address points 4, 5 and 6. Others are either out of the scope of this work (2) or we do not have the possibility to carry them out at this moment in the lab (1, 3 and 7). However, we will complete these requests/recommendations in other future investigations.

Reviewer #2 (Recommendations for the authors):

Major Revision /insufficient required to address methodological and mechanistic gaps.

(1) Enhance Clinical Relevance

Validate RAP2A's prognostic significance across multiple GBM subtypes (e.g., MES, DNA-methylation subtypes) using datasets like TCGA and Gravendeel to confirm specificity.

Perform multivariate survival analyses to rule out confounding factors (e.g., patient age, treatment history).

(2) Strengthen Mechanistic Insights

Investigate whether the Rap2l-Cno/aPKC pathway is conserved in human GBM through rescue experiments (e.g., RAP2A overexpression with Cno/aPKC knockdown) or interaction assays (e.g., Co-IP).

Use live-cell imaging or lineage tracing to directly validate ACD frequency instead of relying on indirect metrics (odd/even cell clusters, NUMB asymmetry).

(3) Improve Model Systems & Experimental Design

Justify the selection of GB5 cells and include additional GBM cell lines, particularly 3D primary patient-derived cell models, to enhance generalizability.

It is essential to perform xenograft or orthotopic patient-derived models to support translational relevance.

(5) Address Alternative Interpretations

Rule out other potential effects of RAP2A knockdown (e.g., differentiation or apoptosis) using GFAP/Tuj1 staining or Annexin V assays.

Explore the broader ACD regulatory network in GBM, including interactions with NUMB and TRIM3, to contextualize findings within known tumor-suppressive pathways.

(6) Improve Figures & Clarity

Add statistical significance markers (e.g., p-values) in Figure 1A and clarify timepoints/culture conditions for Figure 6A.

Condense redundant Drosophila background in the discussion and ensure consistent terminology (e.g., "neurosphere" vs. "cell cluster").

We have been able to address points 1, partially 3 and 6. Others are either out of the scope of this work or we do not have the possibility to carry them out at this moment in the lab. However, we are very interested in completing these requests/recommendations and we will approach that type of experiments in other future investigations.

A mother's healing touch

Author response:

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

Reviewer #1 (Public review): 

Summary: 

This study builds on previous work demonstrating that several beta connexins (Cx26, Cx30, and Cx32) have a carbamylation motif which renders them sensitive to CO₂. In response to CO₂, hemichannels composed of these connexins open, enabling diffusion of small molecules (such as ATP) between the cytosol and extracellular environment. Here, the authors have identified that an alpha connexin, Cx43, also contains a carbamylation motif, and they demonstrate that CO₂ opens Cx43 hemichannels. Most of the study involves using transfected cells expressing wildtype and mutant Cx43 to define amino acids required for CO₂ sensitivity. Hippocampal tissue slices in culture were used to show that CO₂-induced synaptic transmission was affected by Cx43 hemichannels, providing a physiological context. The authors point out that the Cx43 gene significantly diverges from the beta connexins that are CO₂ sensitive, suggesting that the conserved carbamylation motif was present before the alpha and beta connexin genes diverged. 

Strengths: 

(1) The molecular analysis defining the amino acids that contribute to the CO₂ sensitivity of Cx43 is a major strength of the study. The rigor of analysis was strengthened by using three independent assays for hemichannel opening: dye uptake, patch clamp channel measurements, and ATP secretion. The resulting analysis identified key lysines in Cx43 that were required for CO₂-mediated hemichannel opening. A double K to E Cx43 mutant produced a construct that produced hemichannels that were constitutively open, which further strengthened the analysis. 

(2) Using hippocampal tissue sections to demonstrate that CO₂ can influence field excitatory postsynaptic potentials (fEPSPs) provides a native context for CO₂ regulation of Cx43 hemichannels. Cx43 mutations associated with Oculodentodigital Dysplasia (ODDD) inhibited CO₂-induced hemichannel opening, although the mechanism by which this occurs was not elucidated. 

Weaknesses: 

(1) Cx43 channels are sensitive to cytosolic pH, which will be affected by CO₂. Cytosolic pH was not measured, and how this affects CO₂-induced Cx43 hemichannel activity was not addressed. 

We have now addressed this with intracellular pH measurements and removal of the C-terminal pH sensor from Cx43 -the hemichannel remains CO₂ sensitive.

(2) Cultured cells are typically grown in incubators containing 5% CO₂, which is ~40 mmHg. It is unclear how cells would be viable if Cx43 hemichannels are open at this PCO2. 

The cells look completely healthy with normal morphology and no sign of excessive cell death in the cultures. Presumably they have ways of compensating for the effects of partially open Cx43 hemichannels.

(3) Experiments using Gap26 to inhibit Cx43 hemichannels in fEPSP measurements used a scrambled peptide as a control. Analysis should also include Gap peptides specifically targeting Cx26, Cx30, and Cx32 as additional controls. 

We don’t feel this is necessary given the extensive prior literature in hippocampus showing the effect of ATP release via open Cx43 hemichannels on fEPSP amplitude that used astrocytic specific knockout of Cx43 and Gap26 (doi: 10.1523/jneurosci.0015-14.2014).

(4) The mechanism by which ODDD mutations impair CO2-mediated hemichannel opening was not addressed. Also, the potential roles for inhibiting Cx43 hemichannels in the pathology of ODDD are unclear. 

These pathological mutations that alter CO<SUB>2</SUB> sensitivity are similar to pathological mutation in Cx26 and Cx32, which also remove CO<SUB>2</SUB> sensitivity. Our cryo-EM studies on Cx26 give clues as to why these mutations have this effect -they alter conformational mobility of the channel (Brotherton et al 2022 doi: 10.1016/j.str.2022.02.010 and Brotherton et al 2024 doi: 10.7554/eLife.93686). We assume that similar considerations apply to Cx43, but this requires improved cryoEM structures of Cx43 hemichannels at differing levels of PCO<SUB>2</SUB>.

We agree that the link between loss of CO<SUB>2</SUB> sensitivity of Cx43 and ODDD is not established and have revised the text to make this clear.

(5) CO2 has no effect on Cx43-mediated gap junctional communication as opposed to Cx26 gap junctions, which are inhibited by CO2. The molecular basis for this difference was not determined. 

Cx26 gap junction channels are so far unique amongst CO<SUB>2</SUB> sensitive connexins in being closed by CO<SUB>2</SUB>. We have addressed the mechanism by which this occurs in Nijjar et al 2025 DOI: 10.1113/JP285885 -the requirement of carbamylation of K108 in Cx26 (in addition to K125) for GJC closure.

(6) Whether there are other non-beta connexins that have a putative carbamylation motif was not addressed. Additional discussion/analysis of how the evolutionary trajectory for Cx43 maintaining a carbamylation motif is unique for non-beta connexins would strengthen the study. 

We have performed a molecular phylogenetic survey to show that the carbamylation motif occurs across the alpha connexin clade and have shown that Cx50 is indeed CO<SUB>2</SUB> sensitive (doi: 10.1101/2025.01.23.634273). This is now in Fig 12.

Reviewer #2 (Public review): 

Summary: 

This paper examines the CO<SUB>2</SUB>  sensitivity of Cx43 hemichannels and gap junctional channels in transiently transfected Hela cells using several different assays, including ethidium dye uptake, ATP release, whole cell patch clamp recordings, and an imaging assay of gap junctional dye transfer. The results show that raising pCO₂ from 20 to 70 mmHg (at a constant pH of 7.3) causes an increase in opening of Cx43 hemichannels but does not block Cx43 gap junctions. This study also showed that raising pCO<SUB>2</SUB> from 20 to 35 mm Hg resulted in an increase in synaptic strength in hippocampal rat brain slices, presumably due to downstream ATP release, suggesting that the CO<SUB>2</SUB> sensitivity of Cx43 may be physiologically relevant. As a further test of the physiological relevance of the CO₂ sensitivity of Cx43, it was shown that two pathological mutations of Cx43 that are associated with ODDD caused loss of Cx43 CO₂-sensitivity. Cx43 has a potential carbamylation motif that is homologous to the motif in Cx26. To understand the structural changes involved in CO<SUB>2</SUB> sensitivity, a number of mutations were made in Cx43 sites thought to be the equivalent of those known to be involved in the CO<SUB>2</SUB> sensitivity of Cx26, and the CO<SUB>2</SUB> sensitivity of these mutants was investigated. 

Strengths: 

This study shows that the apparent lack of functional Cx43 hemichannels observed in a number of previous in vitro function studies may be due to the use of HEPES to buffer the external pH. When Cx43 hemichannels were studied in external solutions in which CO<SUB>2</SUB>/bicarbonate was used to buffer pH instead of HEPES, Cx43 hemichannels showed significantly higher levels of dye uptake, ATP release, and ionic conductance. These findings may have major physiological implications since Cx43 hemichannels are found in many organs throughout the body, including the brain, heart, and immune system. 

Weaknesses: 

(1) Interpretation of the site-directed mutation studies is complicated. Although Cx43 has a potential carbamylation motif that is homologous to the motif in Cx26, the results of site-directed mutation studies were inconsistent with a simple model in which K144 and K105 interact following carbamylation to cause the opening of Cx43 hemichannels. 

The mechanism of opening of Cx43 is more complex than that of Cx26, Cx32 and Cx50 and involves more Lys residues. The 4 Lys residues in Cx43 that are involved in opening the hemichannel have their equivalents in Cx26, but in Cx26 these additional residues seem to be involved in the closing of the GJC rather than opening of the hemichannel (see above). Cx50 is simpler and involves only two Lys residues (doi: 10.1101/2025.01.23.634273), which are equivalent to those in Cx26.

(2) Secondly, although it is shown that two Cx43 ODDD-associated mutations show a loss of CO₂ sensitivity, there is no evidence that the absence of CO2 sensitivity is involved in the pathology of ODD

We agree, but this is probably because this has not been directly tested by experiment, as the CO<Sub>2</sub> sensitivity of Cx43 was not previously known. As mentioned above we have revised the text to ensure that this is clear.

Reviewer #3 (Public review): 

In this paper, the authors aimed to investigate carbamylation effects on the function of Cx43-based hemichannels. Such effects have previously been characterized for other connexins, e.g., for Cx26, which display increased hemichannel (HC) opening and closure of gap junction channels upon exposure to increased CO₂ partial pressure (accompanied by increased bicarbonate to keep pH constant). 

The authors used HeLa cells transiently transfected with Cx43 to investigate CO₂ dependent carbamylation effects on Cx43 HC function. In contrast to Cx43-based gap junction channels that are reported here to be insensitive to PCO₂ alterations, they provide evidence that Cx43 HC opening is highly dependent on the PCO2 pressure in the bath solution, over a range of 20 up to 70 mmHg encompassing the physiologically normal resting level of around 40 mmHg. They furthermore identified several Cx43 residues involved in Cx43 HC sensitivity to PCO2: K105, K109, K144 & K234; mutation of 2 or more of these AAs is necessary to abolish CO₂ sensitivity. The subject is interesting and the results indicate that a fraction of HCs is open at a physiological 40 mmHg PCO₂, which differs from the situation under HEPES buffered solutions where HCs are mostly closed under resting conditions. The mechanism of HC opening with CO₂ gassing is linked to carbamylation, and the authors pinpointed several Lys residues involved in this process. 

Overall, the work is interesting as it shows that Cx43 HCs have a significant open probability under resting conditions of physiological levels of CO₂ gassing, probably applicable to the brain, heart, and other Cx43 expressing organs. The paper gives a detailed account of various experiments performed (dye uptake, electrophysiology, ATP release to assess HC function) and results concluded from those. They further consider many candidate carbamylation sites by mutating them to negatively charged Glu residues. The paper ends with hippocampal slice work showing evidence for connexin-dependent increases of the EPSP amplitude that could be inhibited by HC inhibition with Gap26 (Figure 10). Another line of evidence comes from the Cx43-linked ODDD genetic disease, whereby L90V as well as the A44V mutations of Cx43 prevented the CO₂-induced hemichannel opening response (Figure 11). Although the paper is interesting, in its present state, it suffers from (i) a problematic Figure 3, precluding interpretation of the data shown, and (ii) the poor use of hemichannel inhibitors that are necessary to strengthen the evidence in the crucial experiment of Figure 2 and others. 

The panels in Figure 3 were mislabelled in the accompanying legend possibly leading to some confusion. This has now been corrected.

We disagree that hemichannel blockers are needed to strengthen the evidence in Figure 2 and other figures. Our controls show that the CO₂-sensitive responses absolutely requires expression of Cx43 and was modified by mutations of Cx43. It is hard to see how this evidence would be strengthened by use of peptide inhibitors or other blockers of hemichannels that may not be completely selective.

Reviewing Editor Comments:

(1) Improve electrophysiological evidence, addressing concerns about the initial experiment and including peptide inhibitor data where applicable. 

We think the concerns about the electrophysiological evidence arise from a misunderstanding because we gave insufficient information about how we conducted the experiments. We have now provided a much more complete legend, added explanations in the text and given more detail in the Methods. We further respond to the reviewer below.

We do not agree on the necessity of the peptide inhibitor to demonstrate dependence on Cx43.  We have shown that parental HeLa cells do not release ATP to changes in PCO₂ or voltage (Fig 2D; Butler & Dale 2023, 10.3389/fncel.2023.1330983; Lovatt et al 2025, 10.1101/2025.03.12.642803, 10.1101/2025.01.23.634273). Our previous papers have shown many times that parental HeLa cells do not load with dye to CO₂ or zero Ca²⁺ (e.g. Huckstepp et al 2010, 10.1113/jphysiol.2010.192096; Meigh et al 2013, 10.7554/eLife.01213; Meigh et al 2014, 10.7554/eLife.04249), and we have shown that parental HeLa cells do not exhibit the same CO₂ dependent change in whole cell conductance that the Cx43-expressing cells do (Fig 2B). In addition, we shown that mutating key residues in Cx43 alters both CO₂-sensitive release of ATP and the CO₂-dependent dye loading without affecting the respective positive control. To bolster this, we have included data for the K144R mutation as a supplement to Fig 3. Given the expense of Gap26 it is impractical to include this as a standard control and unnecessary given the comprehensive controls outlined.

Collectively, these data show that the responses to CO₂ require expression of Cx43 and can be modified by mutation of Cx43.

(2) Strengthen the manuscript by measuring the effects of CO on cytosolic pH and Cx43 hemichannel opening. Consider using tail truncation mutants to assess the role of the C-terminal pH sensor in CO-mediated channel opening.

We agree and have performed the suggested experiments to address this issue.

(3) Investigate the effect of expressing the K105E/K109E Cx43 double mutant on cell viability.

In our experiments the cells look completely healthy based on their morphology in brightfield microscopy and growth rates. 

(4) Discuss and analyze the uniqueness of Cx43 among alpha connexins in maintaining the carbamylation motif.

now discuss this -Cx43 is not unique. We have added a molecular phylogenetic survey of the alpha connexin clade in Fig 12. Apart from Cx37, the carbamylation motif appears in all the other members of the clade (but not necessarily in the human orthologue). In a different MS, currently posted on bioRxiv, we have documented the CO₂ sensitivity of Cx50 and its dependence on the motif.

(5) Consider omitting data on ODDD-associated mutations unless there is evidence linking CO₂ sensitivity to disease pathology.

This experiment is observational, and we are not making claims that there is a direct causal link. Removing the ODDD mutant findings would lose potentially useful information for anyone studying how these mutations alter channel function. We have reworded the text to ensure that we say that the link between loss of CO₂ sensitivity and ODDD remains unproven.

(6) Justify the choice of high K^⁺ and low external calcium as a positive control in ATP release experiments.

These two manipulations can open the hemichannel independently of the CO₂ stimulus. Extracellular Ca²⁺ is well known to block all connexin hemichannels, and Cx43 is known to be voltage sensitive. The depolarisation from high K⁺ is effective at opening the hemichannel and we preferred this as a more physiological way of opening the Cx43 hemichannel. We have added some explanatory text.

(7) Clarify whether Cx43A44V or Cx43L90V mutations block gap junctional coupling.

This is an interesting point. Since Cx43 GJCs are not CO₂ sensitive we feel this is beyond the scope of our paper. 

(8) Discuss the potential implications of pCO₂ changes on myocardial function through alterations in intracellular pH.

We have modified the discussion to consider this point.

Reviewer #1 (Recommendations for the authors):

(1) Measurements of the effects of CO₂ on cytosolic pH/Cx43 hemichannel opening would strengthen the manuscript. Since the pH sensor of Cx43 is on the C terminus, the authors could consider making tail truncation mutants to see how this affects CO₂-mediated Cx43 channel opening.

We have done this (truncating after residue 256) -the channel remains highly CO₂ and voltage sensitive. We have also documented the effect of the  hypercapnic solutions on intracellular pH measured with BCECF. These new data are now included as figure supplements to Figure 2.

(2) What is the impact of expressing the K105E / K109E Cx43 double mutant on cell viability?

There was no obvious observed impact, cell density was as expected (no evidence of increased cell death), brightfield and fluorescence visualisation indicated normal healthy cells. We have added a movie (Fig 9, movie supplement 1) to show the effect of La³⁺ on the GRAB_ATP signal in cells expressing Cx43^{K105E, K109E} so readers can appreciate the morphology and its stability during the recording.

(3) A quick look at other alpha connexins suggested that Cx43 was unique among alpha connexins in maintaining the carbamylation motif. This merits additional discussion/ analysis.

This is an interesting point. Cx43 is not unique in the alpha clade in having the carbamylation motif as a number of other human alpha connexins also possess: Cx50, Cx59 and Cx62, and non-human alpha connexins (Cx40, Cx59, Cx46) also possess the motif. We have shown that Cx50 is CO₂ sensitive. We have performed a brief molecular phylogenetic analysis of the alpha connexon clade to highlight the occurrence of the carbamylation motif. This is now presented as Fig 12 to go with the accompanying discussion.

(4) There were some minor writing issues that should be addressed. For instance, fEPSP is not defined. Also, insets showing positive controls in some experiments were not described in the figure legends.

We have corrected these issues.

Reviewer #2 (Recommendations for the authors):

(1) I would omit the data on the ODDD-associated mutations since there is no evidence that loss of CO₂ sensitivity plays an important role in the underlying disease pathology.

We are not making the claim CO₂ loss leads to the underlying pathology and have reviewed the text to ensure that we clearly express that this is a correlation not a cause. We think this is worth retaining as many pathological mutations in other CO₂ sensitive connexins (Cx26, Cx32 and Cx50) cause loss of CO₂ sensitivity, and this information may be helpful to other researchers.

(2) Why is high K+ rather than low external calcium used as a positive control in ATP release experiments?

We used of high K⁺ and depolarisation as a positive control as regard this as a more physiological stimulus than the low external Ca²⁺.

(3) Does Cx43A44V or Cx43L90V block gap junctional coupling?

An interesting question but we have not examined this.

(4) Provide references for biophysical recordings of Cx43 hemichannels performed in HEPES-buffered salines, which document Cx43 hemichannels as being shut.

have added the original and some later references which examine Cx43 hemichannel gating in HEPES buffer and shows the need for substantial depolarisation to induce channel opening.

(5) In the heart muscle, changes in PCO₂ have long been hypothesized to cause changes in myocardial function by changing pHi.

This is true and we now add some discussion of this point. Now that we know that Cx43 is directly sensitive to CO₂ a direct action of CO₂ cannot be ruled out and careful experimentation is required to test this possibility. 

Reviewer #3 (Recommendations for the authors):

(1) Page 3: "... homologs of K125 and R104 ... ": the context is linked to Cx26, so Cx26 needs to be added here.

Done

(2) Page 4 text and related Figure 2:

(a) Figure 2A&B: PCO2-dependent Cx43 HC opening is clearly present in the carboxy-fluorescein dye uptake experiments (Figure 2A) as well as in the electrophysiological experiments (Figure 2B). The curves look quite different between these two distinct readouts: dye uptake doubles from 20 to 70 mmHg in Figure 2A while the electrophysiological data double from 45 to 70 mmHg in Figure 2B. These responses look quite distinct and may be linked to a non-linearity of the dye uptake assay or a problem in the electrophysiological measurements of Figure 2B discussed in the next point.

Different molecules/ions may have different permeabilities through the channel, which could explain the observed difference. Also, there is some contamination of the whole cell conductance change with another conductance (evident in recordings from parental HeLa cells). This is evident particularly at 70 mmHg. If this contaminating conductance were subtracted from the total conductance in the Cx43 expressing cells, then the dose response relations would be more similar. However, we are reluctant to add this additional data processing step to the paper.

(b) The traces in Figure 2B show that the HC current is inward at 20 mmHg PCO2, while it switches to an outward current at 55mmHg PCO2. HCs are non-selective channels, so their current should switch direction around 0 mV but not at -50 mV. As such, the -50 mV switching point indicates involvement of another channel distinct from non-selective Cx43 hemichannels.

We think that our incomplete description in the legend led to this misunderstanding. We used a baseline of 35 mmHg (where the channels will be slightly open) and changed to 20 mmHg to close them (or to higher PCO₂ to open them from this baseline), hence a decrease in conductance and loss of outward current for 20 mmHg. The holding potential for the recordings and voltage steps were the same in all recordings. We have now edited the legend and added more information into the methods to clarify this and how we constructed the dose response curve.

We agree that Cx43 hemichannels are relatively nonselective and would normally be expected to have a reversal potential around 0 mV, but we are using K-Gluconate and the lowered reversal potential (~-65 mV) is likely due to poor permeation of this anion via Cx43.

(c) A Hill slope of 6 is reported for this curve, which is extremely steep. The paper does not provide any further consideration, making this an isolated statement without any theoretical framework to understand the present finding in such context (i.e., in relation to the PCO2 dependency of Cx channels).

Yes, we agree -it seems to be the case with all CO₂ sensitive connexins that we have looked at that the Hill coefficient versus CO₂ is >4. Hemichannels are of course hexameric so there is potential for 6 CO₂ molecules to be bound and extensive cooperativity. We have modified the text to give greater context.

(d) A further remark to Figure 2 is that it does not contain any experiment showing the effect of Cx43 hemichannel inhibition with a reliable HC inhibitor such as Gap26, which is only used in the penultimate illustration of Figure 10. Gap26 should be used in Figure 2 and most of the other figures to show evidence of HC contribution. The lanthanum ions used in Figure 9 are a very non-specific hemichannel blocker and should be replaced by experiments with Gap26.

We have addressed the first part of this comment above.

We agree that La³⁺ blocks all hemichannels, but in the context of our experiments and the controls we have performed it is entirely adequate and supports our conclusions. Our controls show (mentioned above and below) show that the expression of Cx43 is absolutely required for CO₂-dependent ATP release (and dye loading). In Figure 9 our use of La³⁺ was to show the presence of a constitutively open Cx43 mutant hemichannel. Gap26 would add little to this. Our further controls show that with expression of Cx43^WT La³⁺ did nothing to the ATP signal under baseline conditions (20 mmHg) supporting our conclusion that the mutant channels are constitutively open.

(e) As the experiments of Figure 2 form the basis of what is to follow, the above remarks cast doubt on the robustness of the experiments and the data produced.

We disagree, our results are extremely robust: 1) we have used three independent assays confirm the presence of the response; 2) parental HeLa cells do not release ATP, dye load or show large conductance changes to CO₂ showing the absolute requirement for expression of Cx43; 3) mutations of Cx43 (in the carbamylation motif) alter the CO₂ evoked ATP release and dye loading giving further confirmation of Cx43 as the conduit for ATP release and dye loading; and 4) we use standard positive controls (0 Ca^², high K) to confirm cells still have functional channels for those mutations that modified CO₂ sensitivity.

(f) The sentence "Cells transfected with GRAB-ATP only, showed ... " should be

modified to "In contrast, cells not expressing Cx43 showed no responses to any applied CO2 concentration as concluded from GRAB-ATP experiments"

We have modified the text.

(3) Page 5 and Figures 3 & 4:

(a) Figure 3 illustrates results obtained with mutations of 4 distinct Lys residues. However, the corresponding legend indicates mutations that are different from the ones shown in the corresponding illustrations, making it impossible to reliably understand and interpret the results shown in panels A-E.

Thanks for pointing this out. Our apologies, we modified the figure so that the order of the images matched the order of the graph (and the legend) but then forgot to put the new version of the figure in the text. We have now corrected this so that Figure and legend match.

(b) Figure 4 lacks control WT traces!

The controls for this (showing that parental HeLa cells do not release ATP in response to CO₂ or depolarisation) are shown in Figure 2.

(c) Figure 4, Supplement 1: High Hill coefficients of 10 are shown here, but they are not discussed anywhere, as is also the case for the remark on p.4. A Hill steepness of 10 is huge and points to many processes potentially involved. As reported above, these data are floating around in the manuscript without any connection.

Yes, we agree this is very high and surprising. It may reflect as mentioned above the hexameric nature of the channel and that 4 Lys residues seem to be involved. We have used this equation to give some quantitative understanding of the effect of the mutations on CO₂ sensitivity and still think this is useful. We have no further evidence to interpret these values one way or the other.

(4) Page 6: Carbamate bridges are proposed to be formed between K105 and K144, and between K109 and K234. The first three of these Lysine residues are located in the 55aa long cytoplasmic loop of Cx43, while K234 is in the juxta membrane region involved in tubulin interactions. Both K144 and and K234 are involved in Cx43 HC inhibition: K144 is the last aa of the L2 peptide (D119-K144 sequence) that inhibits Cx43 hemichannels while K234 is the first aa of the TM2 peptide that reduces hemichannel presence in the membrane (sequence just after TM4, at the start of the C-tail). This context should be added to increase insight and understanding of the CO2 carbamylation effects on Cx43 hemichannel opening.

Thanks for suggesting this. We have added some discussion of CT to CL interactions in the context of regulation by pH and [Ca²⁺].

(5) Page 7: The Cx43 ODDD A44V and L90V mutations lead to loss of pCO2 sensitivity in dye loading and ATP assays. However, A44V located in EL1 is reportedly associated with Cx43 HC activation, while L90V in TM2 is associated with HC inhibition. Remarkably, these mutations are focused on non-Lys residues, which brings up the question of how to link this to the paper's main thread.

This follows the pattern that we have seen for other mutations such as A40V, A88V in Cx26 and several CMTX mutations of Cx32. Our cryoEM structures of Cx26 suggest that these mutations alter the flexibility of the molecule and hence abolish CO₂ sensitivity. We have reworded the text to avoid giving the impression that there is a demonstrated link between loss of CO₂ sensitivity of Cx43 and pathology.

(6) Page 8: HCs constitutively open - 'constutively' perhaps does not have the best connotation as it is not related to HC constitution but CO2 partial pressure.

Yes, we agree and have reworded this.

(7) Page 9: "in all subtypes" -> not clear what is meant - do you mean "in all cell types"?

We agree this is unclear -it refers to all astrocytic subtypes. We have amended the text.

(8) Page 10: Composition of hypocapnic recording solution: bubbling description is incomplete "95%O2/5%" and should be "95%O2/5%CO2".

Changed.

(9) Page 11: Composition of zero Ca^²⁺ hypocapnic recording solution: perhaps better to call this "nominally Ca^²⁺-free hypocapnic recording solution" as no Ca^²⁺ buffer is included in this solution

Thanks for pointing this out. We did in fact add 1 mM EGTA to the solutions but omitted this from the recipe, this has now been corrected.

(10) Page 11: in M&M I found that the NaHCO3- is lowered to 10 mM in the zero Ca^²⁺condition, while the control experimental condition has 26 mM NaHCO3-. The zero Ca condition should be kept at a physiologically normal 26 mM NaHCO3- concentration, so why was this done? Lowering NaHCO3- during hemichannel stimulation may result in smaller responses and introduce non-linearities.

For the dye loading we used 20 mmHg as the baseline condition and increased PCO₂ from this. Hence for the zero Ca²⁺ positive control we modified the 20 mmHg hypocapnic solution by substituting Mg²⁺ for Ca²⁺ and adding EGTA. We have modified the text in the Methods to clarify this.

Further remarks on the figures:

(1) Figure 2A: Add 20 & 70 mmHg to the images, to improve the readability of this illustration.

Done

(2) Figure 3: WT responses are shown in panel F, but experimental data (images and curves) are lacking and should be included in a revised version.

The wild type data is shown in Fig 2A. We have some sympathy for the comment, but we felt that Fig 2 should document CO₂ sensitivity, and then the subsequent Figs should analyse its basis. Hence the separation of Cx43^WT data from the mutant data. In panel F, we state that we have recalculated the WT data from Fig 2A to allow the comparison.

(3) Figures 4, 6, 8: Color codes for mmHg CO₂ pressure make reading these figures difficult; perhaps better to add mmHg values directly in relation to the traces.

We have considered this suggestion but feel that the figures would become very cluttered with the additional labelling.

(4) I wouldn't use colored lines when not necessary, e.g., Figure 9 100 µM La3+; Figure 10 (add 20->35 mmHg PCO2 switch; add scrGap26 above blue bars); Figure 11C & D.

We agree and can see that in Figs 9 and 10 this muddles our colour scheme in other figures so have modified these figures. There was not space to put the suggested labels.

(5) The mechanism of increased HC opening is not clear.

We agree and have discussed various options and the analogy with what we know about Cx26. Ultimately new cryo-EM data is required.

(6) Figure 10: 35G/35S are weird abbreviations for 35 mmHg Gap26 and scrambled Gap26.

Yes, but we used these to fit into the available space.

(7) Figure 11, legend: '20 mmHg PCO2 for each transfection for 70 mmHg PCO2'. It is not clear what is meant here.

Thanks for pointing this out, we have reworded this to ensure clarity.

Synthèse du webinaire : La place du numérique dans le projet associatif en 2025

Résumé Exécutif

Cette synthèse présente les conclusions clés de la 5ème édition du baromètre sur les pratiques numériques des associations, une étude menée conjointement par Solidatech et Recherche et Solidarité au printemps 2025 auprès de 2 285 responsables associatifs.

L'analyse révèle une progression continue de la maturité numérique du secteur, avec 26 % des associations se considérant désormais "expérimentées", soit une hausse de 5 points par rapport à 2022.

L'intelligence artificielle (IA) fait une entrée notable, utilisée par 18 % des associations (26 % pour les employeuses), principalement pour des gains d'efficacité, bien que des craintes éthiques et un manque de compétences demeurent des freins importants.

Les objectifs principaux de l'usage du numérique restent stables et prioritaires : améliorer la communication (80 %), animer le réseau (75 %) et gérer les activités (70 %). Si le nombre d'associations ne rencontrant aucune difficulté a presque doublé depuis 2019 (passant de 16 % à 29 %), les freins humains (manque de compétences, appréhensions) restent la préoccupation majeure pour 44 % des structures.

Enfin, l'étude souligne une professionnalisation croissante, avec une implication plus forte des salariés et des instances dirigeantes dans la stratégie numérique.

1. Contexte et Méthodologie de l'Étude

L'étude "La place du numérique dans le projet associatif en 2025" est la 5ème édition d'un baromètre initié en 2013. Elle est le fruit d'un partenariat historique entre Solidatech, un programme d'aide à la transformation numérique des associations, et Recherche et Solidarité, une association spécialisée dans la connaissance de la vie associative.

• Objectifs du baromètre :

◦ Suivre l'évolution des pratiques numériques dans les associations.    ◦ Fournir des enseignements utiles aux acteurs associatifs pour guider leurs démarches.    ◦ Informer les acteurs du numérique sur les réalités et spécificités du secteur associatif.    ◦ Constituer une ressource majeure pour les structures d'appui à la vie associative (CRDLA, Guid'Asso).

• Méthodologie :

◦ Échantillon : 2 285 responsables d'associations ont répondu à l'enquête.    ◦ Représentativité : Les résultats ont été redressés selon la méthode des quotas pour assurer leur représentativité par rapport au secteur associatif dans son ensemble et spécifiquement pour les associations employeuses.    ◦ Analyse : Les données sont analysées globalement et peuvent être segmentées par secteur d'activité, budget, effectif, contexte géographique (rural, urbain, QPV) et maturité numérique.

2. État des Lieux de la Maturité Numérique en 2025

Perception de la Maturité Numérique

L'étude révèle une progression constante de la maturité numérique des associations. La part des associations se déclarant "expérimentées" a gagné 5 points depuis 2022, principalement au détriment de celles se jugeant "en progrès".

Niveau de Maturité

2019

2022

2025

Peu initiée

~22%

~22%

~22%

En progrès

52%

52%

47%

Expérimentée

21%

21%

26%

Implication et Gouvernance du Numérique

L'étude montre une professionnalisation et une prise en main plus stratégique des sujets numériques au sein des associations.

• Professionnalisation : 30 % des associations employeuses confient désormais la gestion du numérique à un salarié dédié, marquant une tendance à la hausse.

• Implication des dirigeants : Le conseil d'administration ou le bureau s'implique directement sur les sujets numériques dans 24 % des associations, une proportion en augmentation continue depuis 2022, ce qui suggère une approche plus stratégique.

• Dépendance : Un référent unique (bénévole pour 24 %, salarié pour 30 %) gère souvent le numérique, créant un risque de dépendance et de perte de compétences en cas de départ.

Budgets Alloués au Numérique

La moitié des associations (50 %) dispose d'un budget dédié au numérique pour les dépenses courantes (maintenance, abonnements, hébergement).

• Investissement : 21 % des associations ont un budget d'investissement pour l'achat de matériel ou des conseils stratégiques.

• Prise de conscience : 24 % n'ont pas de budget dédié mais considèrent que ce serait une bonne idée.

• Cas spécifiques : 21 % estiment qu'un budget n'est pas utile, souvent car il s'agit de très petites structures s'appuyant sur les outils personnels des bénévoles.

3. Objectifs, Usages et Outils Numériques

Les Objectifs Prioritaires

Le "top 3" des objectifs recherchés via le numérique reste inchangé, mais les usages s'intensifient avec une progression de 5 à 7 points pour chaque item par rapport à 2022.

1. Mieux faire connaître l'association (Communication & Visibilité) : 80 %

2. Améliorer l'animation du réseau (Lien interne et externe) : 75 %

3. Gérer plus efficacement les activités : 70 %

Deux pratiques connaissent une progression particulièrement forte :

• Travailler plus efficacement ensemble : Utilisé par 57 % des associations, soit un gain de 18 points depuis 2019, une tendance accélérée par la crise sanitaire.

• Rechercher des financements / collecter des dons : Concerne 33 % des associations, en hausse de 10 points depuis 2019, reflétant le besoin de diversifier les ressources.

L'Usage des Outils Libres

43 % des associations utilisent des outils libres. Pour la première fois en 2025, les motivations éthiques dépassent les raisons pratiques.

• Pour des raisons éthiques : 23 % (transparence, partage, liberté de l'information).

• Pour des raisons pratiques : 20 %.

• Besoin d'accompagnement : 14 % n'en utilisent pas mais souhaiteraient être accompagnées.

• Ne sait pas / Ne se prononce pas : 22 % des répondants, indiquant une méconnaissance persistante de cet écosystème.

4. Focus Spécifique : L'Intelligence Artificielle (IA)

Taux d'Adoption et Potentiel

L'IA est une réalité émergente dans le secteur associatif, avec un potentiel de développement significatif.

• Taux d'utilisation actuel :

◦ 18 % pour l'ensemble des associations.    ◦ 26 % pour les associations employeuses.

• Potentiel à court terme : 13 % des associations réfléchissent à son utilisation (18 % des employeuses), portant le potentiel total à 31 % (44 % pour les employeuses).

• Comparaison : Les associations employeuses (26 %) sont légèrement en retrait par rapport aux PME et ETI, qui affichent un taux d'adoption de 32 % (source : BPI France, 2025).

Principaux Usages de l'IA

Les associations se tournent vers l'IA principalement pour optimiser leurs opérations et leur communication.

Usages de l'IA (utilisateurs actuels et potentiels)

Ensemble des associations

Associations employeuses

Gagner en efficacité dans les tâches quotidiennes (ex: comptes-rendus)

70 %

>70%

Créer des supports de communication internes ou externes (ex: images, vidéos)

59 %

>59%

Créer des documents pédagogiques adaptés aux publics

41 %

>41%

Faciliter l'analyse de données

39 %

>39%

Faciliter les réponses aux appels à projets / demandes de subvention

27 %

>27%

Appréhensions et Risques Identifiés

Malgré leur intérêt, les associations expriment de fortes appréhensions, notamment les employeuses qui, bien que plus utilisatrices, sont aussi plus conscientes des risques.

Appréhensions liées à l'IA

Ensemble des associations

Associations employeuses

Craintes éthiques (perte de lien humain, désinformation)

47 %

>47%

Manque de compétences en interne

45 %

>45%

Risques et impact environnemental

36 %

>36%

Risques liés à la confidentialité des données

36 %

>36%

Risque de déstabiliser l'organisation (disparition de fonctions, etc.)

8 %

>8%

Le faible score (8 %) du risque organisationnel suggère que les usages sont encore perçus comme ponctuels et que l'impact structurel de l'IA est sous-estimé.

5. Difficultés Rencontrées et Leviers d'Action

Évolution des Difficultés

Une nette amélioration est observée : en 2025, 29 % des responsables déclarent ne rencontrer aucune difficulté particulière, contre seulement 16 % en 2019. Pour les 71 % qui en rencontrent, la hiérarchie des freins reste stable.

1. Difficultés humaines (44 %) : Reste la préoccupation principale (lever les appréhensions, trouver les compétences, maintenir le lien).

2. Difficultés techniques (33 %) : Stables, en lien avec l'évolution rapide des technologies et les risques (cybersécurité).

3. Difficultés financières (24 %) : En forte baisse (vs. 41 % en 2019), mais ce chiffre est à nuancer car 81 % des associations financent le numérique sur fonds propres, ce qui peut créer des tensions de trésorerie.

4. Difficultés stratégiques (21 %) : Considérées comme souvent sous-estimées par les analystes de l'étude.

Témoignages d'Acteurs Associatifs (Verbatims)

• Sur le manque de temps : "Le problème [c'est] surtout de temps, des idées mais pas le temps de les mettre en place, de former et d'informer."

• Sur la dépendance : "Ancien bénévole qui maîtrise part. Le risque est de n'avoir personne pour assurer la continuité."

• Sur les financements : "Nous multiplions des comptes gratuits ou à bas coût qui ne sont pas reliés entre eux."

• Sur la cybersécurité : "Nous subissons du phishing de plus en plus évolué."

Attentes pour Progresser

Pour surmonter ces obstacles, les associations expriment plusieurs attentes :

• Meilleure connaissance des outils existants (47 %).

• Montée en compétences des équipes.

• Partage d'expériences avec d'autres associations.

• Accompagnement pour définir une stratégie numérique ou un diagnostic personnalisé (20 %).

6. Les Clés de la Réussite de la Transformation Numérique

L'étude conclut en rappelant quatre principes fondamentaux pour mener à bien un projet numérique :

1. Ne pas perdre de vue le projet associatif : Le numérique doit rester un outil au service des missions de l'association, et non une fin en soi.

2. Considérer la singularité de chaque projet : Prendre en compte les spécificités de l'association (valeurs, contraintes budgétaires, parties prenantes) pour orienter le choix des solutions et la conduite du changement.

3. Instaurer une culture numérique partagée : Fournir un bagage minimum à tous les membres pour éviter les fractures numériques internes et favoriser l'adoption collective des outils.

4. Suivre un cheminement par étape : Aborder la mise en place d'un nouvel outil comme un projet à part entière, avec une méthodologie claire (nommer un responsable, impliquer les utilisateurs, tester, former, déployer).

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Ce document est une synthèse du webinaire "La place du numérique dans le projet associatif en 2025", diffusé par Solidatech. Les données et analyses proviennent exclusivement des propos tenus par les intervenants (Lauren Gouin, Cécile Basin, Boris) durant la présentation.

Synthèse du webinaire : IA & Associations

Résumé Exécutif

Ce document synthétise les enseignements clés du webinaire "IA & Associations : une bonne idée ?", présenté par Solidatech en collaboration avec des experts de la société Advent. L'intelligence artificielle (IA), et plus particulièrement les agents conversationnels génératifs comme ChatGPT, Claude ou Mistral, représente une opportunité majeure pour les associations, leur permettant d'optimiser leur efficacité opérationnelle et leur prise de décision stratégique. Le webinaire a mis en lumière trois axes principaux : les applications pratiques concrètes (rédaction de demandes de subvention, organisation d'événements), les risques inhérents à leur utilisation (fuites de données, biais, hallucinations) et les meilleures pratiques pour formuler des requêtes efficaces ("prompt engineering"). L'approche préconisée est celle d'une adoption mesurée et stratégique, en utilisant l'IA pour des tâches répondant à la méthode des "3 C" : Chronophages, Compliquées et peu motivantes. Enfin, des organisations de soutien comme Solidatech et le programme Cyber Forgood, ainsi que des outils spécifiques, ont été présentés comme des ressources clés pour accompagner les associations dans cette transition.

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1. Contexte et Acteurs de Soutien

Le webinaire visait à démystifier l'usage de l'IA pour le secteur associatif en fournissant des clés de compréhension, des exemples pratiques et des stratégies de mitigation des risques.

Solidatech

Présenté par Lauren Guouin, Solidatech est un programme de solidarité numérique qui accompagne plus de 45 000 associations dans leur transition numérique depuis 2008. Porté par la coopérative d'insertion Les Ateliers du Bocage (mouvement Emmaüs), le programme agit sur trois fronts :

• Équipements numériques : Accès à des logiciels (Microsoft, Adobe, etc.) et du matériel informatique (neuf ou reconditionné) à tarifs solidaires.

• Montée en compétences : Mise à disposition de ressources (articles, newsletters, autodiagnostic numérique), formations certifiées Qualiopi et accompagnements personnalisés.

• Production de savoirs : Diffusion d'études, comme "La place du numérique dans le projet associatif".

Cyber Forgood

Animé par Julio de la société Advent, Cyber Forgood est un programme dédié à la protection et à l'accompagnement des acteurs de l'économie sociale et solidaire face aux cyber-risques. Une nouvelle plateforme, cyberforgood.org, sera lancée le 3 novembre et proposera dès janvier :

• Une académie en ligne de 5 mois sur l'hygiène numérique, le RGPD et l'IA.

• Un "boot camp" en présentiel à Paris pour échanger avec des experts.

• Des accompagnements pro bono en cybersécurité.

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2. Comprendre l'Intelligence Artificielle Générative

Léonard Kip, expert en cybersécurité et IA chez Advent, a défini l'IA comme un programme autonome capable d'imiter des actions humaines (prédiction, génération de contenu, prise de décision). L'explosion récente concerne l'IA générative, qui crée du contenu original à partir d'une requête.

Comment fonctionnent les agents conversationnels ? Ces outils ne "comprennent" pas une question au sens humain. Ils s'appuient sur des réseaux de neurones artificiels entraînés sur des quantités astronomiques de données. Leur fonction principale est de prédire le mot suivant le plus probable en fonction du contexte fourni par la requête de l'utilisateur. Chaque nouveau mot généré enrichit le contexte, permettant de prédire le suivant, et ainsi de suite, pour construire une réponse cohérente. Cette mécanique explique pourquoi la précision et la richesse de la requête initiale sont cruciales pour obtenir un résultat pertinent.

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3. Analyse des Risques Majeurs et Stratégies de Mitigation

L'utilisation de l'IA comporte des risques significatifs qu'il est essentiel de maîtriser. Un sondage réalisé durant le webinaire a révélé que la fuite de données confidentielles est la principale préoccupation (67 % des répondants).

Risque Identifié

Description

Stratégies de Mitigation

Hallucinations

L'IA présente des informations factuellement incorrectes mais de manière très convaincante, car elle a tendance à vouloir satisfaire l'utilisateur plutôt que d'admettre son ignorance.

- Vérifier systématiquement les réponses, surtout les plus surprenantes.<br>- Demander à l'IA de confirmer ou de détailler son raisonnement.<br>- Découper une requête complexe en plusieurs tâches plus simples.

Biais Cognitifs

L'IA reproduit les stéréotypes et préjugés présents dans ses données d'entraînement (internet, ouvrages), ce qui peut mener à des réponses discriminatoires.

- Demander explicitement à l'IA d'éviter les biais et d'être "ouverte d'esprit".<br>- Relire sa propre requête pour s'assurer qu'elle n'induit pas de biais.<br>- Demander à l'IA de corriger une réponse si un biais est identifié.

Fuite de Données Confidentielles

Les conversations peuvent être utilisées par les éditeurs pour entraîner les futures versions de leurs modèles. Des fuites massives ont déjà eu lieu (ex: 370 000 conversations de l'IA Grok).

- Ne jamais fournir d'informations sensibles (dossiers médicaux, données personnelles identifiables).<br>- Généraliser ou approximer les données (ex: "une femme dans la quarantaine" au lieu d'un âge précis).<br>- Utiliser les modes de "conversation éphémère" (disponibles sur Claude, Mistral) qui effacent les échanges.<br>- Dans les paramètres du compte, refuser l'utilisation des données pour l'amélioration de l'IA et programmer la suppression de l'historique.

Génération de Contenu Dangereux

L'IA peut être utilisée pour créer des contenus malveillants, bien que les plateformes majeures renforcent leurs garde-fous.

- Signaler tout contenu inapproprié à l'éditeur de l'outil.<br>- Pour les associations proposant des services basés sur l'IA, mettre en place des systèmes de modération.

Utilisation à des Fins Illégales

Le risque le plus médiatisé est le "deepfake" (hypertrucage) : la création de fausses vidéos, images ou audios pour usurper l'identité d'une personne, une technique devenue très accessible.

- Sensibiliser les membres et bénéficiaires aux risques légaux.<br>- Contrôler les usages si l'association met un service d'IA à disposition.

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4. L'Art de la Requête : Comment Dialoguer Efficacement avec une IA

Pour dépasser le stade de la simple question-réponse et obtenir des résultats à haute valeur ajoutée, il est nécessaire de pratiquer l'ingénierie de requête ("prompt engineering"). Une requête efficace se compose de plusieurs éléments.

La Formule d'une Requête Complète :

1. Instruction : La tâche principale à effectuer.

2. Contexte : Le "pourquoi" de la demande, le public cible, les objectifs et les enjeux. Cet élément est crucial pour guider l'IA.

3. Format : La structure de la réponse souhaitée (tableau, liste à puces, résumé, nombre de mots). Avec le contexte, c'est l'ajout qui apporte le plus de valeur.

4. Ton : Le style rédactionnel attendu (formel, créatif, empathique, etc.).

5. Rôle/Persona : Demander à l'IA d'incarner un expert (ex: "Agis en tant que spécialiste de la collecte de fonds").

6. Exemple : Fournir un ou plusieurs exemples du résultat attendu pour guider la génération.

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5. Cas d'Usage Concrets pour les Associations

Les démonstrations réalisées avec l'outil Claude illustrent le potentiel de l'IA pour des tâches complexes.

• Aide à la Rédaction de Dossiers (ex: Demande de Subvention) :

◦ Scénario : Une association de recyclage d'ordinateurs veut répondre à un appel à projet pour obtenir 500 000 €.    ◦ Méthode : La requête incluait le contexte de l'association, l'objectif et l'intégralité du texte de l'appel à projet.    ◦ Résultat : L'IA a d'abord posé des questions pour obtenir des informations complémentaires (budget, effectifs), puis a généré un plan détaillé du dossier de réponse, des arguments alignés sur les axes de l'appel à projet et une première ébauche de contenu.

• Organisation d'Événements :

◦ Scénario : L'association souhaite organiser une soirée mémorable pour ses 20 ans.    ◦ Méthode : La requête demandait 5 idées d'activités originales.    ◦ Résultat : L'IA a proposé des concepts créatifs (ex: un "mur des 10 000 histoires" de bénéficiaires). Dans un second temps, elle a aidé à élaborer un rétroplanning et des estimations budgétaires pour mettre en œuvre les idées choisies.

• Aide à la Décision Stratégique :

◦ Scénario : L'association, basée à Paris, doit choisir deux nouvelles villes pour implanter des antennes.    ◦ Méthode : La requête demandait de proposer 10 villes et de les comparer selon trois critères : efficacité contre la fracture numérique, coût d'exploitation et potentiel de recrutement de bénévoles.    ◦ Résultat : L'IA a fourni une analyse comparative chiffrée et a recommandé Marseille et Lille en justifiant ce choix par une couverture géographique Nord-Sud optimale, dépassant la simple analyse des scores individuels.

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6. Outils Recommandés et Approche Stratégique

Sélection d'Outils Pertinents

• Agents Conversationnels :

◦ Claude : Recommandé pour son alignement éthique (fondé par d'anciens d'OpenAI pour des raisons éthiques).    ◦ Mistral : Une alternative française/européenne de premier plan, privilégiée pour des enjeux de souveraineté numérique.

• Assistant de Réunion :

◦ Nuta : Solution française qui s'intègre aux outils collaboratifs pour générer des transcriptions, des comptes-rendus et des résumés de réunion.

• Création Marketing :

◦ Canva : Intègre désormais des fonctionnalités IA pour aider à la création de campagnes marketing (vigilance requise sur les questions de propriété intellectuelle).

Définir une Stratégie d'Adoption : La Méthode des "3 C"

Pour éviter un usage excessif et énergivore de l'IA, il est conseillé de l'adopter de manière ciblée. La première étape pour une association est d'identifier collectivement les tâches qui répondent aux trois critères suivants :

1. Chronophage : Une tâche qui consomme beaucoup de temps.

2. Compliquée : Une tâche qui demande une réflexion ou une expertise non triviale.

3. Peu motivante : Une tâche répétitive ou administrative qui pèse sur les équipes.

Si une tâche répond à ces trois critères, alors l'utilisation d'une IA pour l'assister ou l'automatiser est justifiée. Cette approche permet de commencer par un cas d'usage à fort impact et d'habituer progressivement les équipes.

Versions Gratuites vs. Payantes

Le passage à une version payante se justifie si l'outil est utilisé très fréquemment et que les limites de la version gratuite sont atteintes. Les versions payantes donnent généralement accès à des modèles plus performants, réduisant les risques de biais et d'hallucinations, sans toutefois les éliminer complètement.

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7. Conclusion : Vers une Utilisation Maîtrisée et Bénéfique

L'IA doit être considérée comme un assistant puissant et non comme une solution magique ou un substitut à l'expertise humaine. La clé réside dans le maintien du contrôle et de l'esprit critique sur les contenus générés. Comme le souligne Léonard Kip : "Maîtriser l'IA, c'est pour votre épanouissement, pas votre paresse." Une approche progressive, axée sur des besoins réels et menée avec une conscience aiguë des risques, permettra aux associations de tirer le meilleur parti de cette révolution technologique.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

One of the most novel things of the manuscript is the use of a relatively quick photoablation system. Could this technique be applied in other laboratories? While the revised manuscript includes more technical details as requested, the description remains difficult to follow for readers from a biology background. I recommend revising this section to improve clarity and accessibility for a broader scientific audience.

As suggested, we have adapted the paragraph related to the photoablation technique in the Material & Method section, starting line 1147. We believe it is now easier to follow.

The authors suggest that in the animal model, early 3h infection with Neisseria do not show increase in vascular permeability, contrary to their findings in the 3D in vitro model. However, they show a non-significant increase in permeability of 70 KDa Dextran in the animal xenograft early infection. As a bioengineer this seems to point that if the experiment would have been done with a lower molecular weight tracer, significant increases in permeability could have been detected. I would suggest to do this experiment that could capture early events in vascular disruption.

Comparing permeability under healthy and infected conditions using Dextran smaller than 70 kDa is challenging. Previous research (1) has shown that molecules below 70 kDa already diffuse freely in healthy tissue. Given this high baseline diffusion, we believe that no significant difference would be observed before and after N. meningitidis infection, and these experiments were not carried out. As discussed in the manuscript, bacteria-induced permeability in mice occurs at later time points, 16h post-infection, as shown previously (2). As discussed in the manuscript, this difference between the xenograft model and the chip could reflect the absence of various cell types present in the tissue parenchyma or simply vessel maturation time.

One of the great advantages of the system is the possibility of visualizing infection-related events at high resolution. The authors show the formation of actin in a honeycomb structure beneath the bacterial microcolonies. This only occurred in 65% of the microcolonies. Is this result similar to in vitro 2D endothelial cultures in static and under flow? Also, the group has shown in the past positive staining of other cytoskeletal proteins, such as ezrin, in the ERM complex. Does this also occur in the 3D system?

We imaged monolayers of endothelial cells in the flat regions of the chip (the two lateral channels) using the same microscopy conditions (i.e., Obj. 40X N.A. 1.05) that have been used to detect honeycomb structures in the 3D vessels in vitro. We showed that more than 56% of infected cells present these honeycomb structures in 2D, which is 13% less than in 3D, and is not significant due to the distributions of both populations. Thus, we conclude that under both in vitro conditions, 2D and 3D, the amount of infected cells exhibiting cortical plaques is similar. These results are in Figure 4E and S4B.

We also performed staining of ezrin in the chip and imaged both the 3D and 2D regions. Although ezrin staining was visible in 3D (Author response image 1), it was not as obvious as other markers under these infected conditions, and we did not include it in the main text. Interpretation of this result is not straightforward, as the substrate of the cells is different, and it would require further studies on the behavior of ERM proteins in these different contexts.

Author response image 1.

F-actin (red) and ezrin (yellow) staining after 3h of infection with N. meningitidis (green) in 2D (top) and 3D (bottom) vessel-on-chip models.

Recommendation to the authors:

Reviewer #1 (Recommendation to the authors):

I appreciate that the authors addressed most of my comments, of special relevance are the change of the title and references to infection-on-chip. I think that the current choice of words better acknowledges the incipient but strong bioengineering infection community. I also appreciate the inclusion of a limitation paragraph that better frames the current work and proposes future advancements.

The addition of more methodological details has improved the manuscript. Although as mentioned earlier the wording needs to be accessible for the biology community. I also appreciated the addition of the quantification of binding under the WSS gradient in the different geometries and shown in Fig 3H. However, the description of the figure and the legend is not clear. What does "vessel" mean on the graph and "normalized histograms ...(blue)" in the figure legend. Could the authors rephrase it?

In Figure 3F, we investigated whether Neisseria meningitidis exhibits preferential sites of infection. We hypothesized that, if bacteria preferentially adhered to specific regions, the local shear stress at these sites would differ from the overall distribution. To test this, we compared the shear stress at bacterial adhesion sites in the VoC (orange dots and curve) with the shear stress along the entire vascular edges (blue dots and curve). The high Spearman correlation indicates that there is no distinct shear stress value associated with bacterial adhesion. This suggests that bacteria can adhere across all regions, independently of local shear stress. To enhance clarity, the legend of Figure 3 and the related text have been rephrased in the revised manuscript (L289-314).

Line 415. Should reference to Fig S5B, not Fig 5B. Also, the titles in Supplementary Figure 4 and 5 are duplicated, and the description of the legend inf Fig S5 seems a bit off. A and B seem to be swapped.

Indeed, the reference to the right figure has been corrected. Also, the title of Figure S4 has been adapted to its contents, and the legend of Figure S5 has been corrected.

Reviewer #2 (Recommendation to the authors):

Minor comments to the authors:

Line 163 "they formed" instead of "formed".

Line 212 "two days" instead of "two day"

Line 269 a space between two words is missing.

These three comments have been addressed in the revised manuscript.

In addition, I appreciate answering the comments, especially those requiring hypothesizing about including further cells. However, when discussing which other cells could be relevant for the model (lines 631 to 632) it would be beneficial to discuss not only the role of those cells but also how could they be included in the model. I think for the reader, inclusion of further cells could be seen as a challenge or limitation, and addressing these technical points in the discussion could be helpful.

We thank Reviewer #2 for the insightful suggestion. Indeed, the method of introducing cells into the VoC depends on their type. Fibroblasts and dendritic cells, which are resident tissue cells, should be embedded in the collagen gel before polymerization and UV carving. This requires careful optimization to preserve chip integrity, as these cells exert pulling forces while migrating within the collagen matrix. In contrast, T cells and macrophages should be introduced through the vessel lumen to mimic their circulation in vivo. Pericytes can be co-seeded with endothelial cells, as they have been shown to self-organize within a few hours post-seeding. These important informations are now included in the manuscript (L577-587).

Reviewer #3 (Recommendation to the authors):

Suggestions and Recommendations

Some suggestions related to the VOC itself:

Figure 1, Fig S1, paragraph starting line 1071: More information would be helpful for the laser photoablation. For instance, is a non-standard UV laser needed? Which form of UV light is used? What is the frequency of laser pulsing? How many pulses/how long is needed to ablate the region of interest?

The photoablation process requires a focused UV-laser, with high frequency (10 kHz) to lower the carving time while providing the required intensity to degrade collagen gel. To carve a reproducible number of 30 µm-large vessels, we used a 2 µm-large laser beam at an energy of 10 mW and moved the stage (i.e., sample) at a maximum speed of 1 mm/s. This information has been added to the related paragraph starting on line 1147 of the revised manuscript.

It is difficult to understand the geometry of the VOC. In Figure 1C, is the light coloration representing open space through which medium can flow, and the dark section the collagen? On a single chip, how many vessels are cut through the collagen? It looks as if at least two are cut in Figure 1C in the righthand photo.

In Figure 1C, the light coloration is the Factin staining. The horizontal upper and lower parts are the 2D lateral channels that also contain endothelial cells, and are connected to inlets and outlets, respectively. In the middle, two vertically carved 3D vessels are shown in the confocal image.

Technically, we designed the PDMS structures to allow carving of 1 to 3 channels, maximizing the number of vessels that can be imaged while minimizing any loss of permeability at the PDMS/collagen/cells interface. This information has been added in the revised manuscript (L. 1147).

If multiple vessels are cut in the center channel between the lateral channels, how do you ensure that medium flow is even between all vessels? A single chip with multiple different vessel architectures through the center channel would be expected to have different hydrostatic resistance with different architectures, thereby causing differences in flow rates in each vessel.

To ensure a consistent flow rate regardless of the number of carved vessels, we opted to control the flow rate directly across the chip with a syringe pump. During experiments, one inlet and one outlet were closed, and a syringe pump was used. Because the carved vessels are arranged in parallel (derivation), the flow rate remains the same in each vessel. If a pressure controller had been used instead, the flow would have been distributed evenly across the different channels. This has been added to the revised manuscript in the paragraph starting on line 1210.

The figures imply that the laser ablation can be performed at depth within the collagen gel, rather than just etching the surface. If this is the case, it should be stated explicitly. If not, this needs to be clarified.

One of the main advantages of the photoablation technique is carving the collagen gel in volume, and not only etching the surface. Thanks to the 3D UV degradation, we can form the 3D architecture surrounded by the bulk collagen. This has been added to the revised manuscript, lines 154-155.

Is the in-vivo-like vessel architecture connected to the lateral channel at an oblique angle, or is the image turned to fit the entire structure? (Figure 1F and 3E). Is that why there is high shear stress at its junction with the lateral channel depicted in Figure 3E?

All structures require connection to the lateral channels to ensure media circulation and nutrient supply. The in vivo-like design must be rotated to allow the upper and lower branches of the complex structure to pass between the fixed PDMS pillars. To remain consistent with the image and the flow direction, we have kept the same orientation as in the COMSOL simulation. This leads to a locally higher shear stress at the top of the architecture. This has been added in the revised manuscript, in the paragraph starting on line 1474.

Figure S1F,G: In the legend, shapes are circles, not squares. On the graphs, what do the numbers in parentheses mean?

Indeed, the terms "squares" have been replaced by "circles" in Figure 1. (1) and (2) refer to the providers of the collagen, FujiFilm and Corning, respectively. We have added this mention in the legend in Figure S1.

Figure 3B: how do the images on the left and right differ? Each of the 4 images needs to be explained.

The four images represent the infected VoC from different viewing angles, illustrating the three-dimensional spread of infection throughout the vessel. A more detailed description has been added in the legend of Figure 3.

Figure S3C is not referenced but should be, likely before sentence starting on line 299.

Indeed, the reference to Figure S3C has been added line 301 of the revised manuscript.

Results in Figure 3 with the pilD mutant are very interesting. It is worth commenting in the Discussion about how T4P functionality in addition to the presence of T4P contributes to Nm infection, and how in the future this could be probed with pilT mutants.

We thank Reviewer #3 for this relevant insight. Following adhesion, a key functionality of Neisseria meningitidis for colony formation and enhanced infection is twitching motility. As suggested, we have added in the Discussion the idea of using a PilT mutant, which can adhere but cannot retract its pili, in the VoC model to investigate the role of motility in colonization in vitro under flow conditions (L611–623).

Which vessel design was used for the data presented in Figures 4, 5, and 6 and associated supplemental figures?

Straight channels have been mostly used in figures 4, 5, and 6. Rarely, we used the branched in vivo-like designs to observe potential similar infection patterns to in vivo, and related neutrophil activity. This has been added in the revised manuscript, lines 1435-1439.

Figure 4B-D: the images presented in Figure 4C are not representative of the averages presented in Figures 4B,D. For instance, the aggregates appear much larger and more elongated in the animal model in Figure 4C, but the animal model and VOC have the colony doubling time (implying same size) in Figure 4B, and same average aggregate elongation in Figure 4D.

The images in Figure 4C were selected to illustrate the elongation of colonies quantified in Figure 4D. The elongation angles are consistent between both images and align with the channel orientation. Representative images of colony expansion over time, corresponding to Figure 4A and 4B, are provided in Figure S4A.

Figures 4E-F: dextran does not appear to diffuse in the VOC in response to histamine in these images, yet there is a significant increase in histamine-induced permeability in Figure 4F. Dotted lines should be used to indicate vessel walls for histamine, and/or a more representative image should be selected. A control set of images should also be included for comparison.

We thank Reviewer #3 for the insightful comment. We confirm that we have carefully selected representative images for the histamine condition and adjusted them to display the same range of gray levels. The apparent increase in permeability with histamine is explained by a slight rise in background fluorescence, combined with the smaller channel size shown in Figure 4E.

Figure S4 title is a duplicate of Figure S5 and is unrelated to the content of Figure S4. Suggest rewording to mention changes in permeability induced by Nm infection in the VOC and animal model.

Indeed, the title of Figure S4 did not correspond to its content. We have, thus, changed it in the revised manuscript.

Line 489 "...our Vessel-on-Chip model has the potential to fully capture the human neutrophil response during vascular infections, in a species-matched microenvironment", is an overstatement. As presented, the VOC model only contains endothelial cells and neutrophils. Many other cell types and structures can affect neutrophil activity. Thus, it is an overstatement to claim that the model can fully capture the human neutrophil response.

We agree with the Reviewer #3, that neutrophil activity is fully recapitulated with other cell types, such as platelets, pericytes, macrophages, dendritic cells, and fibroblasts, that secrete important molecules such as cytokines, chemokines, TNF-α, and histamine. In our simplified model we were able to reconstitute the complex interaction of neutrophils with endothelial cells and with bacteria. The text was modified accordingly.

Supplemental Figure 6 - Does CD62E staining overlap with sites of Nm attachment

E-selectin staining does not systematically colocalize with Neisseria meningitidis colonies although bacterial adhesion is required. Its overall induced expression is heterogeneous across the tissue and shows heterogeneity from cell to cell as seen in vivo.

Line 475, Figure 6E- Phagocytosis of Nm is described, but it is difficult to see. An arrow should be added to make this clear. Perhaps the reference should have been to Figure 6G? Consider changing the colors in Figure 6G away from red/green to be more color-blind friendly.

Indeed, the reference to the right figure is Figure 6G, where the phagocytosis event is zoomed in. We have changed it in the text. Adapting the color of this figure 6G would imply to also change all the color codes of the manuscript, as red has been used for actin and green for Neisseria meningitidis.

Lines 621-632 - This important discussion point should be reworked. Some suggested references to cite and discuss include PMID: 7913984, 15186399, 17991045, 18640287, 19880493.

We have introduced in the discussion parts the following references as suggested (3–7), and discussed more the importance of introducting of immune cells to study immune cell-bacteria interaction and related immune response (L659-678).

Minor corrections:

•  Line 8 - suggest "photoablation-generated" instead of "photoablation-based"

•  Line 57- remove the word "either", or modify the sentence

•  Sentence on lines 162-165 needs rewording

•  Lines 204-205- "loss of vascular permeability" should read "increase in vascular permeability"

•  Line 293- "Measured" shear stress, should be "computed", since it was not directly measured (according to the Materials & Methods)

•  Line 304- "consistently" should be "consistent"

•  Fig. 3 legend, second line: replace "our" with "the VoC"

•  Line 371, change "our" to "the"

•  Line 415- Figure 5B doesn’t appear to show 2-D data. Is this in Figure S5B? Some clarification is needed. The quantification of Nm vessel association in both the VOC and the animal model should be shown in Figure 5, for direct comparison.

•  Supplementary Figure 5C: correlation coefficient with statistical significance should be calculated.

•  Figure 6 title, rephrase to "The infected VOC model"

•  Line 450, replace "important" with "statistically significant"

•  Line 459, suggest rephrasing to "bacterial pilus-mediated adhesion"

•  Line 533- grammar needs correction

•  Line 589- should be "sheds"

•  Line 1106- should be "pellet"

•  Lines 1223-1224 - is the antibody solution introduced into the inlet of the VOC for staining? Please clarify.

•  Line 1295-unclear why Figure 2B is being referenced here

All the suggested minor corrections have been taken into account in the revised manuscript.

References

(1) Gyohei Egawa, Satoshi Nakamizo, Yohei Natsuaki, Hiromi Doi, Yoshiki Miyachi, and Kenji Kabashima. Intravital analysis of vascular permeability in mice using two-photon microscopy. Scientific Reports, 3(1):1932, Jun 2013. ISSN 2045-2322. doi: 10.1038/srep01932.

(2) Valeria Manriquez, Pierre Nivoit, Tomas Urbina, Hebert Echenique-Rivera, Keira Melican, Marie-Paule Fernandez-Gerlinger, Patricia Flamant, Taliah Schmitt, Patrick Bruneval, Dorian Obino, and Guillaume Duménil. Colonization of dermal arterioles by neisseria meningitidis provides a safe haven from neutrophils. Nature Communications, 12(1):4547, Jul 2021. ISSN 2041-1723. doi: 10.1038/s41467-021-24797-z.

(3) Katherine A. Rhodes, Man Cheong Ma, María A. Rendón, and Magdalene So. Neisseria genes required for persistence identified via in vivo screening of a transposon mutant library. PLOS Pathogens, 18(5):1–30, 05 2022. doi: 10.1371/journal.ppat.1010497.

(4) Heli Uronen-Hansson, Liana Steeghs, Jennifer Allen, Garth L. J. Dixon, Mohamed Osman, Peter Van Der Ley, Simon Y. C. Wong, Robin Callard, and Nigel Klein. Human dendritic cell activation by neisseria meningitidis: phagocytosis depends on expression of lipooligosaccharide (los) by the bacteria and is required for optimal cytokine production. Cellular Microbiology, 6(7):625–637, 2004. doi: https://doi.org/10.1111/j.1462-5822.2004.00387.x.

(5) M. C. Jacobsen, P. J. Dusart, K. Kotowicz, M. Bajaj-Elliott, S. L. Hart, N. J. Klein, and G. L. Dixon. A critical role for atf2 transcription factor in the regulation of e-selectin expression in response to non-endotoxin components of neisseria meningitidis. Cellular Microbiology, 18(1):66–79, 2016. doi: https://doi.org/10.1111/cmi.12483.

(6) Andrea Villwock, Corinna Schmitt, Stephanie Schielke, Matthias Frosch, and Oliver Kurzai. Recognition via the class a scavenger receptor modulates cytokine secretion by human dendritic cells after contact with neisseria meningitidis. Microbes and Infection, 10(10):1158–1165, 2008. ISSN 1286-4579. doi: https://doi.org/10.1016/j.micinf.2008.06.009.

(7) Audrey Varin, Subhankar Mukhopadhyay, Georges Herbein, and Siamon Gordon. Alternative activation of macrophages by il-4 impairs phagocytosis of pathogens but potentiates microbial-induced signalling and cytokine secretion. Blood, 115(2):353–362, Jan 2010. ISSN 0006-4971. doi: 10.1182/blood-2009-08-236711.

① : Bristles should be 0.2 mm in diameter, 10 mm in length and have rounded tips. ① : Kıllar 0,2 mm çapında, 10 mm uzunluğunda ve yuvarlatılmış uçlu olmalıdır.

② : Handle of the brush should be straight and long enough for the palm to grasp. ② : Fırçanın sapı düz ve avucun kavrayabileceği kadar uzun olmalıdır.

③ : There should be 3-4 rows, each consisting of 5-12 bristle clusters. ③ : Her biri 5-12 kıl demetinden oluşan 3-4 sıra bulunmalıdır.

secrets.choice([1, 2, 3, 4, 5]) を実行すればintが返ってくるはずなので、ここはシーケンスの要素の型が返ってくるのだと思います。

Author response:

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

Reviewer #1 (Public review):

The manuscript by Choi and colleagues investigates the impact of variation in cortical geometry and growth on cortical surface morphology. Specifically, the study uses physical gel models and computational models to evaluate the impact of varying specific features/parameters of the cortical surface. The study makes use of this approach to address the topic of malformations of cortical development and finds that cortical thickness and cortical expansion rate are the drivers of differences in morphogenesis.

The study is composed of two main sections. First, the authors validate numerical simulation and gel model approaches against real cortical postnatal development in the ferret. Next, the study turns to modelling malformations in cortical development using modified tangential growth rate and cortical thickness parameters in numerical simulations. The findings investigate three genetically linked cortical malformations observed in the human brain to demonstrate the impact of the two physical parameters on folding in the ferret brain.

This is a tightly presented study that demonstrates a key insight into cortical morphogenesis and the impact of deviations from normal development. The dual physical and computational modeling approach offers the potential for unique insights into mechanisms driving malformations. This study establishes a strong foundation for further work directly probing the development of cortical folding in the ferret brain. One weakness of the current study is that the interpretation of the results in the context of human cortical development is at present indirect, as the modelling results are solely derived from the ferret. However, these modelling approaches demonstrate proof of concept for investigating related alterations more directly in future work through similar approaches to models of the human cerebral cortex.

We thank the reviewer for the very positive comments. While the current gel and organismal experiments focus on the ferret only, we want to emphasize that our analysis does consider previous observations of human brains and morphologies therein (Tallinen et al., Proc. Natl. Acad. Sci. 2014; Tallinen et al., Nat. Phys. 2016), which we compare and explain. This allows us to analyze the implications of our study broadly to understand the explanations of cortical malformations in humans using the ferret to motivate our study. Further analysis of normal human brain growth using computational and physical gel models can be found in our companion paper (Yin et al., 2025), now also published to eLife: S. Yin, C. Liu, G. P. T. Choi, Y. Jung, K. Heuer, R. Toro, L. Mahadevan, Morphogenesis and morphometry of brain folding patterns across species. eLife, 14, RP107138, 2025. doi:10.7554/eLife.107138

In future work, we plan to obtain malformed human cortical surface data, which would allow us to further investigate related alterations more directly. We have added a remark on this in the revised manuscript (please see page 8–9).

Reviewer 2 (Public review):

Summary:

Based on MRI data of the ferret (a gyrencephalic non-primate animal, in whom folding happens postnatally), the authors create in vitro physical gel models and in silico numerical simulations of typical cortical gyrification. They then use genetic manipulations of animal models to demonstrate that cortical thickness and expansion rate are primary drivers of atypical morphogenesis. These observations are then used to explain cortical malformations in humans.

Strengths:

The paper is very interesting and original, and combines physical gel experiments, numerical simulations, as well as observations in MCD. The figures are informative, and the results appear to have good overall face validity.

We thank the reviewer for the very positive comments.

Weaknesses:

On the other hand, I perceived some lack of quantitative analyses in the different experiments, and currently, there seems to be rather a visual/qualitative interpretation of the different processes and their similarities/differences. Ideally, the authors also quantify local/pointwise surface expansion in the physical and simulation experiments, to more directly compare these processes. Time courses of eg, cortical curvature changes, could also be plotted and compared for those experiments. I had a similar impression about the comparisons between simulation results and human MRI data. Again, face validity appears high, but the comparison appeared mainly qualitative.

We thank the reviewer for the comments. Besides the visual and qualitative comparisons between the models, we would like to point out that we have included the quantification of the shape difference between the real and simulated ferret brain models via spherical parameterization and the curvature-based shape index as detailed in main text Fig. 4 and SI Section 3. We have also utilized spherical harmonics representations for the comparison between the real and simulated ferret brains at different maximum order N. In our revision, we have included more calculations for the comparison between the real and simulated ferret brains at more time points in the SI (please see SI page 6). As for the comparison between the malformation simulation results and human MRI data in the current work, since the human MRI data are two-dimensional while our computational models are threedimensional, we focus on the qualitative comparison between them. In future work, we plan to obtain malformed human cortical surface data, from which we can then perform the parameterization-based and curvature-based shape analysis for a more quantitative assessment.

I felt that MCDs could have been better contextualized in the introduction.

We thank the reviewer for the comment. In our revision, we have revised the description of MCDs in the introduction (please see page 2).

Reviewer #1 (Recommendations for the authors):

The study is beautifully presented and offers an excellent complement to the work presented by Yin et al. In its current form, the malformation portion of the study appears predominantly reliant on the numerical simulations rather than the gel model. It might be helpful, therefore, to further incorporate the results presented in Figure S5 into the main text, as this seems to be a clear application of the physical gel model to modelling malformations. Any additional use of the gel models in the malformation portion of the study would help to further justify the necessity and complementarity of the dual methodological approaches.

We thank the reviewer for the suggestion. We have moved Fig. S5 and the associated description to the main text in the revised manuscript (please see the newly added Figure 5 on page 6 and the description on page 5–7). In particular, we have included a new section on the physical gel and computational models for ferret cortical malformations right before the section on the neurology of ferret and human cortical malformations.

One additional consideration is that the analyses in the current study focus entirely on the ferret cortex. Given the emphasis in the title on the human brain, it may be worthwhile to either consider adding additional modelling of the human cortex or to consider modifying the title to more accurately align with the focus of the methods/results.

We thank the reviewer for the suggestion. While the current gel and organismal experiments focus on the ferret only, we want to emphasize that our analysis does consider previous observations of human brains and morphologies therein (Tallinen et al., Proc. Natl. Acad. Sci. 2014; Tallinen et al., Nat. Phys. 2016), which we compare and explain. This allows us to analyze the implications of our study broadly to understand the explanations of cortical malformations in humans using the ferret to motivate our study. Therefore, we think that the title of the paper seems reasonable. To further highlight the connection between the ferret brain simulations and human brain growth, we have included an additional comparison between human brain surface reconstructions adapted from a prior study and the ferret simulation results in the SI (please see SI Section S4 and SI Fig. S5 on page 9–10).

Two additional minor points:

Table S1 seems sufficiently critical to the motivation for the study and organization of the results section to justify inclusion in the main text. Of course, I would leave any such minor changes to the discretion of the authors.

We thank the reviewer for the suggestion. We have moved Table S1 and the associated description to the main text in the revised manuscript (please see Table 1 on page 7).

Page 7, Column 1: “macacques” → “macaques”.

We thank the reviewer for pointing out the typo. We have fixed it in the revised manuscript (please see page 8).

Reviewer #2 (Recommendations for the authors):

The methods lack details on the human MRI data and patients.

We thank the reviewer for the comment. Note that the human MRI data and patients were from prior works (Smith et al., Neuron 2018; Johnson et al., Nature 2018; Akula et al., Proc. Natl. Acad. Sci. 2023) and were used for the discussion on cortical malformations in Fig. 6. In the revision, we have included a new subsection in the Methods section and provided more details and references of the MRI data and patients (please see page 9–10).

Reviewer #1 (Public review):

The authors investigated tactile spatial perception on the breast using discrimination, categorization, and direct localization tasks. They reach four main conclusions:

(1) The breast has poor tactile spatial resolution.

This conclusion is based on comparing just noticeable differences, a marker of tactile spatial resolution, across four body regions, two on the breast. The data compellingly support the conclusion; the study outshines other studies on tactile spatial resolution that tend to use problematic measures of tactile resolution, such as two-point-discrimination thresholds. The result will interest researchers in the field and possibly in other fields due to the intriguing tension between the finding and the sexually arousing function of touching the breast.

The manuscript incorrectly describes the result as poor spatial acuity. Acuity measures the average absolute error, and acuity is good when response biases are absent. Precision relates to the error variance. It is common to see high precision with low acuity or vice versa. Just noticeable differences assess precision or spatial resolution, while points of subjective equality evaluate acuity or bias. Similar confusions between these terms appear throughout the manuscript.<br /> A paragraph within the next section seems to follow up on this insight by examining the across-participant consistency of the differences in tactile spatial resolution between body parts. To this aim, pairwise rank correlations between body sites are conducted. This analysis raises red flags from a statistical point of view. 1) An ANOVA and its follow-up tests assume no variation in the size of the tested effect but varying base values across participants. Thus, if significant differences between conditions are confirmed by the original statistical analysis, most participants will have better spatial resolution in one condition than the other condition, and the difference between body sites will be similar across participants. 2) Correlations are power-hungry, and non-parametric tests are power-hungry. Thus, the number of participants needed for a reliable rank correlation analysis far exceeds that of the study. In sum, a correlation should emerge between body sites associated with significantly different tactile JNDs; however, these correlations might only be significant for body sites with pronounced differences due to the sample size.

(2) Larger breasts are associated with lower tactile spatial resolution

This conclusion is based on a strong correlation between participants' JNDs and the size of their breasts. The depicted correlation convincingly supports the conclusion. The sample size is below that recommended for correlations based on power analyses, but simulations show that spurious correlations of the reported size are extremely unlikely at N=18. Moreover, visual inspection rules out that outliers drive these correlations. Thus, they are convincing. This result is of interest to the field, as it aligns with the hypothesis that nerve fibers are more sparsely distributed across larger body parts.

(3) The nipple is a unit

The data do not support this conclusion. The conclusion that the nipple is perceived as a unit is based on poor tactile localization performance for touches on the nipple compared to the areola. The problem is that the localization task is a quadrant identification task with the center being at the nipple. Quadrants for the areola could be significantly larger due to the relative size of the areola and the nipple; the results section seems to suggest this was accounted for when placing the tactile stimuli within the quadrants, but the methods section suggests otherwise. Additionally, the areola has an advantage because of its distance from the nipple, which leads to larger Euclidean distances between the centers of the quadrants than for the nipple. Thus, participants should do better for the areola than for the nipple even if both sites have the same tactile resolution.

To justify the conclusion that the nipple is a unit, additional data would be required. 1) One could compare psychometric curves with the nipple as the center and psychometric curves with a nearby point on the areola as the center. 2) Performance in the quadrant task could be compared for the nipple and an equally sized portion of the areola and tactile locations that have the same distance to the border between quadrants in skin coordinates. 3) Tactile resolution could be directly measured for both body sites using a tactile orientation task with either a two-dot probe or a haptic grating.

Categorization accuracy in each area was tested against chance using a Monte Carlo test, which is fine, though the calculation of the test statistic, Z, should be reported in the Methods section, as there are several options. Localization accuracies are then compared between areas using a paired t-test. It is a bit confusing that once a distribution-approximating test is used, and once a test that assumes Gaussian distributions when the data is Bernoulli/Binomial distributed. Sampling-based and t-tests are very robust, so these surprising choices should have hardly any effect on the results.

A correlation based on N=4 participants is dangerously underpowered. A quick simulation shows that correlation coefficients of randomly sampled numbers are uniformly distributed at such a low sample size. This likely spurious correlation is not analyzed, but quite prominently featured in a figure and discussed in the text, which is worrisome.

(4) Localization of tactile events on the breast is biased towards the nipple

The conclusion that tactile percepts are drawn toward the nipple is based on localization biases for tactile stimuli on the breast compared to the back. Unfortunately, the way participants reported the tactile locations introduces a major confound. Participants indicated the perceived locations of the tactile stimulus on 3D models of these body parts. The nipple is a highly distinctive and cognitively represented landmark, far more so than the scapula, making it very likely that responses were biased toward the nipple regardless of the actual percepts. One imperfect but better alternative would have been to ask participants to identify locations on a neutral grey patch and help them relate this patch to their skin by repeatedly tracing its outline on the skin.

Participants also saw their localization responses for the previously touched locations. This is unlikely to induce bias towards the nipple, but it renders any estimate of the size and variance of the errors unreliable. Participants will always make sure that the marked locations are sufficiently distant from each other.

The statistical analysis is again a homebrew solution and hard to follow. It remains unclear why standard and straightforward measures of bias, such as regressing reported against actual locations, were not used.

Null-hypothesis significance testing only lets scientists either reject the null hypothesis or not. The latter does NOT mean the Null hypothesis is true, i.e., it can never be concluded that there is no effect. This rule applies to every NHST test. However, it raises particular concerns with distribution tests. The only conclusion possible is that the data are unlikely from a population with the tested distribution; these tests do not provide insight into the actual distribution of the data, regardless of whether the result is significant or not.

Author response:

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

Reviewer #1 (Public review):

The statistically adequate way of testing the biases is a hierarchical regression model (LMM) with a distance of the physical location from the nipple as a predictor, and a distance of the reported location from the nipple as a dependent variable. Either variable can be unsigned or signed for greater power, for example, coding the lateral breast as negative and the medial breast as positive. The bias will show in regression coefficients smaller than 1.

Thank you for this suggestion. We have subsequently replaced the relevant ANOVA analyses with LMM analyses. Specifically, we use an LMM for breast and back separately to show the different effects of distance, then use a combined LMM to compare the interaction. Finally, we use an LMM to assess the differences between precision and bias on the back and breast. The new analysis confirms earlier statements and do not change the results/interpretation of the data.

Moreover, any bias towards the nipple could simply be another instance of regression to the mean of the stimulus distribution, given that the tested locations were centered on the nipple. This confound can only be experimentally solved by shifting the distribution of the tested locations. Finally, given that participants indicated the locations on a 3D model of the body part, further experimentation would be required to determine whether there is a perceptual bias towards the nipple or whether the authors merely find a response bias.

A localization bias toward the nipple in this context does not show that the nipple is the anchor of the breast's tactile coordinate system. The result might simply be an instance of regression to the mean of the stimulus distribution (also known as experimental prior). To convincingly show localization biases towards the nipple, the tested locations should be centered at another location on the breast.

Another problem is the visual salience of the nipple, even though Blender models were uniformly grey. With this type of direct localization, it is very difficult to distinguish perceptual from response biases even if the regression to the mean problem is solved. There are two solutions to this problem: 1) Varying the uncertainty of the tactile spatial information, for example, by using a pen that exerts lighter pressure. A perceptual bias should be stronger for more uncertain sensory information; a response bias should be the same across conditions. 2) Measure bias with a 2IFC procedure by taking advantage of the fact that sensory information is noisier if the test is presented before the standard.

We believe that the fact that we explicitly tested two locations with equally distributed test locations, both of which had landmarks, makes this unlikely. Indeed, testing on the back is exactly what the reviewer suggests. It would also be impossible to test this “on another location on the breast” as we are sampling across the whole breast. Moreover, as markers persisted on the model within each block, the participants were generating additional landmarks on each trial. Thus, if there were any regression to the mean, this would be observed for both locations. Nevertheless, we recognize that this test cannot distinguish between a sensory bias towards the nipple and consistent response bias that is always in the direction of the nipple, though to what extent these are the same thing is difficult to disentangle. That said, if we had restricted testing to half of the breast such that the distribution of points was asymmetrical this would allow us to test the hypothesis put forward by the reviewer. We recognize that this is a limitation of the data and have downplayed statements and added caveats accordingly.

We have changed the appropriate heading and text in the discussion to downplay the finding:

“Reports are biased towards the nipple”

“suggesting that the nipple plays a pivotal role in the mental representation of the breast.”

it might be harder to learn the range of locations on the back given that stimulation is not restricted to an anatomically defined region as it is the case for the breast.

We apologize for any confusion but the point distribution is identical between tasks, as described in the methods.

The stability of the JND differences between body parts across subjects is already captured in the analysis of the JNDs; the ANOVA and the post-hoc testing would not be significant if the order were not relatively stable across participants. Thus, it is unclear why this is being evaluated again with reduced power due to improper statistics.

We apologize for any confusion here. Only one ANOVA with post-hoc testing was performed on the data. The second parenthetical describing the test was perhaps redundant and confusing, so I have removed it.

“(Error! Reference source not found.A, B, 1-way ANOVA with Tukey’s HSD post-hoc t-test: p = 0.0284)”

The null hypothesis of an ANOVA is that at least one of the mean values is different from the others; adding participants as a factor does not provide evidence for similarity.

We agree with this statement and have removed the appropriate text.

The pairwise correlations between body parts seem to be exploratory in nature. Like all exploratory analyses, the question arises of how much potential extra insights outweigh the risk of false positives. It would be hard to generate data with significant differences between several conditions and not find any correlations between pairs of conditions. Thus, the a priori chance of finding a significant correlation is much higher than what a correction accounts for.

We broadly agree with this statement. However, we believe that the analyses were important to determine if participants were systematically more or less acute across body parts. Moreover, both the fact that we actually did not observe any other significant relationships and that we performed post-hoc correction imply that no false positives were observed. Indeed, in the one relationship that was observed, we would need to have an assumed FDR over 10x higher than the existing post hoc correction required implying a true relationship.

If the JND at mid breast (measured with locations centered at the nipple) is roughly the same size as the nipple, it is not surprising that participants have difficulty with the categorical localization task on the nipple but perform better than chance on the significantly larger areola.

We agree that it is not surprising given the previously shown data, however, the initial finding is surprising to many and this experiment serves to reinforce the previous finding.

Neither signed nor absolute localization error can be compared to the results of the previous experiments. The JND should be roughly proportional to the variance of the errors.

We apologize for any confusion, however we are not comparing the values, merely observing that the results are consistent.

Reviewer #2 (Public review):

I had a hard time understanding some parts of the report. What is meant by "broadly no relationship" in line 137?

We have removed the qualifier to simplify the text.

It is suggested that spatial expansion (which is correlated with body part size) is related between medial breast and hand - is this to say that women with large hands have large medial breast size? Nipple size was measured, but hand size was not measured, is this correct?

Correct. We have added text to state as such.

It is furthermore unclear how the authors differentiate medial breast and NAC. The sentence in lines 140-141 seems to imply the two terms are considered the same, as a conclusion about NAC is drawn from a result about the medial breast. This requires clarification.

Thank you for catching this, we have corrected it in the text.

Finally, given that the authors suspect that overall localization ability (or attention) may be overshadowed by a size effect, would not an analysis be adequate that integrates both, e.g. a regression with multiple predictors?

If the reviewer means that participants would be consistently “acute” then we believe that SF1 would have stronger correlations. Consequently, we see no reason to add “overall tactile acuity” as a predictor.

In the paragraph about testing quadrants of the nipple, it is stated that only 3 of 10 participants barely outperformed chance with a p < 0.01. It is unclear how a significant ttest is an indication of "barely above chance".

We have adjusted the text to clarify our meaning.

“On the nipple, however, participants were consistently worse at locating stimuli on the nipple than the breast (paired t-test, t = 3.42, p < 0.01) where only 3 of the 10 participants outperformed chance, though the group as a whole outperformed chance (Error! Reference source not found.B, 36% ± 13%; Z = 5.5, p < 0.01).”

The final part of the paragraph on nipple quadrants (starting line 176) explains that there was a trend (4 of 10 participants) for lower tactile acuity being related to the inability to differentiate quadrants. It seems to me that such a result would not be expected: The stated hypothesis is that all participants have the same number of tactile sensors in their nipple and areola, independent of NAC size. In this section, participants determine the quadrant of a single touch. Theoretically, all participants should be equally able to perform this task, because they all have the same number of receptors in each quadrant of nipple and areola. Thus, the result in Figure 2C is curious.

We agree that this result seemingly contradicts observations from the previous experiment, however we believe that it relates to the distinction between the ability to perform relative distinctions and absolute localizations. In the first experiment, the presentation of two sequential points provides an implicit reference whereas in the quadrant task there is no reference. With the results of the third experiment in mind, biases towards the nipple would effectively reduce the ability of participants to identify the quadrant. What this result may imply is that the degree of bias is greater for women with greater expansion. We have added text to the discussion to lay this out.

“This negative trend implicitly contradicts the previous result where one might expect equal performance regardless of size as the location of the stimuli was scaled to the size of the nipple and areola. However, given the absence of a reference point, systematic biases are more likely to occur and thus may reflect a relationship between localization bias and breast size.”

This section reports an Anova (line 193/194) with a factor "participant". This doesn't appear sensible. Please clarify. The factor distance is also unclear; is this a categorical or a continuous variable? Line 400 implies a 6-level factor, but Anovas and their factors, respectively, are not described in methods (nor are any of the other statistical approaches).

We believe this comment has been addressed above with our replacement of the ANOVA with an LMM. We have also added descriptions of the analysis throughout the methods.

The analysis on imprecision using mean pairwise error (line 199) is unclear: does pairwise refer to x/y or to touch vs. center of the nipple?

We have clarified this to now read:

“To measure the imprecision, we computed the mean pairwise distance between each of the reported locations for a given stimulus location and the mean reported location.”

p8, upper text, what is meant by "relative over-representation of the depth axis"? Does this refer to the breast having depth but the equivalent area on the back not having depth? What are the horizontal planes (probably meant to be singular?) - do you simply mean that depth was ignored for the calculation of errors? This seems to be implied in Figure 3AB.

This is indeed what we meant. We have attempted to clarify in the text.

“Importantly, given the relative over-representation of the depth axis for the breast, we only considered angles in the horizontal planes such that the shape of the breast did not influence the results.” Became:

“Importantly, because the back is a relatively flat surface in comparison to the breast, errors were only computed in the horizontal plane and depth was excluded when computing the angular error.”

Lines 232-241, I cannot follow the conclusions drawn here. First, it is not clear to a reader what the aim of the presented analyses is: what are you looking for when you analyze the vectors? Second, "vector strength" should be briefly explained in the main text. Third, it is not clear how the final conclusion is drawn. If there is a bias of all locations towards the nipple, then a point closer to the nipple cannot exhibit a large bias, because the nipple is close-by. Therefore, one would expect that points close to the nipple exhibit smaller errors, but this would not imply higher acuity - just less space for localizing anything. The higher acuity conclusion is at odds with the remaining results, isn't it: acuity is low on the outer breast, but even lower at the NAC, so why would it be high in between the two?

Thank you for pointing out the circular logic. We have replaced this sentence with a more accurate statement.

“Given these findings, we conclude that the breast has lower tactile acuity than the hand and is instead comparable to the back. Moreover, localization of tactile events to both the back and breast are inaccurate but localizations to the breast are consistently biased towards the nipple.”

The discussion makes some concrete suggestions for sensors in implants (line 283). It is not clear how the stated numbers were computed. Also, why should 4 sensors nipple quadrants receive individual sensors if the result here was that participants cannot distinguish these quadrants?

Thank you for catching this, it should have been 4 sensors for the NAC, not just the nipple. We have fixed this in the text.

I would find it interesting to know whether participants with small breast measurement delta had breast acuity comparable to the back. Alternatively, it would be interesting to know whether breast and back acuity are comparable in men. Such a result would imply that the torso has uniform acuity overall, but any spatial extension of the breast is unaccounted for. The lowest single participant data points in Figure 1B appear similar, which might support this idea.

We agree that this is an interesting question and as you point out, the data does indicate that in cases of minimal expansion acuity may be constant on the torso. However, in the comparison of the JNDs, post-hoc testing revealed no significant difference between the back and either breast region. Consequently, subsampling the group would result in the same result. We have added a sentence to the discussion stating this.

“Consequently, the acuity of the breast is likely determined initially by torso acuity and then any expansion.”

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Reviewer #1 (Public review):

General assessment of the work:

In this manuscript, Mohr and Kelly show that the C1 component of the human VEP is correlated with binary choices in a contrast discrimination task, even when the stimulus is kept constant and confounding variables are considered in the analysis. They interpret this as evidence for the role V1 plays during perceptual decision formation. Choice-related signals in single sensory cells are enlightening because they speak to the spatial (and temporal) scale of the brain computations underlying perceptual decision-making. However, similar signals in aggregate measures of neural activity offer a less direct window and thus less insight into these computations. For example, although I am not a VEP specialist, it seems doubtful that the measurements are exclusively picking up (an unbiased selection of) V1 spikes. Moreover, although this is not widely known, there is in fact a long history to this line of work. In 1972, Campbell and Kulikowski ("The Visual Evoked Potential as a function of contrast of a grating pattern" - Journal of Physiology) already showed a similar effect in a contrast detection task (this finding inspired the original Choice Probability analyses in the monkey physiology studies conducted in the early 1990's). Finally, it is not clear to me that there is an interesting alternative hypothesis that is somehow ruled out by these results. Should we really consider that simple visual signals such as spatial contrast are *not* mediated by V1? This seems to fly in the face of well-established anatomy and function of visual circuits. Or should we be open to the idea that VEP measurements are almost completely divorced from task-relevant neural signals? Why would this be an interesting technique then? In sum, while this work reports results in line with several single-cell and VEP studies and perhaps is technically superior in its domain, I find it hard to see how these findings would meaningfully impact our thinking about the neural and computational basis of spatial contrast discrimination.

Summary of substantive concerns:

(1) The study of choice probability in V1 cells is more extensive than portrayed in the paper's introduction. In recent years, choice-related activity in V1 has also been studied by Nienborg & Cumming (2014), Goris et al (2017), Jasper et al (2019), Lange et al (2023), and Boundy-Singer et al (2025). These studies paint a complex picture (a mixture of positive, absent, and negative results), but should be mentioned in the paper's introduction.

(2) The very first study to conduct an analysis of stimulus-conditioned neural activity during a perceptual decision-making task was, in fact, a VEP study: Campbell and Kulikowski (1972). This study never gained the fame it perhaps deserves. But it would be appropriate to weave it into the introduction and motivation of this paper.

(3) What are interesting alternative hypotheses to be considered here? I don't understand the (somewhat implicit) suggestion here that contrast representations late in the system can somehow be divorced from early representations. If they were, they would not be correlated with stimulus contrast.

(4) I find the arguments about the timing of the VEP signals somewhat complex and not very compelling, to be honest. It might help if you added a simulation of a process model that illustrated the temporal flow of the neural computations involved in the task. When are sensory signals manifested in V1 activity informing the decision-making process, in your view? And how is your measure of neural activity related to this latent variable? Can you show in a simulation that the combination of this process and linking hypothesis gives rise to inverted U-shaped relationships, as is the case for your data?

Reviewer #2 (Public review):

Summary:

Mohr and Kelly report a high-density EEG study in healthy human volunteers in which they test whether correlations between neural activity in the primary visual cortex and choice behavior can be measured non-invasively. Participants performed a contrast discrimination task on large arrays of Gabor gratings presented in the upper left and lower right quadrants of the visual field. The results indicate that single-trial amplitudes of C1, the earliest cortical component of the visual evoked potential in humans, predict forced-choice behavior over and beyond other behavioral and electrophysiological choice-related signals. These results constitute an important advance for our understanding of the nature and flexibility of early visual processing.

Strengths:

(1) The findings suggest a previously unsuspected role for aggregate early visual cortex activity in shaping behavioral choices.

(2) The authors extend well-established methods for assessing covariation between neural signals and behavioral output to non-invasive EEG recordings.

(3) The effects of initial afferent information in the primary visual cortex on choice behavior are carefully assessed by accounting for a wide range of potential behavioral and electrophysiological confounds.

(4) Caveats and limitations are transparently addressed and discussed.

Weaknesses:

(1) It is not clear whether integration of contrast information across relatively large arrays is a good test case for decision-related information in C1. The authors raise this issue in the Discussion, and I agree that it is all the more striking that they do find C1 choice probability. Nevertheless, I think the choice of task and stimuli should be explained in more detail.

(2) In a similar vein, while C1 has canonical topographical properties at the grand-average level, these may differ substantially depending on individual anatomy (which the authors did not assess). This means that task-relevant information will be represented to different degrees in individuals' single-trial data. My guess is that this confound was mitigated precisely by choosing relatively extended stimulus arrays. But given the authors' impressive track record on C1 mapping and modeling, I was surprised that the underlying rationale is only roughly outlined. For example, given the topographies shown and the electrode selection procedure employed, I assume that the differences between upper and lower targets are mainly driven by stimulus arms on the main diagonal. Did the authors run pilot experiments with more restricted stimulus arrays? I do not mean to imply that such additional information needs to be detailed in the main article, but it would be worth mentioning.

(3) Also, the stimulus arrangement disregards known differences in conduction velocity between the upper and lower visual fields. While no such differences are evident from the maximal-electrode averages shown in Figure 1B, it is difficult to assess this issue without single-stimulus VEPs and/or a dedicated latency analysis. The authors touch upon this issue when discussing potential pre-C1 signals emanating from the magnocellular pathway.

(4) I suspect that most of these issues are at least partly related to a lack of clarity regarding levels of description: the authors often refer to 'information' contained in C1 or, apparently interchangeably, to 'visual representations' before, during, or following C1. However, if I understand correctly, the signal predicting (or predicted by) behavioral choice is much cruder than what an RSA-primed readership may expect, and also cruder than the other choice-predictive signals entered as control variables: namely, a univariate difference score on single-trial data integrated over a 10 ms window determined on the basis of grand-averaged data. I think it is worth clarifying and emphasizing the nature of this signal as the difference of aggregate contrast responses that *can* only be read out at higher levels of the visual system due to the limited extent of horizontal connectivity in V1. I do not think that this diminishes the importance of the findings - if anything, it makes them more remarkable.

(5) Arguably even more remarkable is the finding that C1 amplitudes themselves appear to be influenced by choice history. The authors address this issue in the Discussion; however, I'm afraid I could not follow their argument regarding preparatory (and differential?) weighting of read-outs across the visual hierarchy. I believe this point is worth developing further, as it bears on the issue of whether C1 modulations are present and ecologically relevant when looking (before and) beyond stimulus-locked averages.

Reviewer #3 (Public review):

Summary:

Fengwen Huang et al. used multiple neuroscience techniques (transgenetic mouse, immunochemistry, bulk calcium recording, neural sensor, hippocampal-dependent task, optogenetics, chemogenetics, and interfer RNA technique) to elucidate the role of the excitatory cholecystokinin-positive pyramidal neurons in the hippocampus in regulating the hippocampal functions, including navigation and neuroplasticity.

Strengths:

(1) The authors provided the distribution profiles of excitatory cholecystokinin in the dorsal hippocampus via the transgenetic mice (Ai14::CCK Cre mice), immunochemistry, and retrograde AAV.

(2) The authors used the neural sensor and light stimulation to monitor the CCK release from the CA3 area, indicating that CCK can be secreted by activation of the excitatory CCK neurons.

(3) The authors showed that the activity of the excitatory CCK neurons in CA3 is necessary for navigation learning.

(4) The authors demonstrated that inhibition of the excitatory CCK neurons and knockdown of the CCK gene expression in CA3 impaired the navigation learning and the neuroplasticity of CA3-CA1 projections.

Weaknesses:

(1) The causal relationship between navigation learning and CCK secretion?

(2) The effect of overexpression of the CCK gene on hippocampal functions?

(3) What are the functional differences between the excitatory and inhibitory CCK neurons in the hippocampus?

(4) Do CCK sources come from the local CA3 or entorhinal cortex (EC) during the high-frequency electrical stimulation?

Reviewer #2 (Public review):

Summary:

This highly novel and significant manuscript re-analyzes behavioral QTL data derived from morphine locomotor activity in the BXD recombinant inbred panel. The combination of interacting behavioral-pharmacology (morphine and naltrexone) time course data, high-resolution mouse genetic analyses, genetic analysis of gene expression (eQTLs), cross-species analysis with human gene expression and genetic data, and molecular modeling approaches with Bayesian network analysis produces new information on loci modulating morphine locomotor activity.

Furthermore, the identification of time-wise epistatic interactions between the Oprm1 and Fgf12 loci is highly novel and points to methodological approaches for identifying other epistatic interactions using animal model genetic studies.

Strengths:

(1) Use of state-of-the art genetic tools for mapping behavioral phenotypes in mouse models.

(2) Adequately powered analysis incorporating both sexes and time course analyses.

(3) Detection of time and sex-dependent interactions of two QTL loci modulating morphine locomotor activity.

(4) Identification of putative candidate genes by combined expression and behavioral genetic analyses.

(5) Use of Bayesian analysis to model causal interactions between multiple genes and behavioral time points.

Weaknesses:

(1) There is a need for careful editing of the text and figures to eliminate multiple typographical and other compositional errors.

(2) There are multiple examples of overstating the possible significance of results that should be corrected or at least directly pointed out as weaknesses in the Discussion. These include:

a) Assumption that the Oprm1 gene is the causal candidate gene for the major morphine locomotor Chr10 QTL at the early time epochs. Oprm1 is 400,000 bp away from the support interval of the Mor10a QTL locus, and there is no mention as to whether the Oprm1 mRNA eQTL overlaps with Mor10a.

b) Although the Bayesian analysis of possible complex interactions between Oprm1, Fgf12, other interacting genes, and behaviors is very innovative and produces testable hypotheses, a more straightforward mediation analysis of causal relationships between genotype, gene expression, and phenotype would have added strength to the arguments for the causal role of these individual genes.

c) The GWAS data analysis for Oprm1 and Fgf12 is incomplete in not mentioning actual significance levels for Oprm1 and perhaps overstating the nominal significance findings for Fgf12.

Appraisal:

The authors largely succeeded in reaching goals with novel findings and methodology.

Significance of Findings:

This study will likely spur future direct experimental studies to test hypotheses generated by this complex analysis. Additionally, the broad methodological approach incorporating time course genetic analyses may encourage other studies to identify epistatic interactions in mouse genetic studies.

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

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Reply to the reviewers

Response to referee comments: ____RC-2025-03008

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary In this article, the authors used the synthetic TALE DNA binding proteins, tagged with YFP, which were designed to target five specific repeat elements in Trypanosoma brucei genome, including centromere and telomeres-associated repeats and those of a transposon element. This is in order to detect and identified, using YFP-pulldown, specific proteins that bind to these repetitive sequences in T. brucei chromatin. Validation of the approach was done using a TALE protein designed to target the telomere repeat (TelR-TALE) that detected many of the proteins that were previously implicated with telomeric functions. A TALE protein designed to target the 70 bp repeats that reside adjacent to the VSG genes (70R-TALE) detected proteins that function in DNA repair and the protein designed to target the 177 bp repeat arrays (177R-TALE) identified kinetochore proteins associated T. brucei mega base chromosomes, as well as in intermediate and mini-chromosomes, which imply that kinetochore assembly and segregation mechanisms are similar in all T. brucei chromosome.

Major comments: Are the key conclusions convincing? The authors reported that they have successfully used TALE-based affinity selection of protein-associated with repetitive sequences in the T. brucei genome. They claimed that this study has provided new information regarding the relevance of the repetitive region in the genome to chromosome integrity, telomere biology, chromosomal segregation and immune evasion strategies. These conclusions are based on high-quality research, and it is, basically, merits publication, provided that some major concerns, raised below, will be addressed before acceptance for publication. 1. The authors used TALE-YFP approach to examine the proteome associated with five different repetitive regions of the T. brucei genome and confirmed the binding of TALE-YFP with Chip-seq analyses. Ultimately, they got the list of proteins that bound to synthetic proteins, by affinity purification and LS-MS analysis and concluded that these proteins bind to different repetitive regions of the genome. There are two control proteins, one is TRF-YFP and the other KKT2-YFP, used to confirm the interactions. However, there are no experiment that confirms that the analysis gives some insight into the role of any putative or new protein in telomere biology, VSG gene regulation or chromosomal segregation. The proteins, which have already been reported by other studies, are mentioned. Although the author discovered many proteins in these repetitive regions, their role is yet unknown. It is recommended to take one or more of the new putative proteins from the repetitive elements and show whether or not they (1) bind directly to the specific repetitive sequence (e.g., by EMSA); (2) it is recommended that the authors will knockdown of one or a small sample of the new discovered proteins, which may shed light on their function at the repetitive region, as a proof of concept.

Response

The main request from Referee 1 is for individual evaluation of protein-DNA interaction for a few candidates identified in our TALE-YFP affinity purifications, particularly using EMSA to identify binding to the DNA repeats used for the TALE selection. In our opinion, such an approach would not actually provide the validation anticipated by the reviewer. The power of TALE-YFP affinity selection is that it enriches for protein complexes that associate with the chromatin that coats the target DNA repetitive elements rather than only identifying individual proteins or components of a complex that directly bind to DNA assembled in chromatin.

The referee suggests we express recombinant proteins and perform EMSA for selected candidates, but many of the identified proteins are unlikely to directly bind to DNA - they are more likely to associate with a combination of features present in DNA and/or chromatin (e.g. specific histone variants or histone post-translational modifications). Of course, a positive result would provide some validation but only IF the tested protein can bind DNA in isolation - thus, a negative result would be uninformative.

In fact, our finding that KKT proteins are enriched using the 177R-TALE (minichromosome repeat sequence) identifies components of the trypanosome kinetochore known (KKT2) or predicted (KKT3) to directly bind DNA (Marciano et al., 2021; PMID: 34081090), and likewise the TelR-TALE identifies the TRF component that is known to directly associate with telomeric (TTAGGG)n repeats (Reis et al 2018; PMID: 29385523). This provides reassurance on the specificity of the selection, as does the lack of cross selectivity between different TALEs used (see later point 3 below). The enrichment of the respective DNA repeats quantitated in Figure 2B (originally Figure S1) also provides strong evidence for TALE selectivity.

It is very likely that most of the components enriched on the repetitive elements targeted by our TALE-YFP proteins do not bind repetitive DNA directly. The TRF telomere binding protein is an exception - but it is the only obvious DNA binding protein amongst the many proteins identified as being enriched in our TelR-TALE-YFP and TRF-YFP affinity selections.

The referee also suggests that follow up experiments using knockdown of the identified proteins found to be enriched on repetitive DNA elements would be informative. In our opinion, this manuscript presents the development of a new methodology previously not applied to trypanosomes, and referee 2 highlights the value of this methodological development which will be relevant for a large community of kinetoplastid researchers. In-depth follow-up analyses would be beyond the scope of this current study but of course will be pursued in future. To be meaningful such knockdown analyses would need to be comprehensive in terms of their phenotypic characterisation (e.g. quantitative effects on chromosome biology and cell cycle progression, rates and mechanism of recombination underlying antigenic variation, etc) - simple RNAi knockdowns would provide information on fitness but little more. This information is already publicly available from genome-wide RNAi screens (www.tritrypDB.org), with further information on protein location available from the genome-wide protein localisation resource (Tryptag.org). Hence basic information is available on all targets selected by the TALEs after RNAi knock down but in-depth follow-up functional analysis of several proteins would require specific targeted assays beyond the scope of this study.

NonR-TALE-YFP does not have a binding site in the genome, but YFP protein should still be expressed by T. brucei clones with NLS. The authors have to explain why there is no signal detected in the nucleus, while a prominent signal was detected near kDNA (see Fig.2). Why is the expression of YFP in NonR-TALE almost not shown compared to other TALE clones?

Response

The NonR-TALE-YFP immunolocalisation signal indeed is apparently located close to the kDNA and away from the nucleus. We are not sure why this is so, but the construct is sequence validated and correct. However, we note that artefactual localisation of proteins fused to a globular eGFP tag, compared to a short linear epitope V5 tag, near to the kinetoplast has been previously reported (Pyrih et al, 2023; PMID: 37669165),

The expression of NonR-TALE-YFP is shown in Supplementary Fig. S2 in comparison to other TALE proteins. Although it is evident that NonR-TALE-YFP is expressed at lower levels than other TALEs (the different TALEs have different expression levels), it is likely that in each case the TALE proteins would be in relative excess.

It is possible that the absence of a target sequence for the NonR-TALE-YFP in the nucleus affects its stability and cellular location. Understanding these differences is tangential to the aim of this study.

However, importantly, NonR-TALE-YFP is not the only control for used for specificity in our affinity purifications. Instead, the lack of cross-selection of the same proteins by different TALEs (e.g. TelR-TALE-YFP, 177R-TALE-YFP) and the lack of enrichment of any proteins of interest by the well expressed ingiR-TALE-YFP or 147R-TALE-YFP proteins each provide strong evidence for the specificity of the selection using TALEs, as does the enrichment of similar protein sets following affinity purification of the TelR-TALE-YFP and TRF-YFP proteins which both bind telomeric (TTAGGG)n repeats. Moreover, control affinity purifications to assess background were performed using cells that completely lack an expressed YFP protein which further support specificity (Figure 6).

We have added text to highlight these important points in the revised manuscript:

Page 8:

"However, the expression level of NonR-TALE-YFP was lower than other TALE-YFP proteins; this may relate to the lack of DNA binding sites for NonR-TALE-YFP in the nucleus."

Page 8:

"NonR-TALE-YFP displayed a diffuse nuclear and cytoplasmic signal; unexpectedly the cytoplasmic signal appeared to be in the vicinity the kDNA of the kinetoplast (mitochrondria). We note that artefactual localisation of some proteins fused to an eGFP tag has previously been observed in T. brucei (Pyrih et al, 2023)."

Page 10:

Moreover, a similar set of enriched proteins was identified in TelR-TALE-YFP affinity purifications whether compared with cells expressing no YFP fusion protein (No-YFP), the NonR-TALE-YFP or the ingiR-TALE-YFP as controls (Fig. S7B, S8A; Tables S3, S4). Thus, the most enriched proteins are specific to TelR-TALE-YFP-associated chromatin rather than to the TALE-YFP synthetic protein module or other chromatin.

As a proof of concept, the author showed that the TALE method determined the same interacting partners enrichment in TelR-TALE as compared to TRF-YFP. And they show the same interacting partners for other TALE proteins, whether compared with WT cells or with the NonR-TALE parasites. It may be because NonR-TALE parasites have almost no (or very little) YFP expression (see Fig. S3) as compared to other TALE clones and the TRF-YFP clone. To address this concern, there should be a control included, with proper YFP expression.

Response

See response to point 2, but we reiterate that the ingi-TALE -YFP and 147R-TALE-YFP proteins are well expressed (western original Fig. S3 now Fig. S2) but few proteins are detected as being enriched or correspond to those enriched in TelR-TALE-YFP or TRF-YFP affinity purifications (see Fig. S9). Therefore, the ingi-TALE -YFP and 147R-TALE-YFP proteins provide good additional negative controls for specificity as requested. To further reassure the referee we have also included additional volcano plots which compare TelR-TALE-YFP, 70R-TALE-YFP or 177R-TALE-YFP to the ingiR-TALE-YFP affinity selection (new Figure S8). As with No-YFP or NonR-TALE-YFP controls, the use of ingiR-TALE-YFP as a negative control demonstrates that known telomere associated proteins are enriched in TelR-TALE-YFP affinity purification, RPA subunits enriched with 70R-TALE-YFP and Kinetochore KKT poroteins enriched with 177R-TALE-YFP. These analyses demonstrate specificity in the proteins enriched following affinity purification of our different TALE-YFPs and provide support to strengthen our original findings.

We now refer to use of No-YFP, NonR-TALE-YFP, and ingiR-TALE -YFP as controls for comparison to TelR-TALE-YFP, 70R-TALE-YFP or 177R-TALE-YFP in several places:

Page10:

"Moreover, a similar set of enriched proteins was identified in TelR-TALE-YFP affinity purifications whether compared with cells expressing no YFP fusion protein (No-YFP), the NonR-TALE-YFP or the ingiR-TALE-YFP as controls (Fig. S7B, S8A; Tables S3, S4)."

Page 11:

"Thus, the nuclear ingiR-TALE-YFP provides an additional chromatin-associated negative control for affinity purifications with the TelR-TALE-YFP, 70R-TALE-YFP and 177R-TALE-YFP proteins (Fig. S8)."

"Proteins identified as being enriched with 70R-TALE-YFP (Figure 6D) were similar in comparisons with either the No-YFP, NonR-TALE-YFP or ingiR-TALE-YFP as negative controls."

Top Page 12:

"The same kinetochore proteins were enriched regardless of whether the 177R-TALE proteomics data was compared with No-YFP, NonR-TALE or ingiR-TALE-YFP controls."

Discussion Page 13:

"Regardless, the 147R-TALE and ingiR-TALE proteins were well expressed in T. brucei cells, but their affinity selection did not significantly enrich for any relevant proteins. Thus, 147R-TALE and ingiR-TALE provide reassurance for the overall specificity for proteins enriched TelR-TALE, 70R-TALE and 177R-TALE affinity purifications."

After the artificial expression of repetitive sequence binding five-TALE proteins, the question is if there is any competition for the TALE proteins with the corresponding endogenous proteins? Is there any effect on parasite survival or health, compared to the control after the expression of these five TALEs YFP protein? It is recommended to add parasite growth curves, for all the TALE-proteins expressing cultures.

Response

Growth curves for cells expressing TelR-TALE-YFP, 177R-TALE-YFP and ingiR-TALE-YFP are now included (New Fig S3A). No deficit in growth was evident while passaging 70R-TALE-YFP, 147R-TALE-YFP, NonR-TALE-YFP cell lines (indeed they grew slightly better than controls).

The following text has been added page 8:

"Cell lines expressing representative TALE-YFP proteins displayed no fitness deficit (Fig. S3A)."

Since the experiments were performed using whole-cell extracts without prior nuclear fractionation, the authors should consider the possibility that some identified proteins may have originated from compartments other than the nucleus. Specifically, the detection of certain binding proteins might reflect sequence homology (or partial homology) between mitochondrial DNA (maxicircles and minicircles) and repetitive regions in the nuclear genome. Additionally, the lack of subcellular separation raises the concern that cytoplasmic proteins could have been co-purified due to whole cell lysis, making it challenging to discern whether the observed proteome truly represents the nuclear interactome.

Response

In our experimental design, we confirmed bioinformatically that the repeat sequences targeted were not represented elsewhere in the nuclear or mitochondrial genome (kDNA). The absence of subcellular fractionation could result in some cytoplasmic protein selection, but this is unlikely since each TALE targets a specific DNA sequence but is otherwise identical such that cross-selection of the same contaminating protein set would be anticipated if there was significant non-specific binding. We have previously successfully affinity selected 15 chromatin modifiers and identified associated proteins without major issues concerning cytoplasmic protein contamination (Staneva et al 2021 and 2022; PMID: 34407985 and 36169304). Of course, the possibility that some proteins are contaminants will need to be borne in mind in any future follow-up analysis of proteins of interest that we identified as being enriched on specific types of repetitive element in T. brucei. Proteins that are also detected in negative control, or negative affinity selections such as No-YFP, NoR-YFP, IngiR-TALE or 147R-TALE must be disregarded.

'6'. Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? As mentioned earlier, the author claimed that this study has provided new information concerning telomere biology, chromosomal segregation mechanisms, and immune evasion strategies. But there are no experiments that provides a role for any unknown or known protein in these processes. Thus, it is suggested to select one or two proteins of choice from the list and validate their direct binding to repetitive region(s), and their role in that region of interaction.

Response

As highlighted in response to point 1 the suggested validation and follow up experiments may well not be informative and are beyond the scope of the methodological development presented in this manuscript. Referee 2 describes the study in its current form as "a significant conceptual and technical advancement" and "This approach enhances our understanding of chromatin organization in these regions and provides a foundation for investigating the functional roles of associated proteins in parasite biology."

The Referee's phrase 'validate their direct binding to repetitive region(s)' here may also mean to test if any of the additional proteins that we identified as being enriched with a specific TALE protein actually display enrichment over the repeat regions when examined by an orthogonal method. A key unexpected finding was that kinetochore proteins including KKT2 are enriched in our affinity purifications of the 177R-TALE-YFP that targets 177bp repeats (Figure 6F). By conducting ChIP-seq for the kinetochore specific protein KKT2 using YFP-KKT2 we confirmed that KKT2 is indeed enriched on 177bp repeat DNA but not flanking DNA (Figure 7). Moreover, several known telomere-associated proteins are detected in our affinity selections of TelR-TALE-YFP (Figure 6B, FigS6; see also Reis et al, 2018 Nuc. Acids Res. PMID: 29385523; Weisert et al, 2024 Sci. Reports PMID: 39681615).

Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. The answer for this question depends on what the authors want to present as the achievements of the present study. If the achievement of the paper was is the creation of a new tool for discovering new proteins, associated with the repeat regions, I recommend that they add a proof for direct interactions between a sample the newly discovered proteins and the relevant repeats, as a proof of concept discussed above, However, if the authors like to claim that the study achieved new functional insights for these interactions they will have to expand the study, as mentioned above, to support the proof of concept.

Response

See our response to point 1 and the point we labelled '6' above.

Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments. I think that they are realistic. If the authors decided to check the capacity of a small sample of proteins (which was unknown before as a repetitive region binding proteins) to interacts directly with the repeated sequence, it will substantially add of the study (e.g., by EMSA; estimated time: 1 months). If the authors will decide to check the also the function of one of at least one such a newly detected proteins (e.g., by KD), I estimate the will take 3-6 months.

Response

As highlighted previously the proposed EMSA experiment may well be uninformative for protein complex components identified in our study or for isolated proteins that directly bind DNA in the context of a complex and chromatin. RNAi knockdown data and cell location data (as well as developmental expression and orthology data) is already available through tritrypDB.org and trtyptag.org

Are the data and the methods presented in such a way that they can be reproduced? Yes

Are the experiments adequately replicated, and statistical analysis adequate? The authors did not mention replicates. There is no statistical analysis mentioned.

Response

The figure legends indicate that all volcano plots of TALE affinity selections were derived from three biological replicates. Cutoffs used for significance: PFor ChiP-seq two biological replicates were analysed for each cell line expressing the specific YFP tagged protein of interest (TALE or KKT2). This is now stated in the relevant figure legends - apologies for this oversight. The resulting data are available for scrutiny at GEO: GSE295698.

Minor comments: -Specific experimental issues that are easily addressable. The following suggestions can be incorporated: 1. Page 18, in the material method section author mentioned four drugs: Blasticidine, Phleomycin and G418, and hygromycin. It is recommended to mention the purpose of using these selective drugs for the parasite. If clonal selection has been done, then it should also be mentioned.

Response

We erroneously added information on several drugs used for selection in our labaoratory. In fact all TALE-YFP construct carry the Bleomycin resistance genes which we select for using Phleomycin. Also, clones were derived by limiting dilution immediately after transfection.

We have amended the text accordingly:

Page 17/18:

"Cell cultures were maintained below 3 x 106 cells/ml. Pleomycin 2.5 mg/ml was used to select transformants containing the TALE construct BleoR gene."

"Electroporated bloodstream cells were added to 30 ml HMI-9 medium and two 10-fold serial dilutions were performed in order to isolate clonal Pleomycin resistant populations from the transfection. 1 ml of transfected cells were plated per well on 24-well plates (1 plate per serial dilution) and incubated at 37{degree sign}C and 5% CO2 for a minimum of 6 h before adding 1 ml media containing 2X concentration Pleomycin (5 mg/ml) per well."

In the method section the authors mentioned that there is only one site for binding of NonR-TALE in the parasite genome. But in Fig. 1C, the authors showed zero binding site. So, there is one binding site for NonR-TALE-YFP in the genome or zero?

Response

We thank the reviewer for pointing out this discrepancy. We have checked the latest Tb427v12 genome assembly for predicted NonR-TALE binding sites and there are no exact matches. We have corrected the text accordingly.

Page 7:

"A control NonR-TALE protein was also designed which was predicted to have no target sequence in the T. bruceigenome."

Page 17:

"A control NonR-TALE predicted to have no recognised target in the T. brucei geneome was designed as follows: BLAST searches were used to identify exact matches in the TREU927 reference genome. Candidate sequences with one or more match were discarded."

The authors used two different anti-GFP antibodies, one from Roche and the other from Thermo Fisher. Why were two different antibodies used for the same protein?

Response

We have found that only some anti-GFP antibodies are effective for affinity selection of associated proteins, whereas others are better suited for immunolocalisation. The respective suppliers' antibodies were optimised for each application.

Page 6: in the introduction, the authors give the number of total VSG genes as 2,634. Is it known how many of them are pseudogenes?

Response

This value corresponds to the number reported by Consentino et al. 2021 (PMID: 34541528) for subtelomeric VSGs, which is similar to the value reported by Muller et al 2018 (PMID: 30333624) (2486), both in the same strain of trypanosomes as used by us. Based on the earlier analysis by Cross et al (PMID: 24992042), 80% of the identified VSGs in their study (2584) are pseudogenes. This approximates to the estimation by Consentino of 346/2634 (13%) being fully functional VSG genes at subtelomeres, or 17% when considering VSGs at all genomic locations (433/2872).

I found several typos throughout the manuscript.

Response

Thank you for raising this, we have read through the manuscipt several times and hopefully corrected all outstanding typos.

Fig. 1C: Table: below TOTAL 2nd line: the number should be 1838 (rather than 1828)

Corrected- thank you.

• Are prior studies referenced appropriately? Yes

• Are the text and figures clear and accurate? Yes

• Do you have suggestions that would help the authors improve the presentation of their data and conclusions? Suggested above

Reviewer #1 (Significance (Required)):

Describe the nature and significance of the advance (e.g., conceptual, technical, clinical) for the field: This study represents a significant conceptual and technical advancement by employing a synthetic TALE DNA-binding protein tagged with YFP to selectively identify proteins associated with five distinct repetitive regions of T. brucei chromatin. To the best of my knowledge, it is the first report to utilize TALE-YFP for affinity-based isolation of protein complexes bound to repetitive genomic sequences in T. brucei. This approach enhances our understanding of chromatin organization in these regions and provides a foundation for investigating the functional roles of associated proteins in parasite biology. Importantly, any essential or unique interacting partners identified could serve as potential targets for therapeutic intervention.

• Place the work in the context of the existing literature (provide references, where appropriate). I agree with the information that has already described in the submitted manuscript, regarding its potential addition of the data resulted and the technology established to the study of VSGs expression, kinetochore mechanism and telomere biology.

• State what audience might be interested in and influenced by the reported findings. These findings will be of particular interest to researchers studying the molecular biology of kinetoplastid parasites and other unicellular organisms, as well as scientists investigating chromatin structure and the functional roles of repetitive genomic elements in higher eukaryotes.

• 1Define your field of expertise with a few keywords to help the authors contextualize your point of view. 2Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. (1) Protein-DNA interactions/ chromatin/ DNA replication/ Trypanosomes (2) None

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

Summary

Carloni et al. comprehensively analyze which proteins bind repetitive genomic elements in Trypanosoma brucei. For this, they perform mass spectrometry on custom-designed, tagged programmable DNA-binding proteins. After extensively verifying their programmable DNA-binding proteins (using bioinformatic analysis to infer target sites, microscopy to measure localization, ChIP-seq to identify binding sites), they present, among others, two major findings: 1) 14 of the 25 known T. brucei kinetochore proteins are enriched at 177bp repeats. As T. brucei's 177bp repeat-containing intermediate-sized and mini-chromosomes lack centromere repeats but are stable over mitosis, Carloni et al. use their data to hypothesize that a 'rudimentary' kinetochore assembles at the 177bp repeats of these chromosomes to segregate them. 2) 70bp repeats are enriched with the Replication Protein A complex, which, notably, is required for homologous recombination. Homologous recombination is the pathway used for recombination-based antigenic variation of the 70bp-repeat-adjacent variant surface glycoproteins.

Major Comments

None. The experiments are well-controlled, claims well-supported, and methods clearly described. Conclusions are convincing.

Response Thank you for these positive comments.

Minor Comments

1) Fig. 2 - I couldn't find an uncropped version showing multiple cells. If it exists, it should be linked in the legend or main text; Otherwise, this should be added to the supplement.

Response

The images presented represent reproducible analyses, and independently verified by two of the authors. Although wider field of view images do not provide the resolution to be informative on cell location, as requested we have provided uncropped images in new Fig. S4 for all the cell lines shown in Figure 2A.

In addition, we have included as supplementary images (Fig. S3B) additional images of TelR-TALE-YFP, 177R-TALE-YFP and ingiR-TALE YFP localisation to provide additional support their observed locations presented in Figure 1. The set of cells and images presented in Figure 2A and in Fig S3B were prepared and obtained by a different authors, independently and reproducibly validating the location of the tagged protein.

2) I think Suppl. Fig. 1 is very valuable, as it is a quantification and summary of the ChIP-seq data. I think the authors could consider making this a panel of a main figure. For the main figure, I think the plot could be trimmed down to only show the background and the relevant repeat for each TALE protein, leaving out the non-target repeats. (This relates to minor comment 6.) Also, I believe, it was not explained how background enrichment was calculated.

Response

We are grateful for the reviewer's positive view of original Fig. S1 and appreciate the suggestion. We have now moved these analysis to part B of main Figure 2 in the revised manuscript - now Figure 2B. We have also provided additional details in the Methods section on the approaches used to assess background enrichment.

Page 19:

Background enrichment calculation

The genome was divided into 50 bp sliding windows, and each window was annotated based on overlapping genomic features, including CIR147, 177 bp repeats, 70 bp repeats, and telomeric (TTAGGG)n repeats. Windows that did not overlap with any of these annotated repeat elements were defined as "background" regions and used to establish the baseline ChIP-seq signal. Enrichment for each window was calculated using bamCompare, as log₂(IP/Input). To adjust for background signal amongst all samples, enrichment values for each sample were further normalized against the corresponding No-YFP ChIP-seq dataset.

Note: While revising the manuscript we also noticed that the script had a nomalization error. We have therefore included a corrected version of these analyses as Figure 2B (old Fig. S1)

3) Generally, I would plot enrichment on a log2 axis. This concerns several figures with ChIP-seq data.

Response

Our ChIP-seq enrichment is calculated by bamCompare. The resulting enrichment values are indeed log2 (IP/Input). We have made this clear in the updated figures/legends.

4) Fig. 4C - The violin plots are very hard to interpret, as the plots are very narrow compared to the line thickness, making it hard to judge the actual volume. For example, in Centromere 5, YFP-KKT2 is less enriched than 147R-TALE over most of the centromere with some peaks of much higher enrichment (as visible in panel B), however, in panel C, it is very hard to see this same information. I'm sure there is some way to present this better, either using a different type of plot or by improving the spacing of the existing plot.

Response

We thank the reviewer for this suggestion; we have elected to provide a Split-Violin plot instead. This improves the presentation of the data for each centromere. The original violin plot in Figure 4C has been replaced with this Split-Violin plot (still Figure 4C).

5) Fig. 6 - The panels are missing an x-axis label (although it is obvious from the plot what is displayed). Maybe the "WT NO-YFP vs" part that is repeated in all the plot titles could be removed from the title and only be part of the x-axis label?

Response

In fact, to save space the X axis was labelled inside each volcano plot but we neglected to indicate that values are a log2 scale indicating enrichment. This has been rectified - see Figure 6, and Fig. S7, S8 and S9.

6) Fig. 7 - I would like to have a quantification for the examples shown here. In fact, such a quantification already exists in Suppl. Figure 1. I think the relevant plots of that quantification (YFP-KKT2 over 177bp-repeats and centromere-repeats) with some control could be included in Fig. 7 as panel C. This opportunity could be used to show enrichment separated out for intermediate-sized, mini-, and megabase-chromosomes. (relates to minor comment 2 & 8)

Response

The CIR147 sequence is found exclusively on megabase-sized chromosomes, while the 177 bp repeats are located on intermediate- and mini-sized chromosomes. Due to limitations in the current genome assembly, it is not possible to reliably classify all chromosomes into intermediate- or mini- sized categories based on their length. Therefore, original Supplementary Fig. S1 presented the YFP-KKT2 enrichment over CIR147 and 177 bp repeats as a representative comparison between megabase chromosomes and the remaining chromosomes (corrected version now presented as main Figure 2B). Additionally, to allow direct comparison of YFP-KKT2 enrichment on CIR147 and 177 bp repeats we have included a new plot in Figure 7C which shows the relative enrichment of YFP-KKT2 on these two repeat types.

We have added the following text , page 12:

"Taking into account the relative to the number of CIR147 and 177 bp repeats in the current T.brucei genome (Cosentino et al., 2021; Rabuffo et al., 2024), comparative analyses demonstrated that YFP-KKT2 is enriched on both CIR147 and 177 bp repeats (Figure 7C)."

7) Suppl. Fig. 8 A - I believe there is a mistake here: KKT5 occurs twice in the plot, the one in the overlap region should be KKT1-4 instead, correct?

Response

Thanks for spotting this. It has been corrected

8) The way that the authors mapped ChIP-seq data is potentially problematic when analyzing the same repeat type in different regions of the genome. The authors assigned reads that had multiple equally good mapping positions to one of these mapping positions, randomly. This is perfectly fine when analysing repeats by their type, independent of their position on the genome, which is what the authors did for the main conclusions of the work. However, several figures show the same type of repeat at different positions in the genome. Here, the authors risk that enrichment in one region of the genome 'spills' over to all other regions with the same sequence. Particularly, where they show YFP-KKT2 enrichment over intermediate- and mini-chromosomes (Fig. 7) due to the spillover, one cannot be sure to have found KKT2 in both regions. Instead, the authors could analyze only uniquely mapping reads / read-pairs where at least one mate is uniquely mapping. I realize that with this strict filtering, data will be much more sparse. Hence, I would suggest keeping the original plots and adding one more quantification where the enrichment over the whole region (e.g., all 177bp repeats on intermediate-/mini-chromosomes) is plotted using the unique reads (this could even be supplementary). This also applies to Fig. 4 B & C.

Response

We thank the reviewer for their thoughtful comments. Repetitive sequences are indeed challenging to analyze accurately, particularly in the context of short read ChIP-seq data. In our study, we aimed to address YFP-KKT2 enrichment not only over CIR147 repeats but also on 177 bp repeats, using both ChIP-seq and proteomics using synthetic TALE proteins targeted to the different repeat types. We appreciate the referees suggestion to consider uniquely mapped reads, however, in the updated genome assembly, the 177 bp repeats are frequently immediately followed by long stretches of 70 bp repeats which can span several kilobases. The size and repetitive nature of these regions exceeds the resolution limits of ChIP-seq. It is therefore difficult to precisely quantify enrichment across all chromosomes.

Additionally, the repeat sequences are highly similar, and relying solely on uniquely mapped reads would result in the exclusion of most reads originating from these regions, significantly underestimating the relative signals. To address this, we used Bowtie2 with settings that allow multi-mapping, assigning reads randomly among equivalent mapping positions, but ensuring each read is counted only once. This approach is designed to evenly distribute signal across all repetitive regions and preserve a meaningful average.

Single molecule methods such as DiMeLo (Altemose et al. 2022; PMID: 35396487) will need to be developed for T. brucei to allow more accurate and chromosome specific mapping of kinetochore or telomere protein occupancy at repeat-unique sequence boundaries on individual chromosomes.

Reviewer #2 (Significance (Required)):

This work is of high significance for chromosome/centromere biology, parasitology, and the study of antigenic variation. For chromosome/centromere biology, the conceptual advancement of different types of kinetochores for different chromosomes is a novelty, as far as I know. It would certainly be interesting to apply this study as a technical blueprint for other organisms with mini-chromosomes or chromosomes without known centromeric repeats. I can imagine a broad range of labs studying other organisms with comparable chromosomes to take note of and build on this study. For parasitology and the study of antigenic variation, it is crucial to know how intermediate- and mini-chromosomes are stable through cell division, as these chromosomes harbor a large portion of the antigenic repertoire. Moreover, this study also found a novel link between the homologous repair pathway and variant surface glycoproteins, via the 70bp repeats. How and at which stages during the process, 70bp repeats are involved in antigenic variation is an unresolved, and very actively studied, question in the field. Of course, apart from the basic biological research audience, insights into antigenic variation always have the potential for clinical implications, as T. brucei causes sleeping sickness in humans and nagana in cattle. Due to antigenic variation, T. brucei infections can be chronic.

Response

Thank you for supporting the novelty and broad interest of our manuscript

My field of expertise / Point of view:

I'm a computer scientist by training and am now a postdoctoral bioinformatician in a molecular parasitology laboratory. The laboratory is working on antigenic variation in T. brucei. The focus of my work is on analyzing sequencing data (such as ChIP-seq data) and algorithmically improving bioinformatic tools.

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Referee #2

Evidence, reproducibility and clarity

Summary

Carloni et al. comprehensively analyze which proteins bind repetitive genomic elements in Trypanosoma brucei. For this, they perform mass spectrometry on custom-designed, tagged programmable DNA-binding proteins. After extensively verifying their programmable DNA-binding proteins (using bioinformatic analysis to infer target sites, microscopy to measure localization, ChIP-seq to identify binding sites), they present, among others, two major findings: 1) 14 of the 25 known T. brucei kinetochore proteins are enriched at 177bp repeats. As T. brucei's 177bp repeat-containing intermediate-sized and mini-chromosomes lack centromere repeats but are stable over mitosis, Carloni et al. use their data to hypothesize that a 'rudimentary' kinetochore assembles at the 177bp repeats of these chromosomes to segregate them. 2) 70bp repeats are enriched with the Replication Protein A complex, which, notably, is required for homologous recombination. Homologous recombination is the pathway used for recombination-based antigenic variation of the 70bp-repeat-adjacent variant surface glycoproteins.

Major Comments

None. The experiments are well-controlled, claims well-supported, and methods clearly described. Conclusions are convincing.

Minor Comments

1. Fig. 2 - I couldn't find an uncropped version showing multiple cells. If it exists, it should be linked in the legend or main text; Otherwise, this should be added to the supplement.
2. I think Suppl. Fig. 1 is very valuable, as it is a quantification and summary of the ChIP-seq data. I think the authors could consider making this a panel of a main figure. For the main figure, I think the plot could be trimmed down to only show the background and the relevant repeat for each TALE protein, leaving out the non-target repeats. (This relates to minor comment 6.) Also, I believe, it was not explained how background enrichment was calculated.
3. Generally, I would plot enrichment on a log2 axis. This concerns several figures with ChIP-seq data.
4. Fig. 4C - The violin plots are very hard to interpret, as the plots are very narrow compared to the line thickness, making it hard to judge the actual volume. For example, in Centromere 5, YFP-KKT2 is less enriched than 147R-TALE over most of the centromere with some peaks of much higher enrichment (as visible in panel B), however, in panel C, it is very hard to see this same information. I'm sure there is some way to present this better, either using a different type of plot or by improving the spacing of the existing plot.
5. Fig. 6 - The panels are missing an x-axis label (although it is obvious from the plot what is displayed). Maybe the "WT NO-YFP vs" part that is repeated in all the plot titles could be removed from the title and only be part of the x-axis label?
6. Fig. 7 - I would like to have a quantification for the examples shown here. In fact, such a quantification already exists in Suppl. Figure 1. I think the relevant plots of that quantification (YFP-KKT2 over 177bp-repeats and centromere-repeats) with some control could be included in Fig. 7 as panel C. This opportunity could be used to show enrichment separated out for intermediate-sized, mini-, and megabase-chromosomes. (relates to minor comment 2 & 8)
7. Suppl. Fig. 8 A - I believe there is a mistake here: KKT5 occurs twice in the plot, the one in the overlap region should be KKT1-4 instead, correct?
8. The way that the authors mapped ChIP-seq data is potentially problematic when analyzing the same repeat type in different regions of the genome. The authors assigned reads that had multiple equally good mapping positions to one of these mapping positions, randomly. This is perfectly fine when analyzing repeats by their type, independent of their position on the genome, which is what the authors did for the main conclusions of the work. However, several figures show the same type of repeat at different positions in the genome. Here, the authors risk that enrichment in one region of the genome 'spills' over to all other regions with the same sequence. Particularly, where they show YFP-KKT2 enrichment over intermediate- and mini-chromosomes (Fig. 7) due to the spillover, one cannot be sure to have found KKT2 in both regions. Instead, the authors could analyze only uniquely mapping reads / read-pairs where at least one mate is uniquely mapping. I realize that with this strict filtering, data will be much more sparse. Hence, I would suggest keeping the original plots and adding one more quantification where the enrichment over the whole region (e.g., all 177bp repeats on intermediate-/mini-chromosomes) is plotted using the unique reads (this could even be supplementary). This also applies to Fig. 4 B & C.

Significance

This work is of high significance for chromosome/centromere biology, parasitology, and the study of antigenic variation. For chromosome/centromere biology, the conceptual advancement of different types of kinetochores for different chromosomes is a novelty, as far as I know. It would certainly be interesting to apply this study as a technical blueprint for other organisms with mini-chromosomes or chromosomes without known centromeric repeats. I can imagine a broad range of labs studying other organisms with comparable chromosomes to take note of and build on this study. For parasitology and the study of antigenic variation, it is crucial to know how intermediate- and mini-chromosomes are stable through cell division, as these chromosomes harbor a large portion of the antigenic repertoire. Moreover, this study also found a novel link between the homologous repair pathway and variant surface glycoproteins, via the 70bp repeats. How and at which stages during the process, 70bp repeats are involved in antigenic variation is an unresolved, and very actively studied, question in the field. Of course, apart from the basic biological research audience, insights into antigenic variation always have the potential for clinical implications, as T. brucei causes sleeping sickness in humans and nagana in cattle. Due to antigenic variation, T. brucei infections can be chronic.

My field of expertise / Point of view:

I'm a computer scientist by training and am now a postdoctoral bioinformatician in a molecular parasitology laboratory. The laboratory is working on antigenic variation in T. brucei. The focus of my work is on analyzing sequencing data (such as ChIP-seq data) and algorithmically improving bioinformatic tools.

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

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Referee #1

Evidence, reproducibility and clarity

Summary

In this article, the authors used the synthetic TALE DNA binding proteins, tagged with YFP, which were designed to target five specific repeat elements in Trypanosoma brucei genome, including centromere and telomeres-associated repeats and those of a transposon element. This is in order to detect and identified, using YFP-pulldown, specific proteins that bind to these repetitive sequences in T. brucei chromatin. Validation of the approach was done using a TALE protein designed to target the telomere repeat (TelR-TALE) that detected many of the proteins that were previously implicated with telomeric functions. A TALE protein designed to target the 70 bp repeats that reside adjacent to the VSG genes (70R-TALE) detected proteins that function in DNA repair and the protein designed to target the 177 bp repeat arrays (177R-TALE) identified kinetochore proteins associated T. brucei mega base chromosomes, as well as in intermediate and mini-chromosomes, which imply that kinetochore assembly and segregation mechanisms are similar in all T. brucei chromosome.

Major comments:

Are the key conclusions convincing?

The authors reported that they have successfully used TALE-based affinity selection of protein-associated with repetitive sequences in the T. brucei genome. They claimed that this study has provided new information regarding the relevance of the repetitive region in the genome to chromosome integrity, telomere biology, chromosomal segregation and immune evasion strategies. These conclusions are based on high-quality research and it is, basically, merits publication, provided that some major concerns, raised below, will be addressed before acceptance for publication. 1. The authors used TALE-YFP approach to examine the proteome associated with five different repetitive regions of the T. brucei genome and confirmed the binding of TALE-YFP with Chip-seq analyses. Ultimately, they got the list of proteins that bound to synthetic proteins, by affinity purification and LS-MS analysis and concluded that these proteins bind to different repetitive regions of the genome. There are two control proteins, one is TRF-YFP and the other KKT2-YFP, used to confirm the interactions. However, there are no experiment that confirms that the analysis gives some insight into the role of any putative or new protein in telomere biology, VSG gene regulation or chromosomal segregation. The proteins, which have already been reported by other studies, are mentioned. Although the author discovered many proteins in these repetitive regions, their role is yet unknown. It is recommended to take one or more of the new putative proteins from the repetitive elements and show whether or not they (1) bind directly to the specific repetitive sequence (e.g., by EMSA); (2) it is recommended that the authors will knockdown of one or a small sample of the new discovered proteins, which may shed light on their function at the repetitive region, as a proof of concept. 2. NonR-TALE-YFP does not have a binding site in the genome, but YFP protein should still be expressed by T. brucei clones with NLS. The authors have to explain why there is no signal detected in the nucleus, while a prominent signal was detected near kDNA (see Fig.2). Why is the expression of YFP in NonR-TALE almost not shown compared to other TALE clones? 3. As a proof of concept, the author showed that the TALE method determined the same interacting partners enrichment in TelR-TALE as compared to TRF-YFP. And they show the same interacting partners for other TALE proteins, whether compared with WT cells or with the NonR-TALE parasites. It may be because NonR-TALE parasites have almost no (or very little) YFP expression (see Fig. S3) as compared to other TALE clones and the TRF-YFP clone. To address this concern, there should be a control included, with proper YFP expression. 4. After the artificial expression of repetitive sequence binding five-TALE proteins, the question is if there is any competition for the TALE proteins with the corresponding endogenous proteins? Is there any effect on parasite survival or health, compared to the control after the expression of these five TALEs YFP protein? It is recommended to add parasite growth curves, for all the TALE-proteins expressing cultures. 5. Since the experiments were performed using whole-cell extracts without prior nuclear fractionation, the authors should consider the possibility that some identified proteins may have originated from compartments other than the nucleus. Specifically, the detection of certain binding proteins might reflect sequence homology (or partial homology) between mitochondrial DNA (maxicircles and minicircles) and repetitive regions in the nuclear genome. Additionally, the lack of subcellular separation raises the concern that cytoplasmic proteins could have been co-purified due to whole cell lysis, making it challenging to discern whether the observed proteome truly represents the nuclear interactome.

Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

As mentioned earlier, the author claimed that this study has provided new information concerning telomere biology, chromosomal segregation mechanisms, and immune evasion strategies. But there are no experiments that provides a role for any unknown or known protein in these processes. Thus, it is suggested to select one or two proteins of choice from the list and validate their direct binding to repetitive region(s), and their role in that region of interaction. <br /> Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. The answer for this question depends on what the authors want to present as the achievements of the present study. If the achievement of the paper was is the creation of a new tool for discovering new proteins, associated with the repeat regions, I recommend that they add a proof for direct interactions between a sample the newly discovered proteins and the relevant repeats, as a proof of concept discussed above, However, if the authors like to claim that the study achieved new functional insights for these interactions they will have to expand the study, as mentioned above, to support the proof of concept.

Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

I think that they are realistic. If the authors decided to check the capacity of a small sample of proteins (which was unknown before as a repetitive region binding proteins) to interacts directly with the repeated sequence, it will substantially add of the study (e.g., by EMSA; estimated time: 1 months). If the authors will decide to check the also the function of one of at least one such a newly detected proteins (e.g., by KD), I estimate the will take 3-6 months.

Are the data and the methods presented in such a way that they can be reproduced?

Yes

Are the experiments adequately replicated, and statistical analysis adequate?

The authors did not mention replicates. There is no statistical analysis mentioned.

Minor comments:

Specific experimental issues that are easily addressable.

The following suggestions can be incorporated:

1. Page 18, in the material method section author mentioned four drugs: Blasticidine, Phleomycin and G418, and hygromycin. It is recommended to mention the purpose of using these selective drugs for the parasite. If clonal selection has been done, then it should also be mentioned.
2. In the method section the authors mentioned that there is only one site for binding of NonR-TALE in the parasite genome. But in Fig. 1C, the authors showed zero binding site. So, there is one binding site for NonR-TALE-YFP in the genome or zero?
3. The authors used two different anti-GFP antibodies, one from Roche and the other from Thermo Fisher. Why were two different antibodies used for the same protein?
4. Page 6: in the introduction, the authors give the number of total VSG genes as 2,634. Is it known how many of them are pseudogenes?
5. I found several typos throughout the manuscript.
6. Fig. 1C: Table: below TOTAL 2nd line: the number should be 1838 (rather than 1828)

Are prior studies referenced appropriately?

Yes

Are the text and figures clear and accurate?

Yes

Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

Suggested above

Significance

Describe the nature and significance of the advance (e.g., conceptual, technical, clinical) for the field:

This study represents a significant conceptual and technical advancement by employing a synthetic TALE DNA-binding protein tagged with YFP to selectively identify proteins associated with five distinct repetitive regions of T. brucei chromatin. To the best of my knowledge, it is the first report to utilize TALE-YFP for affinity-based isolation of protein complexes bound to repetitive genomic sequences in T. brucei. This approach enhances our understanding of chromatin organization in these regions and provides a foundation for investigating the functional roles of associated proteins in parasite biology. Importantly, any essential or unique interacting partners identified could serve as potential targets for therapeutic intervention.

Place the work in the context of the existing literature (provide references, where appropriate).

I agree with the information that has already described in the submitted manuscript, regarding its potential addition of the data resulted and the technology established to the study of VSGs expression, kinetochore mechanism and telomere biology.

State what audience might be interested in and influenced by the reported findings.

These findings will be of particular interest to researchers studying the molecular biology of kinetoplastid parasites and other unicellular organisms, as well as scientists investigating chromatin structure and the functional roles of repetitive genomic elements in higher eukaryotes.

1Define your field of expertise with a few keywords to help the authors contextualize your point of view. 2Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

1. Protein-DNA interactions/ chromatin/ DNA replication/ Trypanosomes
2. None

Author response:

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

Reviewer #1 (Public review):

(1) The authors only report the quality of the classification considering the number of videos used for training, but not considering the number of mice represented or the mouse strain. Therefore, it is unclear if the classification model works equally well in data from all the mouse strains tested, and how many mice are represented in the classifier dataset and validation.

We agree that strain-level performance is critical for assessing generalizability. In the revision we now report per-strain accuracy and F1 for the grooming classifier, which was trained on videos spanning 60 genetically diverse strains (n = 1100 videos) and evaluated on the test set videos spanning 51 genetically diverse strains (n=153 videos). Performance is uniform across most strains (median F1 = 0.94, IQR = 0.899–0.956), with only modest declines in albino lines that lack contrast under infrared illumination; this limitation and potential remedies are discussed in the text. The new per-strain metrics are presented in the Supplementary figure (corresponding to Figure 4).

(2) The GUI requires pose tracking for classification, but the software provided in JABS does not do pose tracking, so users must do pose tracking using a separate tool. Currently, there is no guidance on the pose tracking recommendations and requirements for usage in JABS. The pose tracking quality directly impacts the classification quality, given that it is used for the feature calculation; therefore, this aspect of the data processing should be more carefully considered and described.

We have added a section to the methods describing how to use the pose estimation models used in JABS. The reviewer is correct that pose tracking quality will impact classification quality. We recommend that classifiers should only be re-used on pose files generated by the same pose models used in the behavior classifier training dataset. We hope that the combination of sharing classifier training data and making a more unified framework for developing and comparing classifiers will get us closer to having foundational behavior classification models that work in many environments. We also would like to emphasize that deviating from using our pose model will also likely hinder re-using our shared large datasets in JABS-AI (JABS1200, JABS600, JABS-BxD).

(3) Many statistical and methodological details are not described in the manuscript, limiting the interpretability of the data presented in Figures 4,7-8. There is no clear methods section describing many of the methods used and equations for the metrics used. As an example, there are no details of the CNN used to benchmark the JABS classifier in Figure 4, and no details of the methods used for the metrics reported in Figure 8.

We thank the reviewer for bringing this to our attention. We have added a methods section to the manuscript to address this concern. Specifically, we now provide: (1) improved citation visibility of the source of CNN experiments such that the reader can locate the architecture information, (2) mathematical formulations for all performance metrics (precision, recall, F1, …) with explicit equations;  (3) detailed statistical procedures including permutation testing methods, power analysis and multiple testing corrections used throughout Figures 7-8. These additions facilitate reproducibility and proper interpretation of all quantitative results presented in the manuscript.

Reviewer #2 (Public review):

(1) The manuscript as written lacks much-needed context in multiple areas: what are the commercially available solutions, and how do they compare to JABS (at least in terms of features offered, not necessarily performance)? What are other open-source options?

JABS adds to a list of commercial and open source animal tracking platforms. There are several reviews and resources that cover these technologies. JABS covers hardware, behavior prediction, a shared resource for classifiers, and genetic association studies. We’re not aware of another system that encompasses all these components. Commercial packages such as EthoVision XT and HomeCage Scan give users a ready-made camera-plus-software solution that automatically tracks each mouse and reports simple measures such as distance travelled or time spent in preset zones, but they do not provide open hardware designs, editable behavior classifiers, or any genetics workflow. At the open-source end, the >100 projects catalogued on OpenBehavior and summarised in recent reviews (Luxem et al., 2023; Işık & Ünal 2023) usually cover only one link in the chain—DIY rigs, pose-tracking libraries (e.g., DeepLabCut, SLEAP) or supervised and unsupervised behaviour-classifier pipelines (e.g., SimBA, MARS, JAABA, B-SOiD, DeepEthogram). JABS provides an open source ecosystem that integrates all four: (i) top-down arena hardware with parts list and assembly guide; (ii) an active-learning GUI that produces shareable classifiers; (iii) a public web service that enables sharing of the trained classifier and applies any uploaded classifier to a large and diverse strain survey; and (iv) built-in heritability, genetic-correlation and GWAS reporting. We have added a concise paragraph in the Discussion that cites these resources and makes this end-to-end distinction explicit.

(2) How does the supervised behavioral classification approach relate to the burgeoning field of unsupervised behavioral clustering (e.g., Keypoint-MoSeq, VAME, B-SOiD)? 

The reviewer raises an important point about the rapidly evolving landscape of automated behavioral analysis, where both supervised and unsupervised approaches offer complementary strengths for different experimental contexts. Unsupervised methods like Keypoint-MoSeq , VAME , and B-SOiD , which prioritize motif discovery from unlabeled data but may yield less precise alignments with expert annotations, as evidenced by lower F1 scores in comparative evaluations. Supervised approaches (like ours), by contrast, employ fully supervised classifiers to deliver frame-accurate, behavior-specific scores that align directly with experimental hypotheses. Ultimately, a pragmatic hybrid strategy, starting with unsupervised pilots to identify motifs and transitioning to supervised fine-tuning with minimal labels, can minimize annotation burdens and enhance both discovery and precision in ethological studies. This has been added in the discussion section of the manuscript.

(3) What kind of studies will this combination of open field + pose estimation + supervised classifier be suitable for? What kind of studies is it unsuited for? These are all relevant questions that potential users of this platform will be interested in.

This approach is suitable for a wide array of neuroscience, genetics, pharmacology, preclinical, and ethology studies. We have published in the domains of action detection for complex behaviors such as grooming, gait and posture, frailty, nociception, and sleep. We feel these tools are indispensable for modern behavior analysis. 

(4) Throughout the manuscript, I often find it unclear what is supported by the software/GUI and what is not. For example, does the GUI support uploading videos and running pose estimation, or does this need to be done separately? How many of the analyses in Figures 4-6 are accessible within the GUI?

We have now clarified these. The JABS framework comprises two distinct GUI applications with complementary functionalities. The JABS-AL (active learning) desktop application handles video upload, behavioral annotation, classifier training, and inference -- it does not perform pose estimation, which must be completed separately using our pose tracking pipeline (https://github.com/KumarLabJax/mouse-tracking-runtime). If a user does not want to use our pose tracking pipeline, we have provided conversions through SLEAP to convert to our JABS pose format.  The web-based GUI enables classifier sharing and cloud-based inference on our curated datasets (JABS600, JABS1200) and downstream behavioral statistics and genetic analyses (Figures 4-6). The JABS-AL application also supports CLI (command line interface) operation for batch processing.  We have clarified these distinctions and provided a comprehensive workflow diagram in the revised Methods section.

(5) While the manuscript does a good job of laying out best practices, there is an opportunity to further improve reproducibility for users of the platform. The software seems likely to perform well with perfect setups that adhere to the JABS criteria, but it is very likely that there will be users with suboptimal setups - poorly constructed rigs, insufficient camera quality, etc. It is important, in these cases, to give users feedback at each stage of the pipeline so they can understand if they have succeeded or not. Quality control (QC) metrics should be computed for raw video data (is the video too dark/bright? are there the expected number of frames? etc.), pose estimation outputs (do the tracked points maintain a reasonable skeleton structure; do they actually move around the arena?), and classifier outputs (what is the incidence rate of 1-3 frame behaviors? a high value could indicate issues). In cases where QC metrics are difficult to define (they are basically always difficult to define), diagnostic figures showing snippets of raw data or simple summary statistics (heatmaps of mouse location in the open field) could be utilized to allow users to catch glaring errors before proceeding to the next stage of the pipeline, or to remove data from their analyses if they observe critical issues.

These are excellent suggestions that align with our vision for improving user experience and data quality assessment. We recognize the critical importance of providing users with comprehensive feedback at each stage of the pipeline to ensure optimal performance across diverse experimental setups. Currently, we provide end-users with tools and recommendations to inspect their own data quality. In our released datasets (Strain Survey OFA and BXD OFA), we provide video-level quality summaries for coverage of our pose estimation models. 

For behavior classification quality control, we employ two primary strategies to ensure proper operation: (a) outlier manual validation and (b) leveraging known characteristics about behaviors. For each behavior that we predict on datasets, we manually inspect the highest and lowest expressions of this behavior to ensure that the new dataset we applied it to maintains sufficient similarity. For specific behavior classifiers, we utilize known behavioral characteristics to identify potentially compromised predictions. As the reviewer suggested, high incidence rates of 1-3 frame bouts for behaviors that typically last multiple seconds would indicate performance issues.

We currently maintain in-house post-processing scripts that handle quality control according to our specific use cases. Future releases of JABS will incorporate generalized versions of these scripts, integrating comprehensive QC capabilities directly into the platform. This will provide users with automated feedback on video quality, pose estimation accuracy, and classifier performance, along with diagnostic visualizations such as movement heatmaps and behavioral summary statistics.

Reviewer #1 (Recommendations for the authors):

(1) A weakness of this tool is that it requires pose tracking, but the manuscript does not detail how pose tracking should be done and whether users should expect that the data deposited will help their pose tracking models. There is no specification on how to generate pose tracking that will be compatible with JABS. The classification quality is directly linked to the quality of the pose tracking. The authors should provide more details of the requirements of the pose tracking (skeleton used) and what pose tracking tools are compatible with JABS. In the user website link, I found no such information. Ideally, JABS would be integrated with the pose tracking tool into a single pipeline. If that is not possible, then the utility of this tool relies on more clarity on which pose tracking tools are compatible with JABS.

The JABS ecosystem was deliberately designed with modularity in mind, separating the pose estimation pipeline from the active learning and classification app (JABS-AL) to offer greater flexibility and scalability for users working across diverse experimental setups. Our pose estimation pipeline is documented in detail within the new Methods subsection, outlining the steps to obtain JABS-compatible keypoints with our recommended runtime (https://github.com/KumarLabJax/mouse-tracking-runtime) and frozen inference models (https://github.com/KumarLabJax/deep-hrnet-mouse). This pipeline is an independent component within the broader JABS workflow, generating skeletonized keypoint data that are then fed into the JABS-AL application for behavior annotation and classifier training.

By maintaining this separation, users have the option to use their preferred pose tracking tools— such as SLEAP —while ensuring compatibility through provided conversion utilities to the JABS skeleton format. These details, including usage instructions and compatibility guidance, are now thoroughly explained in the newly added pose estimation subsection of our Methods section. This modular design approach ensures that users benefit from best-in-class tracking while retaining the full power and reproducibility of our active learning pipeline.

(2) The authors should justify why JAABA was chosen to benchmark their classifier. This tool was published in 2013, and there have been other classification tools (e.g., SIMBA) published since then.  

We appreciate the reviewer’s suggestion regarding SIMBA. However, our comparisons to JAABA and a CNN are based on results from prior work (Geuther, Brian Q., et al. "Action detection using a neural network elucidates the genetics of mouse grooming behavior." Elife 10 (2021): e63207.), where both were used to benchmark performance on our publicly released dataset. In this study, we introduce JABS as a new approach and compare it against those established baselines. While SIMBA may indeed offer competitive performance, we believe the responsibility to demonstrate this lies with SIMBA’s authors, especially given the availability of our dataset for benchmarking.

(3) I had a lot of trouble understanding the elements of the data calculated in JABS vs outside of JABS. This should be clarified in the manuscript.

(a) For example, it was not intuitive that pose tracking was required and had to be done separately from the JABS pipeline. The diagrams and figures should more clearly indicate that.

(b) In section 2.5, are any of those metrics calculated by JABS? Another software GEMMA, but no citation is provided for this tool. This created ambiguity regarding whether this is an analysis that is separate from JABS or integrated into the pipeline.  

We acknowledge the confusion regarding the delineation between JABS components and external tools, and we have comprehensively addressed this throughout the manuscript. The JABS ecosystem consists of three integrated modules: JABS-DA (data acquisition), JABS-AL (active learning for behavior annotation and classifier training), and JABS-AI (analysis and integration via web application). Pose estimation, while developed by our laboratory, operates as a preprocessing pipeline that generates the keypoint coordinates required for subsequent JABS classifier training and annotation workflows. We have now added a dedicated Methods subsection that explicitly maps each analytical step to its corresponding software component, clearly distinguishing between core JABS modules and external tools (such as GEMMA for genetic analysis). Additionally, we have provided proper citations and code repositories for all external pipelines to ensure complete transparency regarding the computational workflow and enable full reproducibility of our analyses.

(4) There needs to be clearer explanations of all metrics, methods, and transformations of the data reported.

(a) There is very little information about the architecture of the classification model that JABS uses.

(b) There are no details on the CNN used for comparing and benchmarking the classifier in JABS.

(c) Unclear how the z-scoring of the behavioral data in Figure 7 was implemented.

(d) There is currently no information on how the metrics in Figure 8 are calculated.

We have added a comprehensive Methods section that not only addresses the specific concerns raised above but provides complete methodological transparency throughout our study. This expanded section includes detailed descriptions of all computational architectures (including the JABS classifier and grooming benchmark models and metrics), statistical procedures and data transformations (including the z-scoring methodology for Figure 7), downstream genetic analysis (including all measures presented in Figure 8), and preprocessing pipelines. 

(5) The authors talk about their datasets having visual diversity, but without seeing examples, it is hard to know what they mean by this visual diversity. Ideally, the manuscript would have a supplementary figure with a representation of the variety of setups and visual diversity represented in the datasets used to train the model. This is important so that readers can quickly assess from reading the manuscript if the pre-trained classifier models could be used with the experimental data they have collected.

The visual diversity of our training datasets has been comprehensively documented in our previous tracking work (https://www.nature.com/articles/s42003-019-0362-1), which systematically demonstrates tracking performance across mice with diverse coat colors (black, agouti, albino, gray, brown, nude, piebald), body sizes including obese mice, and challenging recording conditions with dynamic lighting and complex environments. Notably, Figure 3B in that publication specifically illustrates the robustness across coat colors and body shapes that characterize the visual diversity in our current classifier training data. To address the reviewer's concern and enable readers to quickly assess the applicability of our pre-trained models to their experimental data, we have now added this reference to the manuscript to ground our claims of visual diversity in published evidence.

(6) All figures have a lot of acronyms used that are not defined in the figure legend. This makes the figures really hard to follow. The figure legends for Figures 1,2, 7, and 9 did not have sufficient information for me to comprehend the figure shown.

We have fixed this in the manuscript. 

(7) In the introduction, the authors talk about compression artifacts that can be introduced in camera software defaults. This is very vague without specific examples.

This is a complex topic that balances the size and quality of video data and is beyond the scope of this paper. We have carefully optimized this parameter and given the user a balanced solution. A more detailed blog post on compression artifacts can be found at our lab’s webpage (https://www.kumarlab.org/2018/11/06/brians-video-compression-tests/). We have also added a comment about keyframes shifting temporal features in the main manuscript. 

(8) More visuals of the inside of the apparatus should be included as supplementary figures. For example, to see the IR LEDs surrounding the camera.

We have shared data from JABS as part of several papers including the tracking paper (Geuther et al 2019), grooming, gait and posture, mouse mass. We have also released entire datasets that as part of this paper (JABS1800, JABS-BXD). We also have step by step assembly guide that shows the location of the lights/cameras and other parts (see Methods, JABS workflow guide, and this PowerPoint file in the GitHub repository (https://github.com/KumarLabJax/JABS-datapipeline/blob/main/Multi-day%20setup%20PowerPoint%20V3.pptx).

(9) Figure 2 suggests that you could have multiple data acquisition systems simultaneously. Do each require a separate computer? And then these are not synchronized data across all boxes?

Each JABS-DA unit has its own edge device (Nvidia Jetson). Each system (which we define as multiple JABS-DA areas associated with one lab/group) can have multiple recording devices (arenas). The system requires only 1 control portal (RPi computer) and can handle as many recording devices as needed (Nvidia computer w/ camera associated with each JABS-DA arena). To collect data, 1 additional computer is needed to visit the web control portal and initiate a recording session. Since this is a web portal, users can use any computer or a tablet. The recording devices are not strictly synchronized but can be controlled in a unified manner.

(10) The list of parts on GitHub seems incomplete; many part names are not there.

We thank referee for bringing this to our attention. We have updated the GitHub repository (and its README) which now links out to the design files. 

(11) The authors should consider adding guidance on how tethers and headstages are expected to impact the use of JABS, as many labs would be doing behavioral experiments combined with brain measurements.

While our pose estimation model was not specifically trained on tethered animals, published research demonstrates that keypoint detection models maintain robust performance despite the presence of headstages and recording equipment. Once accurate pose coordinates are extracted, the downstream behavior classification pipeline operates independently of the pose estimation method and would remain fully functional. We recommend users validate pose estimation accuracy in their specific experimental setup, as the behavior classification component itself is agnostic to the source of pose coordinates.

Reviewer #2 (Recommendations for the authors):

(1) "Using software-defaults will introduce compression artifacts into the video and will affect algorithm performance." Can this be quantified? I imagine most of the performance hit comes from a decrease in pose estimation quality. How does a decrease in pose estimation quality translate to action segmentation? Providing guidelines to potential users (e.g., showing plots of video compression vs classifier performance) would provide valuable information for anyone looking to use this system (and could save many labs countless hours replicating this experiment themselves). A relevant reference for the effect of compression on pose estimation is Mathis, Warren 2018 (bioRxiv): On the inference speed and video-compression robustness of DeepLabCut.

Since our behavior classification approach depends on features derived from keypoint, changes in keypoint accuracy will affect behavior segmentation accuracy. We agree that it is important to try and understand this further, particularly with the shared bioRxiv paper investigating the effect of compression on pose estimation accuracy. Measuring the effect of compression on keypoint and behavior classification is a complex task to evaluate concisely, given the number of potential variables to inspect. To list a few variables that should be investigated are: discrete cosine transform quality (Mathis, Warren experiment), Frame Size (Mathis, Warren experiment), Keyframe Interval (new, unique to video data), inter-frame settings (new, unique to video data), behavior of interest, Pose models with compression-augmentation used in training ( https://arxiv.org/pdf/1506.08316?) and type of CNN used (under active development). The simplest recommendation that we can make at this time is that we know compression will affect behavior predictions and that users should be cautious about using our shared classifiers on compressed video data. To show that we are dedicated in sharing these results as we run those experiments, in a related work ( CV4Animals conference accepted paper (https://www.cv4animals.com/) and can be downloaded here https://drive.google.com/file/d/1UNQIgCUOqXQh3vcJbM4QuQrq02HudBLD/view) we have already begun to inspect how changing some factors affect behavior segmentation performance. In this work, we investigate the robustness of behavior classification across multiple behaviors using different keypoint subsets. Our findings in this work is that classifiers are relatively stable across different keypoint subsets. We are actively working on follow-up effort to investigate the effect of keypoint noise, CNN model architecture, and other factors we've listed above on behavior segmentation tasks.

(2) The analysis of inter-annotator variability is very interesting. I'm curious how these differences compare to two other types of variability:

(a) intra-annotator variability; I think this is actually hard to quantify with the presented annotation workflow. If a given annotator re-annotated a set of videos, but using different sparse subsets of the data, it is not possible to disentangle annotator variability versus the effect of training models on different subsets of data. This can only be rigorously quantified if all frames are labeled in each video.

We propose an alternative approach to behavior classifier development in the text associated with Figure 3C. We do not advocate for high inter-annotator agreement since individual behavior experts have differing labeling style (an intuitive understanding of the behavior). Rather, we allow multiple classifiers for the same behavior and allow the end user to prioritize classifiers based on heritability of the behavior from a classifier.  

(b) In lieu of this, I'd be curious to see the variability in model outputs trained on data from a single annotator, but using different random seeds or train/val splits of the data. This analysis would provide useful null distributions for each annotator and allow for more rigorous statistical arguments about inter-annotator variability. 

JABS allows the user to use multiple classifiers (random forest, XGBoost). We do not expect the user to carry out hyperparameter tuning or other forms of optimization. We find that the major increase in performance comes from optimizing the size of the window features and folds of cross validation. However, future versions of JABS-AL could enable a complete hyper-parameter scan across seeds and data splits to obtain a null distribution for each annotator. 

(c) I appreciate the open-sourcing of the video/pose datasets. The authors might also consider publicly releasing their pose estimation and classifier training datasets (i.e., data plus annotations) for use by method developers.

We thank the referee for acknowledging our commitment to open data sharing practices. Building upon our previously released strain survey dataset, we have now also made our complete classifier training resources publicly available, including the experimental videos, extracted pose coordinates, and behavioral annotations. The repository link has been added to the manuscript to ensure full reproducibility and facilitate community adoption of our methods.  

(3) More thorough discussion on the limitations of the top-down vs bottom-up camera viewpoint; are there particular scientific questions that are much better suited to bottomup videos (e.g., questions about paw tremors, etc.).

Top-down imaging, bottom-up, and multi-view imaging have a variety of pros and cons. Generally speaking, multi-view imaging will provide the most accurate pose models but requires increased resources on both hardware setup as well as processing of data. Top-down provides the advantage of flexibility for materials, since the floor doesn’t need to be transparent. Additionally lighting and potential reflection with the bottom-up perspective. Since the paws are not occluded from the bottom-up perspective, models should have improved paw keypoint precision allowing the model to observe more subtle behaviors. However, the appearance of the arena floor will change over time as the mice defecate and urinate. Care must be taken to clean the arena between recordings to ensure transparency is maintained. This doesn’t impact top-down imaging that much but will occlude or distort from the bottom-up perspective. Additionally, the inclusion of bedding for longer recordings, which is required by IACUC, will essentially render bottom-up imaging useless because the bedding will completely obscure the mouse. Overall, while bottomup may provide a precision benefit that will greatly enhance subtle motion, top-down imaging is overall more robust for obtaining consistent imaging across large experiments for longer periods of time.

(4) More thorough discussion on what kind of experiments would warrant higher spatial or temporal resolution (e.g., investigating slight tremors in a mouse model of neurodegenerative disease might require this greater resolution).

This is an important topic that deserves its own perspective guide. We try to capture some of this in the paper on specifications. However, we only scratch the surface. Overall, there are tradeoffs between frame rate, resolution, color/monochrome, and compression. Labs have collected data at hundreds of frames per second to capture the kinetics of reflexive behavior for pain (AbdoosSaboor lab) or whisking behavior. Labs have also collected data a low 2.5 frames per second for tracking activity or centroid tracking (see Kumar et al PNAS). The data collection specifications are largely dependent on the behaviors being captured. Our rule of thumb is the Nyquist Limit, which states that the data capture rate needs to be twice that of the frequency of the event. For example, certain syntaxes of grooming occur at 7Hz and we need 14FPS to capture this data. JABS collects data at 30FPS, which is a good compromise between data load and behavior rate. We use 800x800 pixel resolution which is a good compromise to capture animal body parts while limiting data size. Thank you for providing the feedback that the field needs guidance on this topic. We will work on creating such guidance documents for video data acquisition parameters to capture animal behavior data for the community as a separate publication.

(5) References 

(a) Should add the following ref when JAABA/MARS are referenced: Goodwin et al.2024, Nat Neuro (SimBA)

(b) Could also add Bohnslav et al. 2021, eLife (DeepEthogram).

(c) The SuperAnimal DLC paper (Ye et al. 2024, Nature Comms) is relevant to the introduction/discussion as well.

We thank the referee for the suggestions. We have added these references.  

(6) Section 2.2:

While I appreciate the thoroughness with which the authors investigated environmental differences in the JABS arena vs standard wean cage, this section is quite long and eventually distracted me from the overall flow of the exposition; might be worth considering putting some of the more technical details in the methods/appendix.

These are important data for adopters of JABS to gain IACUC approval in their home institution. These committees require evidence that any new animal housing environment has been shown to be safe for the animals. In the development of JABS, we spent a significant amount of time addressing the JAX veterinary and IACUC concerns. Therefore, we propose that these data deserve to be in the main text. 

(7) Section 2.3.1:

(a) Should again add the DeepEthogram reference here

(b) Should reference some pose estimation papers: DeepLabCut, SLEAP, Lightning Pose. 

We thank the referee for the suggestions. We have added these references.  

(c) "Pose based approach offers the flexibility to use the identified poses for training classifiers for multiple behaviors" - I'm not sure I understand why this wouldn't be possible with the pixel-based approach. Is the concern about the speed of model training? If so, please make this clearer.

The advantage lies not just in training speed, but in the transferability and generalization of the learned representations. Pose-based approaches create structured, low-dimensional latent embeddings that capture behaviorally relevant features which can be readily repurposed across different behavioral classification tasks, whereas pixel-based methods require retraining the entire feature extraction pipeline for each new behavior. Recent work demonstrates that pose-based models achieve greater data efficiency when fine-tuned for new tasks compared to pixel-based transfer learning approaches [1], and latent behavioral representations can be partitioned into interpretable subspaces that generalize across different experimental contexts [2]. While pixel-based approaches can achieve higher accuracy on specific tasks, they suffer from the "curse of dimensionality" (requiring thousands of pixels vs. 12 pose coordinates per frame) and lack the semantic structure that makes pose-based features inherently reusable for downstream behavioral analysis.

(1) Ye, Shaokai, et al. "SuperAnimal pretrained pose estimation models for behavioral analysis." Nature communications 15.1 (2024): 5165.

(2) Whiteway, Matthew R., et al. "Partitioning variability in animal behavioral videos using semi-supervised variational autoencoders." PLoS computational biology 17.9 (2021): e1009439.  

(d) The pose estimation portion of the pipeline needs more detail. Do users use a pretrained network, or do they need to label their own frames and train their own pose estimator? If the former, does that pre-trained network ship with the software? Is it easy to run inference on new videos from a GUI or scripts? How accurate is it in compliant setups built outside of JAX? How long does it take to process videos?

We have added the guidance on pose estimation in the manuscript (section “2.3.1 Behavior annotation and classifier training” and in the methods section titled “Pose tracking pipeline”)

(e) The final paragraph describing how to arrive at an optimal classifier is a bit confusing - is this the process that is facilitated by the app, or is this merely a recommendation for best practices? If this is the process the app requires, is it indeed true that multiple annotators are required? While obviously good practice, I imagine there will be many labs that just want a single person to annotate, at least in the beginning prototyping stages. Will the app allow training a model with just a single annotator?

We have clarified this in the text. 

(8) Section 2.5:

(a) This section contained a lot of technical details that I found confusing/opaque, and didn't add much to my overall understanding of the system; sec 2.6 did a good job of clarifying why 2.5 is important. It might be worth motivating 2.5 by including the content of 2.6 first, and moving some of the details of 2.5 to the method/appendix.

We moved some of the technical details in section 2.5 to the methods section titled “Genetic analysis”. Furthermore, we have added few statements to motivate the need of genetic analysis and how the webapp can facilitate this (which is introduced in the section 2.6)    

(9) Minor corrections:

(a) Bottom of first page, "always been behavior quantification task" missing "a".

(b) "Type" column in Table S2 is undocumented and unused (i.e., all values are the same); consider removing.

(c) Figure 4B, x-axis: add units.

(d) Page 8/9: all panel references to Figure S1 are off by one

We have fixed them in the updated manuscript.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Public review):

This paper by Poverlein et al reports the substantial membrane deformation around the oxidative phosphorylation super complex, proposing that this deformation is a key part of super complex formation. I found the paper interesting and well-written.

We thank the Reviewer for finding our work interesting. 

Analysis of the bilayer curvature is challenging on the fine lengthscales they have used and produces unexpectedly large energies (Table 1). Additionally, the authors use the mean curvature (Eq. S5) as input to the (uncited, but it seems clear that this is Helfrich) Helfrich Hamiltonian (Eq. S7). If an errant factor of one half has been included with curvature, this would quarter the curvature energy compared to the real energy, due to the squared curvature.

We thank the Reviewer for raising this important issue. We have now clarified in the SI and main manuscript that we employ the Helfrich model. In our initial implementation, we indeed used the mean curvature H, thereby missing a factor of 2. As the Reviewer correctly noted, this resulted in curvature deformation energies that were underestimated by a factor of ~4. We have now corrected for this effect in the revised analysis, and the updated Table 1. Importantly, however, this correction does not alter the general conclusions of our work that supercomplex formation relieves membrane strain and stabilizes the system. We have added an additional paragraph where we discuss the magnitude of the observed bending effects, and compared the previous estimates in literature:

SI: 

“The local mean curvature of the membrane midplane was computed using the Helfrich model (4,5) …”

(4) W. Helfrich, Elastic properties of lipid bilayers theory and possible experiments. Zeitschrift für Naturforschung 28c, 693-703 (1973).

(5) F. Campelo et al., Helfrich model of membrane bending: From Gibbs theory of liquid interfaces to membranes as thick anisotropic elastic layers. Advances in Colloid and Interface Science 208, 25-33 (2014).

Main Text: 

“which measures the energetic cost of deforming the membrane from a flat geometry (ΔG_curv) based on the Helfrich model (45, 46). …

Our analysis suggests that both contributions are substantially reduced upon formation of the SC, with the curvature penalty decreasing by 79.2 ± 5.2 kcal mol^-1 (for a membrane area of ca. 1000 nm²) and the thickness penalty by 2.8 ± 2.0 kcal mol^-1 (Table 1).”

“We note that the magnitude of the estimated bending energies (~10² kcal mol^-1) (Table 1), while seemingly high at first glance, falls within the range expected for large-scale membrane deformation processes induced by large multi-domain proteins. For example, the Piezo mechanosensitive channel performs roughly 150k_BT (≈ 90 kcal mol⁻¹) of work to bend the bilayer into its dome-like shape (65). Comparable energies have also been estimated for the nucleation of small membrane pores (66), while vesicle formation typically requires bending energies on the order of 300 kcal mol^-1, largely independent of vesicle size (67). When normalized by the affected membrane area (~1000 nm²), these values correspond to an energy density of approximately 0.1 kcal mol^-1 nm^-2, which places our estimates within a biophysically reasonable regime. Notably, cryo-EM structures of several supercomplexes shows that such assemblies can impose significant curvature on the surrounding bilayer (36, 50, 68), supporting the notion that respiratory chain organization is closely coupled to local membrane deformation. Nevertheless, we expect that the absolute deformation energies may be overestimated, as the continuum Helfrich model neglects molecular-level effects such as lipid tilt and local rearrangements, which can partially relax curvature stresses and reduce the effective bending penalty near protein–membrane interfaces (69, 70).”

The bending modulus used (ca. 5 kcal/mol) is small on the scale of typically observed biological bending moduli. This suggests the curvature energies are indeed much higher even than the high values reported. Some of this may be due to the spontaneous curvature of the lipids and perhaps the effect of the protein modifying the nearby lipids properties.

The SI initially included an incorrect value for the bending modulus (20 kJ mol^-1 instead of 20k_BT), which has now been corrected. The revised value is consistent with experimentally reported bending moduli from X-ray scattering measurements, although there remains substantial uncertainty in the precise values across different experimental and computational studies.

“The bending deformation energy was computed from the mean curvature field H(x,y), assuming a constant bilayer bending modulus κ (taken as 20k_bT  = 11.85 kcal mol^-1 (6)):”

(6) S. Brown et al., Comparative analysis of bending moduli in one-component membranes via coarsegrained molecular dynamics simulations. Biophysical Journal 124, 1–13 (2025).

It is unclear how CDL is supporting SC formation if its effect stabilizing the membrane deformation is strong or if it is acting as an electrostatic glue. While this is a weakenss for a definite quantification of the effect of CDL on SC formation, the study presents an interesting observation of CDL redistribution and could be an interesting topic for future work.

We agree with the Reviewer that future studies would be important to investigate the relationship between CDL-induced stabilization of membrane and its electrostatic effects.  

In summary, the qualitative data presented are interesting (especially the combination of molecular modeling with simpler Monte Carlo modeling aiding broader interpretation of the results). The energies of the membrane deformations are quite large. This might reflect the roles of specific lipids stabilizing those deformations, or the inherent difficulty in characterizing nanometer-scale curvature.

We thank the Reviewer for appreciating our work and for the help in further improving our findings.

Reviewer #3 (Public review):

Summary:

In this contribution, the authors report atomistic, coarse-grained and lattice simulations to analyze the mechanism of supercomplex (SC) formation in mitochondria. The results highlight the importance of membrane deformation as one of the major driving forces for the SC formation, which is not entirely surprising given prior work on membrane protein assembly, but certainly of major mechanistic significance for the specific systems of interest.

We thank Reviewer 3 for appreciating the importance of our study. 

Strengths:

The combination of complementary approaches, including an interesting (re)analysis of cryo-EM data, is particularly powerful, and might be applicable to the analysis of related systems. The calculations also revealed that SC formation has interesting impacts on the structural and dynamical (motional correlation) properties of the individual protein components, suggesting further functional relevance of SC formation. In the revision, the authors further clarified and quantified their analysis of membrane responses, leading to further insights into membrane contributions. They have also toned down the decomposition of membrane contributions into enthalpic and entropic contributions, which is difficult to do. Overall, the study is rather thorough, highly creative and the impact on the field is expected to be significant.

Weaknesses:

Upon revision, I believe the weakness identified in previous work has been largely alleviated.

We thank the Reviewer for their previous remarks, which allowed us to significantly improve our manuscript.

augmenting what the rest of humanity can do

Author response:

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

Reviewer #1 (Public review):

Circannual timing is a phylogenetically widespread phenomenon in long-lived organisms and is central to the seasonal regulation of reproduction, hibernation, migration, fur color changes, body weight, and fat deposition in response to photoperiodic changes. Photoperiodic control of thyroid hormone T3 levels in the hypothalamus dictates this timing. However, the mechanisms that regulate these changes are not fully understood. The study by Stewart et al. reports that hypothalamic iodothyronine deiodinase 3 (Dio3), the major inactivator of the biologically active thyroid hormone T3, plays a critical role in circannual timing in the Djungarian hamster. Overall, the study yields important results for the field and is well-conducted, with the exception of the CRISPR/Cas9 manipulation.

We appreciate the positive and supportive comment from the Reviewer. We have clarified the oversight in the Crispr/Cas9 data representation below. Our correction should alleviate any concern raised.

Figure 1 lays the foundation for examining circannual timing by establishing the timing of induction, maintenance, and recovery phases of the circannual timer upon exposure of hamsters to short photoperiod (SP) by monitoring morphological and physiological markers. Measures of pelage color, torpor, body mass, plasma glucose, etc, established that the initiation phase occurred by weeks 4-8 in SP, the maintenance by weeks 12-20, and the recovery after week 20, where all morphological and physiological changes started to reverse back to long photoperiod phenotypes.

The statistical analyses look fine, and the results are unambiguous.

We thank the Reviewer for recognizing our attempts to highlight the phenomenon of circannual interval timing.

Their representation could, however, be improved. In Figures 1d and 1e, two different measures are plotted on each graph and differentiated by dots and upward or downward arrowheads. The plots are so small, though, that distinguishing between the direction of the arrows is difficult. Some color coding would make it more reader-friendly. The same comment applies to Figure S4. 

We have increased the panel size for Figure 1d and 1e. We have also changed the colour of the graphs in Figure 1d and 1e to facilitate the differentiation of the two dependent variables. For the circos plots, we attempted different ways to represent the data. We have opted to keep the figures in their current stage. The overall aim is to provide a ‘gestalt’ view of the timing of changes in transcript expression and highlighted only a few key genes. The whole dataset is provided in the supplementary materials for Reviewer/Reader interrogation.

The authors went on to profile the transcriptome of the mediobasal and dorsomedial hypothalamus, paraventricular nucleus, and pituitary gland (all known to be involved in seasonal timing) every 4 weeks over the different phases of the circannual interval timer. A number of transcripts displaying seasonal rhythms in expression levels in each of the investigated structures were identified, including transcripts whose expression peaks during each phase. This included two genes of particular interest due to their known modulation of expression in response to photoperiod, Dio3 and Sst, found among the transcripts upregulated during the induction and maintenance phases, respectively. The experiments are technically sound and properly analyzed, revealing interesting candidates. Again, my main issues lie with the representation in the figure. In particular, the authors should clarify what the heatmaps on the right of Figures 1f and 1g represent. I suspect they are simply heatmaps of averaged expression of all genes within a defined category, but a description is missing in the legend, as well as a scale for color coding near the figure.

We have clarified the heatmap and density maps in the Figure legend. We apologise for the lack of information to describe the figure panels. (see lines 644-648)

Figure 2 reveals that SP-programmed body mass loss is correlated to increased Dio3-dependent somatostatin (Sst) expression. First, to distinguish whether the body mass loss was controlled by rheostatic mechanisms and not just acute homeostatic changes in energy balance, experiments from hamsters fed ad lib or experiencing an acute food restriction in both LP and SP were tested. Unlike plasma insulin, food restriction had no additional effect on SP-driven epididymal fat mass loss (Figure S7). This clearly establishes a rheostatic control of body mass loss across weeks in SP conditions. Importantly, Sst expression in the mediobasal hypothalamus increased in both ad lib fed or restriction fed SP hamsters and this increase in expression could be reduced by a single subcutaneous injection of active T3, clearly suggesting that increase in Sst expression in SP is due to a decrease of active T3 likely via Dio3 increase in expression in the hypothalamus. The results are unambiguous

We thank the Reviewer for the supportive and affirmative feedback.

Figure 3 provides a functional test of Dio3's role in the circannual timer. Mediobasal hypothalamic injections of CRISPR-Cas9 lentiviral vectors expressing two guide RNAs targeting the hamster Dio3 led to a significant reduction in the interval between induction and recovery phases seen in SP as measured by body mass, and diminished the extent of pelage color change by weeks 15-20. In addition, hamsters that failed to respond to SP exposure by decreasing their body mass also had undetectable Dio3 expression in the mediobasal hypothalamus. Together, these data provide strong evidence that Dio3 functions in the circannual timer. I noted, however, a few problems in the way the CRISPR modification of Dio3 in the mediobasal hypothalamus was reported in Figure S8. One is in Figure S8b, where the PAM sites are reported to be 9bp and 11bp downstream of sgRNA1 and sgRNA2, respectively. Is this really the case? If so, I would have expected the experiment to fail to show any effect as PAM sites need to immediately follow the target genomic sequence recognized by the sgRNA for Cas9 to induce a DNA double-stranded break. It seems that each guide contains a 3' NGG sequence that is currently underlined as part of sgRNAs in both Fig S8b and in the method section. If this is not a mistake in reporting the experimental design, I believe that the design is less than optimal and the efficiencies of sgRNAs are rather low, if at all functional.

We apologize for the oversight and indeed the reporting in Figure S8b was a mistake. The PAM site previously indicated was the ‘secondary PAM site’ (which as the Reviewer notes would likely have low efficiency). The PAM site is described within the gRNA in the figure. We use Adobe Illustrator to generate figures, and during the editing process, the layer for PAM text was accidentally moved ‘back’ to a lower level. The oversight was not rectified before submission. We apologise for this unreservedly. The PAM site text has been moved forward, to highlight the location of the primary site (ie immediately following gRNA) and labelled the gRNA and PAM site in the ‘Target region’. The secondary PAM site text was removed to eliminate any confusion.

The authors report efficiencies around 60% (line 325), but how these were obtained is not specified. 

The efficiency provided are based on bioinformatic analyses and not in vivo assays. To reduce any confusion, we have removed the text. The gRNA were clearly effective to induce mutations based on the sequencing analyses.

Another unclear point is the degree to which the mediobasal hypothalamus was actually mutated. Only one mutated (truncated) sequence in Figure S8c is reported, but I would have expected a range of mutations in different cells of the tissue of interest.

The tissue punch would include multiple different cells (e.g., neuronal, glial, etc). We agree with the Reviewer that genomic samples from different cells would be included in the sequencing analyses. Given the large mutation in the target region, the gRNA was effective. We have only shown one representative sequence. If the Reviewer would like to see all mutations, we can easily show the other samples.

Although the authors clearly find a phenotypic effect with their CRISPR manipulation, I suspect that they may have uncovered greater effects with better sgRNA design. These points need some clarification. I would also argue that repeating this experiment with properly designed sgRNAs would provide much stronger support for causally linking Dio3 in circannual timing.

The gRNA was designed using the Gold-standard approach – ChopChop [citation Labon et al., 2019]. If the Reviewer’s concern re design is due to the comment above re PAM site; this issue was clarified and there are no concerns for the gRNA design. The major challenge with the Dio3 gene (single exon) with a very short sequence length (approx.. 412bp). There is limited scope within this sequence length to generate gRNA.

A proposed schematic model for mechanisms of circannual interval timing is presented in Figure S9. I think this represents a nice summary of the findings put in a broader context and should be presented as a main figure in the manuscript itself rather than being relayed in supplementary materials.

We agree with the Reviewer position and moved the figure to the main manuscript. The figure is now Figure 4.

Reviewer #2 (Public review):

Several animals and plants adjust their physiology and behavior to seasons. These changes are timed to precede the seasonal transitions, maximizing chances of survival and reproduction. The molecular mechanisms used for this process are still unclear. Studies in mammals and birds have shown that the expression of deiodinase type-1, 2, and 3 (Dio1, 2, 3) in the hypothalamus spikes right before the transition to winter phenotypes. Yet, whether this change is required or an unrelated product of the seasonal changes has not been shown, particularly because of the genetic intractability of the animal models used to study seasonality. Here, the authors show for the first time a direct link between Dio3 expression and the modulation of circannual rhythms.

We appreciate the clear synthesis and support for the manuscript.

Strengths:

The work is concise and presents the data in a clear manner. The data is, for the most part, solid and supports the author's main claims. The use of CRISPR is a clear advancement in the field. This is, to my knowledge, the first study showing a clear (i.e., causal) role of Dio3 in the circannual rhythms in mammals. Having established a clear component of the circannual timing and a clean approach to address causality, this study could serve as a blueprint to decipher other components of the timing mechanism. It could also help to enlighten the elusive nature of the upstream regulators, in particular, on how the integration of day length takes place, maybe within the components in the Pars tuberalis, and the regulation of tanycytes.

We thank the Reviewer for this positive summary.

Weaknesses:

Due to the nature of the CRISPR manipulation, the low N number is a clear weakness. This is compensated by the fact that the phenotypes shown here are strong enough. Also, this is the only causal evidence of Dio3's role; thus, additional evidence would have significantly strengthened the author's claims. The use of the non-responsive population of hamsters also helps, but it falls within the realm of correlations.

We would also like to remind the Reviewer that one Crispr-Cas9 Dio3^cc treated hamster did not show any mutation in the genome. This hamster was observed to have a change in body mass and pelage colour like controls. This animal provides another positive control.

We also conducted a statistical power analysis to examine whether n=3 is sufficient for the Dio3^cc treatment group. Using the appropriate expected difference in means and standard deviations for an alpha of 0.05; we regularly observed beta >0.8 across the dependent variables. 

Additionally, the consequences of the mutations generated by CRISPR are not detailed; it is not clear if the mutations affect the expression of Dio3 or generate a truncation or deletion, resulting in a shorter protein.

We agree with the Reviewer that transcript and protein assays would strengthen the genome mutation data. Due to the small brain region under investigation, we are limited in the amount of biological material to extract. Dio3 is an intronless gene and very short – approximately 412 base pairs in length. We opted to maximize resources into sequencing the gene as the confirmation of genetic mutation is paramount. Given the large size of the mutation in the treated hamsters, there would be no amplification of transcript or protein translated.

Reviewer #3 (Public review):

The authors investigated SP-induced physiological and molecular changes in Djungarian hamsters and the endogenous recovery from it after circa half a year. The study aimed to elucidate the intrinsic mechanism and included nice experiments to distinguish between rheostatic effects on energy state and homeostatic cues driven by an interval timer. It also aimed to elucidate the role of Dio3 by introducing a targeted mutation in the MBH by ICV. The experiments and analyses are sound, and the amount of work is impressive. The impact of this study on the field of seasonal chronobiology is probably high.

We thank the Reviewer for their positive comments and support for our work.

Even though the general conclusions are well-founded, I have fundamental criticism concerning 3 points, which I recommend revising:

(1) The authors talk about a circannual interval timer, but this is no circannual timer. This is a circasemiannual timer. It is important that the authors use precise wording throughout the manuscript.

We agree with the Reviewer that the change in physiology and behaviour does not approximate a full year (e.g. annual) and only a half of the year. We opted to use circannual timer as this term is established in the field (see doi: 10.1177/0748730404266626; doi: 10.1098/rstb.2007.2143). We cannot identify any publication that has used the term ‘semiannual timer’. We do not feel this manuscript is the appropriate time to introduce a new term to the field; we will endeavour to push the field to consider the use of ‘semiannual timer’. A Review or Opinion paper is best place for this discussion. We hope the Reviewer will understand our position.

(2) The authors put their results in the context of clocks. For example, line 180/181 seasonal clock. But they have described and investigated an interval timer. A clock must be able to complete a full cycle endogenously (and ideally repeatedly) and not only half of it. In contrast, a timer steers a duration. Thus, it is well possible that a circannual clock mechanism and this circa-semiannual timer of photoperiodic species are 2 completely different mechanisms. The argumentation should be changed accordingly.

We agree with the Reviewers definitions of circannual ‘clock’ and ‘timer’. We were careful to distinguish between the two concepts early in the manuscript (lines 41-46). We have added italics to emphasis the different terms. The use of seasonal clock on line 180/191 was imprecise and we appreciate the Reviewer highlighting our oversight and the text was revised. We have also revised the Abstract accordingly.

(3) The authors chose as animal model the Djungarian hamster, which is a predominantly photoperiodic species and not a circannual species. A photoperiodic species has no circannual clock. That is another reason why it is difficult to draw conclusions from the experiment for circannual clocks. However, the Djungarian hamster is kind of "indifferent" concerning its seasonal timing, since a small fraction of them are indeed able to cycle (Anchordoquy HC, Lynch GR (2000), Evidence of an annual rhythm in a small proportion of Siberian hamsters exposed to chronic short days. J Biol Rhythms 15:122-125.). Nevertheless, the proportion is too small to suggest that the findings in the current study might reflect part of the circannual timing. Therefore, the authors should make a clear distinction between timers and clocks, as well as between circa-annual and circa-semiannual durations/periods.

This comment is not clear to us. The Reviewer states the hamsters are not a circannual species, but then highlight one study that shows circannual rhythmicity. We agree that circannual rhythmicity in Djungarian hamsters is dependent on the physiological process under investigation (e.g. body mass versus reproduction) and that photoperiodic response system either dampen or mask robust cycles. We have corrected the text oversight highlighted above and the manuscript is focused on interval timers. We have kept the term circannual over semicircannual due to the prior use in the scientific literature.

Reviewing Editor Comments:

The detailed suggestions of the reviewers are outlined below (or above in case of reviewer 1). In light of the criticism, we ask the authors to especially pay attention to the comments on the Cas9/Crisp experiment, raised by Reviewers 1 and 2. As currently described, there are serious questions on the design of the sgRNAs, and also missing critical methodological details. If the latter are diligently taken care of, they may resolve the questions on the sgRNA design. Please also reconsider the wording along the suggestions of Reviewer 3.

We appreciate the Editors time and support for the manuscript. We have clarified and corrected our oversight for the PAM site. This correction confirms the strength of the Crispr-cas9 gRNA used in the study. The correction should remove all concerns. We have also considered using semicircannual in the text. As there is existing scientific literature using circannual interval timer, and there is no publication to our knowledge for using ‘semicircannual; we have opted to keep with the current approach and use circannual. We feel a subsequent Opinion paper is more suitable to introduce a new term.

Reviewer #2 (Recommendations for the authors):

First, I want to commend the authors for their work. It is a clear advancement for our field. Below are a couple of comments and suggestions I have:

we thank the Review for the positive comment and support. We have endeavoured to incorporate their suggested improvements to the manuscript.

(1) Looking at the results of Figure 1A and Figure S8, the control in S8 showed a lower pelage color score as compared to the hamsters in 1A. Is this a byproduct of the ICV injection?

The difference between Figure 1 and 3 is likely due to the smaller sample sizes. The controls in Figure 1 had a higher proportion of hamsters show complete white fur (score =3) at 1618 weeks compared to controls in Figure 3. It is possible, although unlikely that the ICV injection would reduce the development of winter phenotype. There was no substance in the ICV injection that would impact the prolactin signalling pathway. Our perspective is that the difference between the two figures is due to the different sampling population. Overall, the timing of the change in pelage colour is the same between the figures and suggest that the mechanisms of interval timer were unaffected.

(2) Is there a particular reason why the pelage color for the CRISPR mutants is relegated to the supplemental information? In my opinion, this is also important, even though the results might be difficult to explain. Additionally, did the authors check for food intake and adipose mass in these animals?

We agree with the Reviewer the pelage change is very interesting. We decided to have Figure 3 focus on body mass. The rationale was due to the robust nature of the data collection from Crispr-cas9 study (Fig.3b), in addition to the non-responsive hamsters (Fig.3e). We disagree that the data patterns are hard to explain, as pelage changes was similar to the photoperiodic induced change in body mass. No differences were observed for food intake or adipose tissue. We have added this information in the text (see lines 162-163).

(3) I might have missed it, but did the authors check for the expression of Dio3 on the CRISPR mutants? Does the deletion cause reduced expression or any other mRNA effect, such as those resulting in the truncation of a protein?

Due to the limited biological material extracted from the anatomical punches, we decided to focus on genomic mutations. Dio3 has a very short sequence length and the size of the mutations identified indicate that no RNA could be transcribed.

(4) Could the authors clarify which reference genome or partial CDS (i.e., accession numbers) they used to align the gRNA? Did they use the SSSS strain or the Psun_Stras_1 isolate?

The gRNAs were designed using the online tool CHOPCHOP, using the Mus musculus

Dio3 gene. The generated gRNAs were subsequently aligned via blast with the Phodopus sungorus Dio3 partial cds (GenBank: MF662622.1), to ensure alignment with the species. We are confident that the gRNA designed align 100% in hamsters. Furthermore, we conducted BLAST to ensure there were no off-targets. The only gene identified in the BLAST was the rodent (i.e. hamster, mouse) Dio3 sequence.

(5) Figure 3b. I do agree with the authors in pointing out that the decrease in body mass is occurring earlier in Dio3wt hamsters; however, the shape of the body mass dynamic is also different. Do the authors have any comments on the possible role of Dio3 in the process of exist of overwintering?

This is a very interesting question. We do not have the data to evaluate the role of Dio3 for overwintering. We argue that disruption in Dio3 reduced the circannual interval period. For this interpretation, yes, Dio3 is necessary for overwintering. However, we would need to show the sufficiency of Dio3 to induce the winter phenotype in hamsters housed in long photoperiod. At this time, we do not have the technical ability to conduct this experiment.

(6) In Figure 3d, the Dio3wt group does not show any dispersion. Is this correct? If that's true, and no dispersion is observed, no normality can be assumed, and a t-test can't be performed (Line 692).The Mann-Whitney test might be better suited.

We conducted a Welch’s t-test to compare the difference in body mass period. We used the Welch’s test as the variance were not equal; Mann-Whitney test is best for skewed distributions. To clarify the test used, we have added ‘Welch’s test’ to the Figure legend.

(9) Figure 1 h. It might be convenient to add the words "Induction", "maintenance", and "recovery" over each respective line on the polar graph for easier reading.

We have added the text as suggested by the Reviewer.

Reviewer #3 (Recommendations for the authors):

(1) Figure 1: Please enlarge all partial graphics at least to the size of Figure 2. In the print version, labels are barely readable

we have increased the panels in Figure 1 and 3 by 20% to accommodate the Reviewers suggestion.

(2) Legend Figure 2: Add that the food restriction was 16h.

We have added 16h to the text.

(3) Figure 3b: enlarge font size. In the legend: Dio3cc hamsters delayed.... The delay might have been a week or so, but not more (and even that is unclear since the rise in body mass in that week seems to be rather a disturbance of the curve). Thus 'delay' might not be the most appropriate wording. Instead, the initial decline is slower, but both started at nearly the same week (=> no delay). Minimum body mass is reached at the identical week as in wt (=> no delay). Also, the increase started at the same week but was much faster in Dio3cc than in wt. Figure 3c: How can there be a period when there is no repeated cycle (rhythm)? This is rather a duration. Moreover, according to the displayed data, I am wondering which start point and which endpoint is used. The first and last values are the highest of the graph, but have they been the maximum? Especially for Dio3wt, it can be assumed that animals haven't reached the maximum at the end of the graph.

We have increased the font size in Figure 3b. We have changed ‘delayed’ to ‘slower’ in the text. Period analyses, such as the Lomb-Scargle measure the duration of a cycle (and multiple cycles). The start point and end point used in the analyses were the initial data collection date (week 0) and the final data collection date (week 32). The Lomb-Scargle analyses determines the duration of the period that occurs within these phases of the cycle. We believe the period analyses conducted by the Lomb-Scargle is the most suitable for the scientific question.

(4) Figure S9: This is a very nice graph and summarises your main results. It should appear in the main manuscript and not in the supplements.

We appreciate the positive comment and suggestion. We agree with the Reviewer and have move the graph to the main figure. The revised manuscript indicates the graph as Figure 4.

Synthèse des "Rendez-vous de la techno" : La filière STI2D

Résumé

Ce document synthétise les informations et témoignages présentés lors de l'événement "Les rendez-vous de la techno" consacré à la filière Sciences et Technologies de l'Industrie et du Développement Durable (STI2D).

La filière STI2D se positionne comme une voie d'excellence scientifique et technologique, conçue pour les élèves qui privilégient l'apprentissage par la pratique, la manipulation et la réalisation de projets concrets, en contraste avec l'approche plus théorique de la voie générale.

Elle s'adresse à des profils créatifs, aimant le travail en groupe, la résolution de problèmes et l'innovation.

Le cursus est structuré pour fournir des connaissances solides en sciences, technologie, mathématiques et ingénierie, tout en développant une sensibilité aux enjeux industriels et environnementaux.

La pédagogie, axée sur des projets concrets comme la conception d'une voiture solaire ou la modélisation 3D de châteaux, permet aux élèves de mettre en œuvre leurs compétences de manière tangible.

La filière STI2D se distingue par la grande diversité des poursuites d'études qu'elle autorise.

Elle ouvre aussi bien la voie à des études courtes (BTS, BUT) qu'à des parcours longs et exigeants menant aux plus hautes qualifications (Classes Préparatoires aux Grandes Écoles TSI, écoles d'ingénieurs, licences universitaires).

Les témoignages d'élèves et d'étudiants confirment que la filière constitue un tremplin efficace vers la réussite, y compris pour des élèves se réorientant depuis la voie générale, et que ses diplômés sont recherchés dans de nombreux secteurs d'activité de pointe.

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1. Présentation Générale de la Filière STI2D

1.1. Public Cible et Profil de l'Élève

La filière STI2D est accessible après une classe de seconde générale et technologique.

Elle est particulièrement adaptée aux élèves présentant les caractéristiques suivantes :

• Intérêt pour la technologie et les sciences : Un goût prononcé pour la manipulation, la compréhension des phénomènes physiques et la mise en œuvre de solutions techniques.

• Esprit pratique et créatif : L'envie de travailler en groupe sur des projets, de résoudre des problèmes concrets et de faire preuve de créativité et d'innovation.

• Ambition : La filière attire des élèves qui envisagent des carrières d'ingénieur ou de technicien supérieur.

Selon Mme Amarante, le choix de cette filière correspond à un profil qui "aime la technologie", qui est "plutôt créatif", qui "aime aussi résoudre des problèmes, trouver des solutions".

1.2. Compétences et Connaissances Acquises

Le baccalauréat STI2D est présenté comme un "bac technologique plutôt scientifique" qui permet d'acquérir des compétences solides et variées :

• Connaissances pluridisciplinaires : Sciences, technologie, mathématiques et ingénierie.

• Compétences industrielles et environnementales : Une sensibilisation forte aux enjeux de l'industrie moderne et du développement durable.

• Approche design et innovation : Développement de la créativité et de la capacité à innover.

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2. Structure du Cursus Pédagogique

L'enseignement en STI2D est conçu pour rendre les concepts scientifiques plus accessibles par l'expérimentation et la réalisation.

2.1. Classe de Première

L'objectif est de permettre aux élèves qui "ont du mal à comprendre les enseignements" de manière abstraite de "se rapprocher de la manipulation" et de "comprendre des phénomènes en petit groupe".

Le programme s'articule autour de deux spécialités :

• Ingénierie, Innovation et Développement Durable (I2D) : Acquisition de connaissances scientifiques fondamentales à travers trois domaines : la matière, l'énergie et l'information.

• Innovation Technologique (IT) : Mise en œuvre des connaissances acquises en I2D à travers la réalisation de trois projets concrets durant l'année.

2.2. Classe de Terminale

En terminale, l'enseignement de spécialité I2D se poursuit, complété par un choix parmi quatre approfondissements spécifiques. L'année est marquée par un projet de 72 heures qui couvre l'étude, l'analyse, la conception, la simulation et le prototypage.

Spécialité

Acronyme

Description

Architecture et Construction

AC

Approfondissement des connaissances liées à la matière et à la structure.

Innovation Technologique et Éco-conception

ITEC

Approfondissement des connaissances liées à la conception mécanique et au design.

Systèmes d'Information et Numérique

SIN

Approfondissement des connaissances liées à l'informatique et aux systèmes numériques.

Énergie et Environnement

EE

Approfondissement des connaissances liées à la gestion, au transport et au stockage de l'énergie.

Un exemple de projet pluridisciplinaire cité est celui de la voiture solaire, qui a mobilisé trois spécialités :

• AC pour la conception du châssis.

• EE pour la gestion de l'énergie (panneaux solaires, stockage, alimentation moteur).

• SIN pour la commande et le pilotage de la voiture.

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3. Poursuites d'Études et Débouchés

La filière STI2D offre un large éventail de possibilités après le baccalauréat, permettant aux élèves de choisir entre des études courtes ou longues.

3.1. Panorama des Options Post-Baccalauréat

Type de Parcours

Formations Possibles

Exemples Cités

Études Courtes (Bac+2 / Bac+3)

BTS (Brevet de Technicien Supérieur)

BTS CIEL (Informatique et Réseau), BTS Électrotechnique, CPI, CPRP, CRSA.

BUT (Bachelor Universitaire de Technologie)

BUT Génie Civil Construction Durable, BUT Informatique, BUT Génie Industriel et Maintenance. Il est à noter que les BUT ont des places réservées pour les bacheliers technologiques.

Études Longues (Bac+5 et plus)

Classes Préparatoires aux Grandes Écoles (CPGE)

Prépa TSI (Technologie et Sciences Industrielles), spécifiquement destinée aux bacheliers STI2D/STL, et Prépa TPC (Technologie, Physique et Chimie).

Écoles d'Ingénieurs

Accès direct via le concours GPI Polytech pour STI2D/STL ou après une CPGE ou un BTS/BUT.

Licences Universitaires

Licence Informatique, Mathématiques, Physique, Sciences pour l'Ingénieur.

3.2. Données et Tendances (Parcoursup Janvier 2025)

Les données de Parcoursup indiquent une répartition équilibrée des choix des bacheliers STI2D, avec "autant de jeunes qui s'orientent vers des BTS que sur des BUT".

Un nombre légèrement inférieur d'élèves se dirige directement vers les classes préparatoires, les écoles d'ingénieurs ou les licences universitaires.

3.3. Secteurs d'Activité

Les diplômés peuvent intégrer des secteurs très variés, dont beaucoup sont des "métiers en tension" :

• BTP, architecture

• Énergie, électronique, environnement

• Audiovisuel, informatique, recherche et développement

• Secteurs de pointe : aéronautique, ferroviaire, construction navale

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4. Témoignages et Expériences Pratiques

4.1. L'Atelier de Prototypage : Une Démonstration Concrète

Une visite de l'atelier de prototypage a été organisée pour des élèves de seconde. Guidés par M. René, ils ont découvert :

• Des machines de fabrication complexes : Une voiture de course fabriquée sur place et ayant participé à une course à Albi.

• Des technologies de prototypage rapide : Des imprimantes 3D plastique et métal, ainsi qu'une machine de découpe laser.

La démonstration a mis en évidence la simplicité d'utilisation de certaines machines, incarnant l'esprit "Fablab" du lycée. Un élève a pu utiliser la machine de découpe laser après seulement 10 minutes d'explications pour réaliser une pièce. Cette expérience a souligné l'accessibilité de la technologie et la capacité des élèves à "concevoir et réaliser des pièces" rapidement.

4.2. Paroles d'Élèves de Terminale STI2D

Les témoignages des élèves de terminale illustrent la richesse et la diversité des parcours et des projets au sein de la filière.

• Spécialité Architecture et Construction (AC) :

◦ Jade a travaillé sur la modélisation des conduites d'eaux usées d'une ville fictive (Moeville) et souhaite devenir architecte d'intérieur.  

◦ Albin, réorienté depuis la première générale, ne "regrette pas du tout" son choix.

Il a participé à un projet de visite et de modélisation 3D du château de Jaligny.

Il souligne la valeur de l'approche plus appliquée de la filière et vise une école d'architecture ou un BUT Génie Civil.

• Spécialité Énergie et Environnement (EE) :

◦ Tom a choisi cette filière pour son "attrait relativement particulier pour tout ce qui était les énergies" et le désir "d'améliorer le fonctionnement de la société sur son point énergétique".

Bien qu'il se destine à devenir pilote, il "prend du plaisir à suivre les cours".

• Spécialité Innovation Technologique et Éco-conception (ITEC) :

◦ Will a choisi ITEC car il avait "beaucoup aimé les cours d'innovation technologique" en première.

Il se dirige vers une école d'informatique ou de cybersécurité.  

◦ Zoé, intéressée par le design (automobile, espace, mode), trouve que la spécialité ITEC est une bonne formation polyvalente où "on fait un peu de tout".

• Spécialité Systèmes d'Information et Numérique (SIN) :

◦ Liam apprécie le fait qu'en filière technologique, "il y a plus de pratique que de théorie" et que "on travaille plus souvent en classe qu'à la maison".    ◦ Martin a choisi la filière STI2D pour accéder à la spécialité SIN en vue d'une carrière dans l'informatique. Il n'est "pas déçu" et s'oriente vers les sciences des données.

4.3. Paroles d'Étudiants en Post-Baccalauréat

• BTS :

◦ Les étudiants de BTS CPI (Conception de Produits Industriels) montrent la complémentarité des parcours : Chris vient d'un bac général et y voit "la continuité de la matière science de l'ingénieur", tandis que Gauthier vient d'un bac STI2D ITEC et a été attiré par "le design qu'on faisait en ITECH".  

◦ Paul, en BTS CPRP, a préféré le cadre du BTS à celui du BUT pour son projet de carrière dans l'ingénierie militaire.

Il note que la cohabitation entre bacheliers généraux et STI2D est "plutôt complémentaire", les uns apportant la théorie (maths, physique), les autres la pratique.

• Classe Préparatoire TSI :

◦ Deux étudiants confirment que la prépa est le "meilleur moyen pour faire ingénieur".

Ils décrivent un changement de rythme important par rapport à la terminale : "Ça change de STI2D", "c'est vachement plus intense".

Cependant, l'adaptation est facilitée par une "bonne ambiance" et une "beaucoup de solidarité", notamment à l'internat.

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5. Points Clés et Ressources

5.1. Diversité et Représentation

Il est souligné que la filière STI2D compte "globalement plus de garçons que de filles", tout en insistant sur le fait que "c'est aussi une filière pour les filles".

La présence de plusieurs étudiantes parmi les témoins (Jade, Zoé, Joyce) vient appuyer ce propos.

5.2. Outils d'Orientation

Pour aider les élèves dans leur parcours, deux ressources numériques accessibles via "Mon Bureau Numérique" sont mises en avant :

• La plateforme Avenir : En lien avec l'ONISEP, elle propose de la documentation, des fiches formations et des témoignages.

• Mon projet sup : Un outil d'aide à la préparation du projet d'orientation au lycée, permettant de cibler des secteurs d'activité en fonction des compétences et des intérêts de l'élève.

Reviewer #1 (Public review):

Summary:

The goal of the manuscript was to determine if strenuous exercise negatively impacted regeneration. Indeed, the major conclusion of the manuscript is that elevated exercise during the early stages of regeneration compromises the regenerative process. The authors further conclude that regeneration is disrupted due to defects in blastema formation, which is caused by impaired HA deposition and reduced active (nuclear) Yap.

Strengths:

(1) The paradigm of elevated exercise disrupting ECM and regeneration is significant, and provides an experimental model to better understand connections between the ECM and cell/tissue activities.

(2) The conclusion that exercise intensity correlates with defects in regeneration is supported.

(3) The demonstration for the requirement for HA is well supported via transcriptomics and multiple independent strategies to manipulate HA levels.

(4) The demonstration that nuclear Yap depends on the amount of HA is well-supported.

Weaknesses:

(1) The authors conclude throughout the manuscript that "blastema formation" is disrupted, but they do not provide any insights into how blastema formation is disrupted (reduced de-differentiation? reduced cell migration? both?). While they show that there are fewer dividing cells, the timing of exercise is prior to outgrowth. So, the effect of dividing cells is likely secondary, which is not considered (or not clearly explained).

(2) The authors conclude that patterning is affected, but their analyses of patterns (bifurcations) are very limited. It is also not clear if patterning is believed to be affected by a common exercise-induced mechanism or a different exercise-induced mechanism (or by a secondary mechanism).

(3) The significance of HA in regeneration has been shown before in zebrafish fins, as well as in a handful of other models of regeneration. Although largely cited, explaining some of this work in more detail would give the reader a better picture of how HA is believed to promote regeneration. It may also highlight some emerging questions about the role of HA in regeneration that would permit a richer story and specific future directions.

(4) In general, parts of the text lack specificity/clarity, and in other cases, there seems to be contradictory information.

(5) Overall, many of the conclusions were well supported by the data, and this study is likely to provide a foundation for future research on the role of the ECM in tissue repair and regeneration. The main limitations were in connecting the experimental details with the specific processes required for regeneration, and in clearly explaining the findings.

Author response:

Reviewer #1

We agree that further clarification how elevated exercise disrupts blastema formation would strengthen the manuscript. Our data suggests a major contribution of proliferation. Exercise reduced the fraction of proliferative cells at 3 dpa, consistent with disrupted HA production and downstream Yap signaling. This interpretation aligns with prior studies showing that proliferation contributes to blastema establishment and is not restricted to the outgrowth phase of fin regeneration (Poleo et al, 2001; Poss et al, 2002; Wang et al, 2019; Pfefferli et al, 2014; Hou et al, 2020). We will explore additional experiments to reinforce these insights into the cellular mechanisms underlying exercise-disrupted blastema formation.

We acknowledge that our analysis of ray branching abnormalities is limited in the current manuscript. We focus our study on introducing the zebrafish swimming and regeneration model and then characterizing ECM and signaling changes accounting for disrupted blastema establishment. For completeness, we included the observation of skeletal patterning defects (branching delays and bone fusions) but without detailed analysis. We note that decreased expression of shha and Shh-pathway components following early exercise corresponds with the branching defects. However, we recognize exercise could have additional effects during the outgrowth  phase when branching morphogenesis actively occurs. Therefore, we will expand our discussion to outline future research directions related to exercise impacts on regenerative skeletal patterning.

We will expand the Introduction and/or Discussion sections to provide more context on known HA roles across regeneration contexts, including in zebrafish fins. Finally, we will improve the text’s clarity and specificity throughout the manuscript, including to resolve or explain any apparent contradictions.

Reviewer #2

We appreciate the Reviewer's concern regarding the specificity of forced exercise as a model for mechanical loading. Forced exercise has been widely used in vivo to induce mechanical loading without the requirement for specialized implants or animal restraint, including in mouse (Wallace et al, 2015; Bomer et al, 2016), rat (Honda et al, 2003; Boerckel et al, 2011; Boerckel et al, 2012), and, most relevant to our study, zebrafish models (Fiaz et al, 2012; Fiaz et al, 2014; Suniaga et al, 2018). However, we will expand our discussion of this approach and ensure precise language distinguishing exercise from mechanical loading.

We acknowledge the possibility that early shear stress disrupts the wound epidermis, which we will elaborate on in a revised Discussion. However, exercise-induced disruptions to the fin epidermis of early regenerates (1–2 dpa; Figure 2) typically resolve within one day, whereas fibroblast lineage cells still fail to establish a robust blastema. Therefore, sustained effects of mechanical loading and/or mechanosensation are likely major contributors to the observed regeneration phenotypes.

We will explore whether HA acts as a general enhancer of fin regeneration by comparing blastemal HA supplementation vs. controls in non-exercised regenerating animals, if technically feasible. We will merge Figure S7 (HA supplementation) with Figure 5 (HA depletion) for clarity, as suggested.

We will include a schematic and clear definitions for 'peripheral' and 'central' rays in a revised manuscript.

Reviewer #3

We included Hoechst and eosin fluorescent staining in the manuscript to show changes in tissue architecture following swimming exercise (Supplemental Figure 4). We will extend this histological analysis to include hematoxylin and eosin staining to provide additional tissue visualization.

References

Poleo G, Brown CW, Laforest L, Akimenko MA. Cell proliferation and movement during early fin regeneration in zebrafish. Dev Dyn. 2001 Aug;221(4):380-90.

Poss KD, Nechiporuk A, Hillam AM, Johnson SL, Keating MT. Mps1 defines a proximal blastemal proliferative compartment essential for zebrafish fin regeneration. Development. 2002 Nov;129(22):5141-9.

Wang YT, Tseng TL, Kuo YC, Yu JK, Su YH, Poss KD, Chen CH. Genetic Reprogramming of Positional Memory in a Regenerating Appendage. Curr Biol. 2019 Dec 16;29(24):4193-4207.e4.

Pfefferli C, Müller F, Jaźwińska A, Wicky C. Specific NuRD components are required for fin regeneration in zebrafish. BMC Biol. 2014 Apr 29;12:30.

Hou Y, Lee HJ, Chen Y, Ge J, Osman FOI, McAdow AR, Mokalled MH, Johnson SL, Zhao G, Wang T. Cellular diversity of the regenerating caudal fin. Sci Adv. 2020 Aug 12;6(33):eaba2084.

Wallace IJ, Judex S, Demes B. Effects of load-bearing exercise on skeletal structure and mechanics differ between outbred populations of mice. Bone. 2015 Mar;72:1-8.

Bomer N, Cornelis FM, Ramos YF, den Hollander W, Storms L, van der Breggen R, Lakenberg N, Slagboom PE, Meulenbelt I, Lories RJ. The effect of forced exercise on knee joints in Dio2(-/-) mice: type II iodothyronine deiodinase-deficient mice are less prone to develop OA-like cartilage damage upon excessive mechanical stress. Ann Rheum Dis. 2016 Mar;75(3):571-7.

Honda A, Sogo N, Nagasawa S, Shimizu T, Umemura Y. High-impact exercise strengthens bone in osteopenic ovariectomized rats with the same outcome as Sham rats. J Appl Physiol (1985). 2003 Sep;95(3):1032-7.

Boerckel JD, Kolambkar YM, Stevens HY, Lin AS, Dupont KM, Guldberg RE. Effects of in vivo mechanical loading on large bone defect regeneration. J Orthop Res. 2012 Jul;30(7):1067-75.

Boerckel JD, Uhrig BA, Willett NJ, Huebsch N, Guldberg RE. Mechanical regulation of vascular growth and tissue regeneration in vivo. Proc Natl Acad Sci U S A. 2011 Sep 13;108(37):E674-80.

Fiaz AW, Léon-Kloosterziel KM, Gort G, Schulte-Merker S, van Leeuwen JL, Kranenbarg S. Swim-training changes the spatio-temporal dynamics of skeletogenesis in zebrafish larvae (Danio rerio). PLoS One. 2012;7(4):e34072.

Fiaz AW, Léon‐Kloosterziel KM, van Leeuwen JL, Kranenbarg S. Exploring the molecular link between swim‐training and caudal fin development in zebrafish (Danio rerio) larvae. Journal of Applied Ichthyology. 2014 Aug;30(4):753-61.

Suniaga S, Rolvien T, Vom Scheidt A, Fiedler IAK, Bale HA, Huysseune A, Witten PE, Amling M, Busse B. Increased mechanical loading through controlled swimming exercise induces bone formation and mineralization in adult zebrafish. Sci Rep. 2018 Feb 26;8(1):3646.

Author response:

The following is the authors’ response to the previous reviews

Public Reviews:

Reviewer #1 (Public review):

This study extends the previous interesting work of this group to address the potentially differential control of movement and posture. Their earlier work explored a broad range of data to make the case for a downstream neural integrator hypothesized to convert descending velocity movement commands into postural holding commands. Included in that data were observations from people with hemiparesis due to stroke. The current study uses similar data, but pushes into a different, but closely related direction, suggesting that these data may address the independence of these two fundamental components of motor control. I find the logic laid out in the second sentence of the abstract ("The paretic arm after stroke is notable for abnormalities both at rest and during movement, thus it provides an opportunity to address the relationships between control of reaching, stopping, and stabilizing") less then compelling, but the study does make some interesting observations. Foremost among them, is the relation between the resting force postural bias and the effect of force perturbations during the target hold periods, but not during movement. While this interesting observation is consistent with the central mechanism the authors suggest, it seems hard to me to rule out other mechanisms, including peripheral ones. These limitations should should be discussed.

Thank you for summarizing our work. Note we have improved the logic in our abstract (…”providing an opportunity to ask whether control of these behaviors is independently affected in stroke”) based on your comments as outlined in our previous revision. We now extensively discuss limitations and potential alternative mechanisms in greater detail, in a dedicated section (lines 846-895; see response to reviewer 2 for further details).

Reviewer #2 (Public review):

Summary:

Here the authors address the idea that postural and movement control are differentially impacted with stroke. Specifically, they examined whether resting postural forces influenced several metrics of sensorimotor control (e.g., initial reach angle, maximum lateral hand deviation following a perturbation, etc.) during movement or posture. The authors found that resting postural forces influenced control only following the posture perturbation for the paretic arm of stroke patients, but not during movement. They also found that resting postural forces were greater when the arm was unsupported, which correlated with abnormal synergies (as assessed by the Fugl-Meyer). The authors suggest that these findings can be explained by the idea that the neural circuitry associated with posture is relatively more impacted by stroke than the neural circuitry associated with movement. They also propose a conceptual model that differentially weights the reticulospinal tract (RST) and corticospinal tract (CST) to explain greater relative impairments with posture control relative to movement control, due to abnormal synergies, in those with stroke.

Thank you for the brief but comprehensive summary. We would like to clarify one point: we do not suggest that our findings are necessarily due to the neural circuitry associated with posture being more impacted than the neural circuitry associated with movement. (rather, our conceptual model suggests that increased outflow through the (ipsilateral) RST, involved in posture, compensates for CST damage, at the expense of posture abnormalities spilling over into movement). Instead, we suggest that the neural circuitry for posture vs. movement control remains relatively separate in stroke, with impairments in posture control not substantially explaining impairments in movement control.

Comments on revisions:

The authors should be commended for being very responsive to comments and providing several further requested analyses, which have improved the paper. However, there is still some outstanding issues that make it difficult to fully support the provided interpretation.

Thank you for appreciating our response to your earlier comments. We address the outstanding issues below.

The authors say within the response, "We would also like to stress that these perturbations were not designed so that responses are directly compared to each other ***(though of course there is an *indirect* comparison in the sense that we show influence of biases in one type of perturbation but not the other)***." They then state in the first paragraph of the discussion that "Remarkably, these resting postural force biases did not seem to have a detectable effect upon any component of active reaching but only emerged during the control of holding still after the movement ended. The results suggest a dissociation between the control of movement and posture." The main issue here is relying on indirect comparisons (i.e., significant in one situation but not the other), instead of relying on direct comparisons. Using well-known example, just because one group / condition might display a significant linear relationship (i.e., slope_1 > 0) and another group / condition does not (slope_2 = 0), does not necessarily mean that the two groups / conditions are statistically different from one another [see Figure 1 in Makin, T. R., & Orban de Xivry, J. J. (2019). Ten common statistical mistakes to watch out for when writing or reviewing a manuscript. eLife, 8, e48175.].

We agree and are well aware of the limitation posed by an indirect comparison – hence the language we used to comment on the data (“did not seem”, “suggest”, etc.). To address this limitation, we performed a more direct comparison of how the two types of perturbations (moving vs. holding) interact with resting biases. For this comparison, we calculated a Response Asymmetry Index (RAI):

Above, 𝑟_𝐴 is the response on direction where resting bias is most-aligned with the perturbation, and 𝑟_𝑂 is the response on direction where resting bias is most-opposed to the perturbation.

We calculated RAIs for two response metrics used for both moving and holding perturbations: maximum deviation and time to stabilization/settling time. For these two response metrics, positive RAIs indicate an asymmetry in line with an effect of resting bias.

The idea behind the RAI is that, while the magnitude of responses may well differ between the two types of perturbations, this will be accounted for by the ratio used to calculate the asymmetry. The same approach has been used to assess symmetry/laterality across a variety of different modalities, such as gait asymmetry (Robinson et al., 1987), the relative fMRI activity in the contralateral vs. ipsilateral sensorimotor cortex while performing a motor task (Cramer et al., 1997), or the relative strength of ipsilateral vs. contralateral responses to transcranial magnetic stimulation (McPherson et al., 2018). Notably, the normalization also addresses potential differences in overall stiffness between holding vs. moving perturbations, which would similarly affect aligned and opposing cases (see our response to your following point).

Figure 8 shows RAIs we obtained for holding (red) vs. moving/pulse (blue) perturbations. For the maximum deviation (left), there is more asymmetry for the holding case though the pvalue is marginal (p=0.088) likely due to the large variability in the pulse case (individual values shown in black dots). For time to stabilization/settling time (right) the difference is significant (p=0.0048). Together, these analyses indicate that resting biases interact substantially more with holding compared to movement control, in line with a relative independence between these two control modalities. We now include this panel as Figure 8, and describe it in Results (lines 587-611).

Note that even a direct comparison does not prove that resting biases and active movement control are perfectly independent. We now discuss these issues in more depth, in the new Limitations section suggested by the Reviewer (lines 836-849).

The authors have provided reasonable rationale of why they chose certain perturbation waveforms for different. Yet it still holds that these different waveforms would likely yield very different muscular responses making it difficult to interpret the results and this remains a limitation. From the paper it is unknown how these different perturbations would differentially influence a variety of classic neuromuscular responses, including short-range stiffness and stretch reflexes, which would be at play here.

Much of the results can be interpreted when one considers classic neuromuscular physiology. In Experiment 1, differences in resting postural bias in supported versus unsupported conditions can readily be explained since there is greater muscle activity in the unsupported condition that leads to greater muscle stiffness to resist mechanical perturbations (Rack, P. M., & Westbury, D. R. (1974). The short-range stiffness of active mammalian muscle and its effect on mechanical properties. The Journal of physiology, 240(2), 331-350.). Likewise muscle stiffness would scale with changes in muscle contraction with synergies. Importantly for experiment 2, muscle stiffness is reduced during movement (Rack and Westbury, 1974) which may explain why resting postural biases do not seem to be impacting movement. Likewise, muscle spindle activity is shown to scale with extrafusal muscle fiber activity and forces acting through the tendon (Blum, K. P., Campbell, K. S., Horslen, B. C., Nardelli, P., Housley, S. N., Cope, T. C., & Ting, L. H. (2020). Diverse and complex muscle spindle afferent firing properties emerge from multiscale muscle mechanics. eLife, 9, e55177.). The concern here is that the authors have not sufficiently considered muscle neurophysiology, how that might relate to their findings, and how that might impact their interpretation. Given the differences in perturbations and muscle states at different phases, the concern is that it is not possible to disentangle whether the results are due to classic neurophysiology, the hypothesis they propose, or both. Can the authors please comment.

It is possible that neuromuscular physiology may explain part of our results. However, this would not contradict our conceptual model.

Regarding Experiment 1, it is possible that stiffness would scale with changes in background muscle contraction as the reviewer suggests. Indeed, Bennett and al.(Bennett et al., 1992) used brief perturbations on the wrist to assess elbow stiffness, finding that, during movement, stiffness was increased in positions with a higher gravity load (and, in general, in positions where the net muscle torque was higher). However, during posture maintenance (like in our Experiment 1), they found that stiffness did not vary with (elbow) position or gravity load (two characteristics of our findings in Experiment 1):

“The observed stiffness variation was not simply due to passive tissue or other joint angle dependent properties, as stiffnesses measured during posture were position invariant. Note that the minimum stiffness found in posture was higher than the peak stiffness measured during movement, and did not change much with the gravity load.” (illustrated in Fig. 5 of that paper)

We thus find it very unlikely that stiffness explains the difference between the supported vs. unsupported conditions in Experiment 1.

Even if stiffness modulation between the supported vs. unsupported conditions could explain our finding of stronger posture biases in the latter case, it would not be incompatible with our interpretation of increased RST drive: increased stiffness would potentially magnify the effects of the RST drive we propose to drive these resting biases. It is possible that the increase in resting biases under conditions of increased muscle contraction (lack of arm support) is mediated through an increase in muscle stiffness. In other words, the increase in resting biases may not directly reflect additional RST outflow per se, but the scaling, through stiffness, of the same magnitude of RST outflow. Understanding this interaction was beyond the scope of our experiment design; in line with this, we briefly comment about it in our Limitations section.

Regarding Experiment 2, stiffness has indeed been shown to be lower during movement, and we now comment the potential effect of this on our results in the “Limitations” section (lines 815-830, replicated below). Importantly, for the case of holding perturbations, the increased stiffness associated with holding would increase resistance to both extension and flexion-inducing perturbations. Thus, higher stiffness would be unlikely to explain our finding whereby resting biases resist or aggravate the effects of holding perturbations depending on perturbation direction. In addition, the framework in Blum et al., that describes how interactions between alpha and gramma drive can explain muscle activity patterns, does not rule out central neural control of stiffness: “muscle spindles have a unique muscle-within-muscle design such that their firing depends critically on both peripheral and central factors” (emphasis ours). It may be, for example, that gamma motoneurons controlling muscle spindles and stiffness are modulated from input from the reticular formation, making this a mechanism in line with our conceptual model.

“Moreover, it has been shown that joint stiffness is reduced during movement compared to holding control (Rack and Westbury, 1974; Bennett et al., 1992). Along similar lines, muscle spindle activity – which may modulate stiffness – scales with extrafusal muscle fiber activity (such as muscle exertion involved in holding) and forces acting through the tendon (Blum et al., 2020). Such observations could, in principle, explain why we were unable to detect a relationship between resting biases and active movement control but we readily found a relationship between resting biases and active holding control: reduced joint stiffness during movement could scale down the influence of resting abnormalities. There are two issues with this explanation, however. First, it is debatable whether this should be considered an alternative explanation per se: stiffness modulation could be, in total or in part, the manifestation of a central movement/posture CST/RST mechanism similar to the one we propose in our conceptual model. For example, (Blum et al., 2020) argue that muscle spindle firing depends on both peripheral and central factors. Second, increased stiffness would not necessarily help detect differences in how active postural control responds to within-resting-posture vs. out-of-resting-posture perturbations. This is because an overall increase in stiffness would likely increase resistance to perturbations in any direction.”

The authors should provide a limitations paragraph. They should address 1) how they used different perturbation force profiles, 2) the muscles were in different states which would change neuromuscular responses between trial phase / condition, 3) discuss a lack of direct statistical comparisons that support their hypothesis, and 4) provide a couple of paragraphs on classic neurophysiology, such as muscle stiffness and stretch reflexes, and how these various factors could influence the findings (i.e., whether they can disentangle whether the reported results are due to classic neurophysiology, the hypothesis they propose, or both).

Thank you for your suggestion. We now discuss these points in a separate paragraph (lines 846895), bringing together our previous discussion on stretch reflexes, our description of different perturbation types, and the additional issues raised by the reviewer above.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

The authors have responded well to all my concerns, save two minor points.

Figure 2 appears to be unchanged, although they describe appropriate changes in the response letter.

Thank you for catching this error – we now include the updated figure (further updated to use the terms near/distant in place of proximal/distal).

I still take issue with the use of proximal and distal to describe the locations of targets. Taking definitions somewhat randomly from the internet, "The terms proximal and distal are used in structures that are considered to have a beginning and an end," and "Proximal and distal are anatomical terms used to describe the position of a body part in relation to another part or its origin." In any case, the hand does not become proximal just because you bring it to your chest. Why not simply stick to the common and clearly defined terms "near" and "distant"?

Point taken. We have updated the paper to use the terms near/distant.

Additional changes/corrections not outlined above

We now include a link to the data and code supporting our findings (https://osf.io/hufy8/). In addition, we made several minor edits throughout the text to improve readability, and corrected occasional mislabeling of CCW and CW pulse data. Note that this correction did not alter the (lack of) relationship between resting biases and responses to perturbations during active movement.

Response letter references

Bennett D, Hollerbach J, Xu Y, Hunter I (1992) Time-varying stiffness of human elbow joint during cyclic voluntary movement. Exp Brain Res 88:433–442.

Blum KP, Campbell KS, Horslen BC, Nardelli P, Housley SN, Cope TC, Ting LH (2020) Diverse and complex muscle spindle afferent firing properties emerge from multiscale muscle mechanics. Elife 9:e55177.

Cramer SC, Nelles G, Benson RR, Kaplan JD, Parker RA, Kwong KK, Kennedy DN, Finklestein SP, Rosen BR (1997) A functional MRI study of subjects recovered from hemiparetic stroke. Stroke 28:2518–2527.

McPherson JG, Chen A, Ellis MD, Yao J, Heckman C, Dewald JP (2018) Progressive recruitment of contralesional cortico-reticulospinal pathways drives motor impairment post stroke. J Physiol 596:1211–1225 Available at: https://doi.org/10.1113/JP274968.

Rack PM, Westbury D (1974) The short range stiffness of active mammalian muscle and its effect on mechanical properties. J Physiol 240:331–350.

Robinson R, Herzog W, Nigg BM (1987) Use of force platform variables to quantify the effects of chiropractic manipulation on gait symmetry. J Manipulative Physiol Ther 10:172–176.

Williams PE, Goldspink G (1973) The effect of immobilization on the longitudinal growth of striated muscle fibres. J Anat 116:45.

Reviewer #3 (Public review):

Summary:

The authors set out to extend their previous mapping of Drosophila head mechanosensory neurons (Eichler et al., 2024) by reconstructing their full second-order connectome. Their aim is to reveal how bristle mechanosensory neurons (BMNs) interface with excitatory and inhibitory partners to generate location-specific grooming movements, and to identify the circuit motifs and developmental lineages that support this transformation.

Strengths:

The strengths of this work are clear. The authors present a comprehensive synaptic-resolution connectome for BMNs, identifying nearly all of their pre- and postsynaptic partners. This dataset reveals important circuit motifs:

(1) BMNs provide feedforward excitation to descending neurons, feedforward inhibition to interneurons, and are themselves strongly regulated by GABAergic presynaptic inhibition.

(2) These motifs together support the idea that BMN activity is locally gated and hierarchically suppressed, fitting well with known behavioural sequences of grooming.

(3) The study also shows that connectivity preserves somatotopy, such that BMNs from neighbouring bristle populations converge onto shared partners, while distant BMNs remain segregated.

(4) A developmental analysis reveals both primary and secondary partners, suggesting a layered scaffold plus adult-specific elaborations.

(5) Finally, the identification of hemilineage 23b (LB23) as a core postsynaptic pathway - incorporating previously described antennal grooming neurons (aBN2) - provides a striking link between developmental lineage, anatomical connectivity, and behavioral output.

(6) Together, the dataset represents a valuable resource for the neuroscience community and a foundation for future functional studies.

Weaknesses:

There are also some weaknesses that mostly only limit clarity.

(1) The writing is dense, with results often presented in a cryptic fashion and the functional implications deferred to the discussion. As a result, the significance of circuit motifs such as BMN→motor or reciprocal inhibitory loops is sometimes buried, rather than highlighted when first described.

(2) Some assumptions require more explanation for non-specialist readers - for example, how bristle identity is inferred in EM in the absence of cuticular structures, or what is meant by "ascending" and "descending" in a dataset that does not include the ventral nerve cord. While some of this comes from the earlier paper, it would help readers of this one to explain this.

(3) Visualization choices also sometimes obscure key conclusions: network graphs can be visually appealing but do not clearly convey somatotopy or BMN-type differences; heatmaps or region-level matrices would make the parallel, block-like organization of the circuit more evident.

(4) The data might also speak to roles beyond grooming (e.g., mechanosensory modulation of posture or feeding), and a brief acknowledgement of this would broaden the impact.

(5) The restriction to one hemisphere should be explicitly acknowledged as a limitation when framing this as a 'comprehensive' connectome.

Overall, the authors achieve their main goal: they convincingly show that BMNs connect into parallel, somatotopically organized pathways, with LB23 providing a key lineage-based link from sensory input to grooming output. The dataset is carefully analyzed, and while the presentation could be streamlined, the connectome will be a valuable resource for researchers studying sensory processing, motor control, and the logic of circuit organization.

Reviewer #1 (Public review):

The manuscript presents a compelling new in vitro system based on isogenic co-cultures of human iPSC-derived hepatocytes and macrophages, enabling the modelling of hepatic immune responses with unprecedented physiological relevance. The authors show that co-culture leads to enhanced maturation of hepatocytes and tissue-resident macrophage identity, which cannot be achieved through conditioned media alone. Using this system, they functionally validate immune-driven hepatotoxic responses to a panel of drugs and compare the system's predictive power to that of monocyte-derived macrophages. The results underscore the necessity of macrophage-hepatocyte crosstalk for accurate modelling of liver inflammation and drug toxicity in vitro.

The manuscript is clearly written and addresses a key limitation in liver organoid systems: the lack of immune complexity and tissue-specific macrophage imprinting. Nevertheless, several conclusions would benefit from a more careful interpretation of the data, and some important controls or explanations are missing, particularly in the flow cytometry gating strategies, stress marker validation, and cluster interpretations.

Strengths:

(1) Novelty and Relevance: The study presents a highly innovative co-culture system based on isogenic human iPSCs, addressing an unmet need in modelling immune-mediated hepatotoxicity.

(2) Mechanistic Insight: The reciprocal reprogramming between iHeps and iMacs, including induction of KC-specific pathways and hepatocyte maturation markers, is convincingly demonstrated.

(3) Functional Readouts: The application of the model to detect IL-6 responses to hepatotoxic compounds enhances its translational relevance.

Weaknesses:

(1) Several key claims, particularly those derived from PCA plots and DEG analyses, are overinterpreted and require more conservative language or further validation.

(2) The purity of sorted hepatocytes and macrophages is not convincingly demonstrated; contamination across gates may confound transcriptomic readouts.

(3) Stress response genes and ER stress/apoptosis signatures are not properly assessed, despite being potentially activated in the system.

(4) Some figure panels and legends lack statistical annotations, and microscopy validation of morphological changes is missing.

(5) The co-culture model with monocyte-derived macrophages is not fully characterised, making comparisons less informative.

Reviewer #3 (Public review):

Summary:

In this study, the authors establish a human in vitro liver model by co-culturing induced hepatocyte-like cells (iHEPs) with induced macrophages (iMACs). Through flow cytometry-based sorting of cell populations at days 3 and 7 of co-culture, followed by bulk RNA sequencing, they demonstrate that bidirectional interactions between these two cell types drive functional maturation. Specifically, the presence of iMACs accelerates the hepatic maturation program of iHEPs, while contact-dependent cues from iHEPs enhance the acquisition of Kupffer cell identity in iMACs, indicating that direct cell-cell interactions are critical for establishing tissue-resident macrophage characteristics.

Functionally, the authors show that iMAC-derived Kupffer-like cells respond to pathological stimuli by producing interleukin-6 (IL-6), a hallmark cytokine of hepatic immune activation. When exposed to a panel of clinically relevant hepatotoxic drugs, the co-culture system exhibited concentration-dependent modulation of IL-6 secretion consistent with reported drug-induced liver injury (DILI) phenotypes. Notably, this response was absent when hepatocytes were co-cultured with monocyte-derived macrophages from peripheral blood, underscoring the liver-specific phenotype and functional relevance of the iMAC-derived Kupffer-like cells. Collectively, the study proposes this co-culture platform as a more physiologically relevant model for interrogating macrophage-hepatocyte crosstalk and assessing immune-mediated hepatotoxicity in vitro.

Strengths:

A major strength of this study lies in its systematic dissection of cell-cell interactions within the co-culture system. By isolating each cell type following co-culture and performing comprehensive transcriptomic analyses, the authors provide direct evidence of bidirectional crosstalk between iMACs and iHEPs. The comparison with single-culture controls is particularly valuable, as it clearly demonstrates how co-culture enhances functional maturation and lineage-specific gene expression in both cell types. This approach allows for a more mechanistic understanding of how hepatocyte-macrophage interactions contribute to the acquisition of tissue-specific phenotypes.

Weaknesses:

(1) Overreliance on bulk RNA-seq data:

The primary evidence supporting cell maturation is derived from bulk RNA sequencing, which has inherent limitations in resolving heterogeneous cellular states and functional maturation. The conclusions regarding hepatocyte maturation are based largely on increased expression of a subset of CYP genes and decreased AFP levels - markers that, while suggestive, are insufficient on their own to substantiate functional maturation. Additional phenotypic or functional assays (e.g., metabolic activity, protein-level validation) would significantly strengthen these claims.

(2) Insufficient characterization of input cell populations:

The manuscript lacks adequate validation of the cellular identities prior to co-culture. Although the authors reference previously published protocols for generating iHEPs and iMACs, it remains unclear whether the cells used in this study faithfully retain expected lineage characteristics. For example, hepatocyte preparations should be characterized by flow cytometry for ALB and AFP expression, while iMACs should be assessed for canonical macrophage markers such as CD45, CD11b, and CD14 before co-culture. Without these baseline data, it is difficult to interpret the magnitude or significance of any co-culture-induced changes.

(3) Quantitative assessment of IL-6 production is insufficient:

The analysis of drug-induced IL-6 responses is based primarily on relative changes compared to control conditions. However, percentage changes alone are inadequate to capture the biological relevance of these responses. Absolute cytokine production levels - particularly in response to LPS stimulation - should be reported and directly compared to PBMC-derived macrophages to determine whether iMAC-derived Kupffer-like cells exhibit enhanced cytokine output. Moreover, the Methods section should clearly describe how ELISA results were normalized or corrected to account for potential differences in cell number, viability, or culture conditions.

(4) Unclear mechanistic interpretation of IL-6 modulation:

The observed changes in IL-6 production upon drug treatment cannot be interpreted solely as evidence of Kupffer cell-specific functionality. For instance, IL-6 suppression by NSAIDs such as diclofenac is well known to result from altered prostaglandin synthesis due to COX inhibition, while leflunomide's effects are linked to metabolite-induced modulation of immune cell proliferation and broader cytokine networks. These mechanisms are distinct from Kupffer cell identity and may not directly reflect liver-specific macrophage function. Consequently, changes in IL-6 secretion alone - particularly without additional mechanistic evidence or analysis of other cytokines - are insufficient to conclude that co-culture with hepatocytes drives the acquisition of bona fide Kupffer cell maturity.

1,Eu pagei o aluguel 2,Ele tomou em casa 3,Eu jantei no resturante ontem à noite 4,Ela saiu com uns amigos 5,eu almocei às onze e meia 6,Eles deitaram à meia-noite 7,Eu li um jornal 8,Ela se levantou às seis e quinze 9,Eu me-levantei às oito e vinte 10.Nós fomos ao cinema

Reviewer #2 (Public review):

Summary:

The authors generate an optimized small molecule inhibitor of SMARCA2/4 and test it in a panel of cell lines. All uveal melanoma (UM) cell lines in the panel are growth inhibited by the inhibitor making the focus of the paper. This inhibition is correlated with loss of promoter occupancy of key melanocyte transcription factors e.g. SOX10. SOX10 overexpression and a point mutation in SMARCA4 can rescue growth inhibition exerted by the SMARCA2/4 inhibitor. Treatment of a UM xenograft model results in growth inhibition and regression which correlates with reduced expression of SOX10 but not discernible toxicity in the mice. Collectively, the data suggest a novel treatment of uveal melanoma.

Strengths:

There are many strengths of the study, including the strong challenge of the on-target effect, the assays used and the mechanistic data. The results are compelling as are the effects of the inhibitor. The in vivo data is dose-dependent and doses are low enough to be meaningful and associated with evidence of target engagement.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Public review): 

Summary: 

The presented study by Centore and colleagues investigates the inhibition of BAF chromatin remodeling complexes. The study is well written and includes comprehensive datasets, including compound screens, gene expression analysis, epigenetics, as well as animal studies. This is an important piece of work for the uveal melanoma research field, and sheds light on a new inhibitor class, as well as a mechanism that might be exploited to target this deadly cancer for which no good treatment options exist. 

Strengths: 

This is a comprehensive and well-written study. 

Weaknesses: 

There are minimal weaknesses. 

Reviewer #2 (Public review): 

Summary: 

The authors generate an optimized small molecule inhibitor of SMARCA2/4 and test it in a panel of cell lines. All uveal melanoma (UM) cell lines in the panel are growth inhibited by the inhibitor making the focus of the paper. This inhibition is correlated with loss of promoter occupancy of key melanocyte transcription factors e.g. SOX10. SOX10 overexpression and a point mutation in SMARCA4 can rescue growth inhibition exerted by the SMARCA2/4 inhibitor. Treatment of a UM xenograft model results in growth inhibition and regression which correlates with reduced expression of SOX10 but not discernible toxicity in the mice. Collectively, the data suggest a novel treatment of uveal melanoma. 

Strengths: 

There are many strengths of the study, including the strong challenge of the on-target effect, the assays used and the mechanistic data. The results are compelling as are the effects of the inhibitor. The in vivo data is dose-dependent and doses are low enough to be meaningful and associated with evidence of target engagement. 

Weaknesses: 

The authors have addressed weaknesses in the revised version. 

Reviewer #3 (Public review): 

Summary: 

This manuscript reports the discovery of new compounds that selectively inhibit SMARCA4/SMARCA2 ATPase activity and have pronounced effects on uveal melanoma cell proliferation. They induce apoptosis and suppress tumor growth, with no toxicity in vivo. The report provides biological significance by demonstrating that the drugs alter chromatin accessibility at lineage specific gene enhancer regions and decrease expression of lineage specific genes, including SOX10 and SOX10 target genes. 

Strengths: 

The study provides compelling evidence for the therapeutic use of these compounds and does a thorough job at elucidating the mechanisms by which the drugs work. The study will likely have a high impact on the chromatin remodeling and cancer fields. The datasets will be highly useful to these communities. 

Weaknesses: 

The authors have addressed all my concerns. 

Recommendations for the authors: 

We would, however, like to draw the authors attention to 2 comments by the referees. 

Referee 1 comments: While BAP1 mutant UM cell lines were included for some of the experiments, it seems the in-vivo data mentioned in the response to the reviewers comment is missing? The authors stated that "MP46 (Supplementary Fig. 3a) is BAP1null uveal melanoma cell line with no detectable protein expression (AmiroucheneAngelozzi et al., Mol Oncol 2014), and we have observed strong tumor growth inhibition in this CDX model with our BAF ATPase inhibitor." But the CDX model data shown in Figure 4 is from 92.1 cells. If this data is available, then the manuscript would benefit from its addition. 

We thank the reviewer for bringing this to our attention. As the reviewer mentioned, we show 92-1 CDX model in our manuscript. Additionally, strong tumor growth inhibition was observed in MP-46  CDX model treated with our BAF ATPase inhibitor and can be found in Vaswani et al., 2025 (PMID:39801091, https://pubmed.ncbi.nlm.nih.gov/39801091/).

Referee 3 comments: 

Supplementary Figure 2C 

Is the T910M mutation in the parental MP41 cells heterozygous? If so, the authors should indicate this in the figure legend. If this is a homozygous mutation, the authors should explain how the inhibitors suppress SMARCA4 activity in cells that have a LOF mutation. 

Could the authors please comment on these issues before a final version is posted online? 

We thank the reviewer for bringing this to our attention. T910M mutation is heterozygous and the variant allele frequency for that mutation is 0.5. We updated the figure legend accordingly to reflect the genotype of the mutations highlighted in the table.

Reviewer #1 (Recommendations for the authors): 

The authors have addressed most of the questions in their review. 

While BAP1 mutant UM cell lines were included for some of the experiments, it seems the in-vivo data mentioned in the response to the reviewers comment is missing? The authors stated that "MP46 (Supplementary Fig. 3a) is BAP1-null uveal melanoma cell line with no detectable protein expression (Amirouchene-Angelozzi et al., Mol Oncol 2014), and we have observed strong tumor growth inhibition in this CDX model with our BAF ATPase inhibitor." But the CDX model data shown in Figure 4 is from 92.1 cells. If this data is available, then the manuscript would benefit from its addition. 

Reviewer #3 (Recommendations for the authors): 

Supplementary Figure 2C 

Is the T910M mutation in the parental MP41 cells heterozygous? If so, the authors should indicate this in the figure legend. If this is a homozygous mutation, the authors should explain how the inhibitors suppress SMARCA4 activity in cells that have a LOF mutation.

Author response:

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

Reviewer #1 (Public review): 

Summary: 

In this manuscript, the authors performed an integration of 48 scRNA-seq public datasets and created a single-cell transcriptomic atlas for AML (222 samples comprising 748,679 cells). This is important since most AML scRNA-seq studies suffer from small sample size coupled with high heterogeneity. They used this atlas to further dissect AML with t(8;21) (AML-ETO/RUNX1-RUNX1T1), which is one of the most frequent AML subtypes in young people. In particular, they were able to predict Gene Regulatory Networks in this AML subtype using pySCENIC, which identified the paediatric regulon defined by a distinct group of hematopoietic transcription factors (TFs) and the adult regulon for t(8;21). They further validated this in bulk RNA-seq with AUCell algorithm and inferred prenatal signature to 5 key TFs (KDM5A, REST, BCLAF1, YY1, and RAD21), and the postnatal signature to 9 TFs (ENO1, TFDP1, MYBL2, KLF1, TAGLN2, KLF2, IRF7, SPI1, and YXB1). They also used SCENIC+ to identify enhancer-driven regulons (eRegulons), forming an eGRN, and found that prenatal origin shows a specific HSC eRegulon profile, while a postnatal origin shows a GMP profile. They also did an in silico perturbation and found AP-1 complex (JUN, ATF4, FOSL2), P300, and BCLAF1 as important TFs to induce differentiation. Overall, I found this study very important in creating a comprehensive resource for AML research. 

Strengths: 

(1) The generation of an AML atlas integrating multiple datasets with almost 750K cells will further support the community working on AML. 

(2) Characterisation of t(8;21) AML proposes new interesting leads. 

We thank the reviewer for a succinct summary of our work and highlighting its strengths.

Weaknesses: 

Were these t(8;21) TFs/regulons identified from any of the single datasets? For example, if the authors apply pySCENIC to any dataset, would they find the same TFs, or is it the increase in the number of cells that allows identification of these? 

We implemented pySCENIC on individual datasets and compared the TFs (defining the regulons) identified to those from the combined AML scAtlas analysis. There were some common TFs identified, but these vary between individual studies. The union of all TFs identified makes a very large set - comprising around a third of all known TFs. AML scAtlas provides a more refined repertoire of TFs, perhaps as the underlying network inference approach is more robust with a higher number of cells. The findings of these investigations are included in Supplementary Figure 4DE, we hope this is useful for other users of pySCENIC.

Reviewer #2 (Public review): 

Summary: 

The authors assemble 222 publicly available bone marrow single-cell RNA sequencing samples from healthy donors and primary AML, including pediatric, adolescent, and adult patients at diagnosis. Focusing on one specific subtype, t(8;21), which, despite affecting all age classes, is associated with better prognosis and drug response for younger patients, the authors investigate if this difference is reflected also in the transcriptomic signal. Specifically, they hypothesize that the pediatric and part of the young population acquires leukemic mutations in utero, which leads to a different leukemogenic transformation and ultimately to differently regulated leukemic stem cells with respect to the adult counterpart. The analysis in this work heavily relies on regulatory network inference and clustering (via SCENIC tools), which identifies regulatory modules believed to distinguish the pre-, respectively, post-natal leukemic transformation. Bulk RNA-seq and scATAC-seq datasets displaying the same signatures are subsequently used for extending the pool of putative signature-specific TFs and enhancer elements. Through gene set enrichment, ontology, and perturbation simulation, the authors aim to interpret the regulatory signatures and translate them into potential onset-specific therapeutic targets. The putative pre-natal signature is associated with increased chemosensitivity, RNA splicing, histone modification, stemness marker SMARCA2, and potentially maintained by EP300 and BCLAF1. 

Strengths: 

The main strength of this work is the compilation of a pediatric AML atlas using the efficient Cellxgene interface. Also, the idea of identifying markers for different disease onsets, interpreting them from a developmental angle, and connecting this to the different therapy and relapse observations, is interesting. The results obtained, the set of putative up-regulated TFs, are biologically coherent with the mechanisms and the conclusions drawn. I also appreciate that the analysis code was made available and is well documented. 

We thank the reviewer for evaluating our work, and highlighting its key features, including creation of AML atlas, downstream analysis and interpretation for t(8;21) subtype.

Weaknesses:

There were fundamental flaws in how methods and samples were applied, a general lack of critical examination of both the results and the appropriateness of the methods for the data at hand, and in how results were presented. In particular: 

(1) Cell type annotation: 

(a) The 2-phase cell type annotation process employed for the scRNA-seq sample collection raised concerns. Initially annotated cells are re-labeled after a second round with the same cell types from the initial label pool (Figure 1E). The automatic annotation tools were used without specifying the database and tissue atlases used as a reference, and no information was shown regarding the consensus across these tools. 

Cell type annotations are heavily influenced by the reference profiles used and vary significantly between tools. To address this, we used multiple cell type annotation tools which predominantly encompassed healthy peripheral blood cell types and/or healthy bone marrow populations. This determined the primary cluster cell types assigned. 

Existing tools and resources are not leukemia specific, thus, to identify AMLassociated HSPC subpopulations we created a custom SingleR reference, using a CD34 enriched AML single-cell dataset. This was not suitable for the annotation of the full AML scAtlas, as it is derived from CD34 sorted cell types so is biased towards these populations. 

We have made this much clearer in the revised manuscript, by splitting Figure 1 into two separate figures (now Figure 1 and Figure 2) reflecting both different analyses performed. The methods have also been updated with more detail on the cell type annotations, and we have included the automated annotation outputs as a supplementary table, as this may be useful for others in the single-cell community. 

(b) Expression of the CD34 marker is only reported as a selection method for HSPCs, which is not in line with common practice. The use of only is admitted as a surface marker, while robust annotation of HSPCs should be done on the basis of expression of gene sets. 

Most of the cells used in the HSPC analysis were in fact annotated as HSPCs with some exceptions. In line with this feedback, we have re-worked this analysis and simply taken HSPC annotated clusters forward for the subsequent analysis, yielding the same findings. 

(c) During several analyses, the cell types used were either not well defined or contradictory, such as in Figure 2D, where it is not clear if pySCENIC and AUC scores were computed on HSPCs alone or merged with CMPs. In other cases, different cell type populations are compared and used interchangeably: comparing the HSPCderived regulons with bulk (probably not enriched for CD34+ cells) RNA samples could be an issue if there are no valid assumptions on the cell composition of the bulk sample. 

We apologize for the lack of clarity regarding which cell types were used, the text has been updated to clarify that in the pySCENIC analysis all myeloid progenitor cells were included. 

The bulk RNA-seq samples were used only to test the enrichment of our AML scAtlas derived regulons in an unbiased and large-scale way. While CD34 enriched samples could be preferable, this was not available to us. 

We agree that more effort could be made to ensure the single-cell/myeloid progenitor derived regulons are comparable to the bulk-RNA sequencing data. In the original bulk RNA-seq validation analysis, we used all bulk-RNA sequencing timepoints (diagnostic, on-treatment, relapse) and included both bone marrow and peripheral blood. Upon reflection, and to better harmonize the bulk RNA-seq selection strategy with that of AML scAtlas, we revised our approach to include only diagnostic bone marrow samples. We expect that, since the leukemia blast count for pediatric AML is typically high at diagnosis, these samples will predominantly contain leukemic blasts. 

(2) Method selection: 

(a) The authors should explain why they use pySCENIC and not any other approach.They should briefly explain how pySCENIC works and what they get out in the main text. In addition they should explain the AUCell algorithm and motivate its usage. 

pySCENIC is state-of-the-art method for network inference from scRNA data and is widely used within the single-cell community (over 5000 citations for both versions of the SCENIC pipeline). The pipeline has been benchmarked as one of the top performers for GRN analysis (Nguyen et al, 2021. Briefings in Bioinformatics). AUCELL is a module within the pySCENIC pipeline to summarize the activity of a set of genes (a regulon) into a single number which helps compare and visualize different regulons.  We have modified the manuscript (Results section 2 paragraph 2) to better explain this method and provided some rationale and accompanying citations to justify its use for this analysis. We thank the reviewer for highlighting this and hope our updates add some clarity.

(b) The obtained GRN signatures were not critically challenged on an external dataset. Therefore, the evidence that supports these signatures to be reliable and significant to the investigated setting is weak. 

These signatures were inferred using the most suitable AML single-cell RNA datasets currently available. To validate our findings, we used two independent datasets (the TARGET AML bulk RNA sequencing cohort, and the Lambo et al. scRNA-seq dataset). To clarify this workflow in the manuscript, we have added a panel to Figure 3 outlining the analytical process. To our knowledge, there are no other better-suited datasets for validation. Experimental validations on patient samples, while valuable, are beyond the scope of this study.

(3) There are some issues with the analysis & visualization of the data. 

Based on this feedback, we have improved several aspects of the analysis, changed some visualizations, and improved figure resolution throughout the manuscript. 

(4) Discussion: 

(a) What exactly is the 'regulon signature' that the authors infer? How can it be useful for insights into disease mechanisms? 

The ’regulon signature’ here refers to a gene regulatory program (multiple gene modules, each defined by a transcription factor and its targets) which are specific to different age groups. Further investigation into this can be useful for understanding why patients of different ages confer a different clinical course. We have amended the text to explain this.  

(b) The authors write 'Together this indicates that EP300 inhibition may be particularly effective in t(8;21) AML, and that BCLAF1 may present a new therapeutic target for t(8;21) AML, particularly in children with inferred pre-natal origin of the driver translocation.' I am missing a critical discussion of what is needed to further test the two targets. Put differently: Would the authors take the risk of a clinical study given the evidence from their analysis? 

Indeed, many extensive studies would be required before these findings are clinically translatable. We have included a discussion paragraph (discussion paragraph 7) detailing what further work is required in terms of experimental validation and potential subsequent clinical study.

Reviewer #1 (Recommendations for the authors): 

In addition to the point raised above, Cytoscape files for the GRNs and eGRNs inferred would be useful to have. 

We have now provided Cytoscape/eGRN tables in supplementary materials.

Reviewer #2 (Recommendations for the authors): 

(1) Figures 1F and 1G: You show the summed-up frequencies for all patients, right? It would be very interesting to see this per patient, or add error bars, since the shown frequencies might be driven by single patients with many cells. 

While this type of plot could be informative, the large number of samples in the AML scAtlas rendered the output difficult to interpret. As a result, we decided not to include it in the manuscript.

(2) An issue of selection bias has to be raised when only the two samples expressing the expected signatures are selected from the external scRNA dataset. Similarly, in the DepMap analysis, the age and nature of the other cell lines sensitive to EP300 and BCLAF1 should be reported. 

Since the purpose of this analysis was to build on previously defined signatures, we selected the two samples which we had preliminary hypotheses for. It would indeed be interesting to explore those not matching these signatures; however, samples numbers are very small, so without preliminary findings robust interpretation and validation would be difficult. An expanded validation would be more appropriate once more data becomes available in the future. 

We agree that investigating the age and nature of other BCLAF1/EP300 sensitive cell lines is a very valuable direction. Our analysis suggests that our BCLAF1 findings may also be applicable to other in-utero origin cancers, and we have now summarized these observations in Supplementary Figure 7H. 

(3) Is there statistical evidence for your claim that "This shows that higher-risk subtypes have a higher proportion of LSCs compared to favorable risk disease."? At least intermediate and adverse look similar to me. How does this look if you show single patients?  

We are grateful to the reviewer for noticing this oversight and have now included an appropriate statistical test in the revised manuscript. As before, while showing single patients may be useful, the large number of patients makes such plot difficult to interpret. For this reason, we have chosen not to include them.

(4) Specify the statistical test you used to 'identify significantly differentially expressed TFs' (line 192). 

The methods used for differential expression analysis are now clearly stated in the text as well as in the methods section. We hope this addition improves clarity for the reader.

(5) Figure 2B: You show the summed up frequencies for all patients, right? It would be intriguing to see this figure per patient, since the shown frequencies might be driven by single patients with many cells. 

Yes, the plot includes all patients. Showing individual patients on a single plot is not easily interpretable. 

(6) Y axis in 2D is not samples, but single cells? Please specify. 

We thank the reviewer for bringing this to our attention and have now updated Figure 3D accordingly. 

(7) Figure 3A: I don't get why the chosen clusters are designated as post- and prenatal, given the occurrence of samples in them. 

This figure serves to validate the previously defined regulon signatures, so the cluster designations are based on this. We have amended the text to elaborate on this point, which will hopefully provide greater clarity.

(8) Figure 3E: What is shown on the y axis? Did you correct your p-values for multiple testing? 

We apologize for this oversight and have now added a y axis label. P values were not corrected for multiple testing, as there are only few pairwise T tests performed.

(9) Robustness: You find some gene sets up- and down-regulated. How would that change if you used an eg bootstrapped number of samples, or a different analysis approach? 

To address this, we implemented both edgeR and DESeq2 for DE testing. Our findings (Supplementary Figure 5B) show that 98% of edgeR genes are also detected by DESeq2. We opted to use the smaller edgeR gene list for our analysis, due to the significant overlap showing robust findings. We thank the reviewer for this helpful suggestion, which has strengthened our analysis

(10) Multiomics analysis:

(a) Why only work on 'representative samples'? The idea of an integrated atlas is to identify robust patterns across patients, no? I'd love to see what regulons are robust, ie,  shared between patients.

As discussed in point 2, there are very few samples available for the multiomics analysis. Therefore, we chose to focus on those samples which we had a working hypothesis for, as a validation for our other analyses. 

(b) I don't agree that finding 'the key molecular processes, such as RNA splicing, histone modification, and TF binding' expressed 'further supports the stemness signature in presumed prenatal origin t(8;21) AML'.

Following the improvements made on the bulk RNA-Seq analysis in response to the previous reviewer comments, we ended up with a smaller gene set. Consequently, the ontology results have changed. The updated results are now more specific and indicate that developmental processes are upregulated in presumed prenatal origin t(8;21) AML. 

(c) Please clarify if the multiome data is part of the atlas.

The multiome data is not a part of AML scAtlas, as it was published at a later date. We used this dataset solely for validation purposes and have updated the figures and text to clearly indicate that it is used as a validation dataset.  

(d) Please describe the used data with respect to the number of patients, cells, age, etc.

We clarified this point in the text and have also included supplementary tables detailing all samples used in the atlas and validation datasets. 

(e) The four figures in Figure 4E look identical to me. What is the take-home message here? Do all perturbations have the same impact on driving differentiation? Please elaborate.

The perturbation figure is intended to illustrate that other genes can behave similarly to members of the AP-1 complex (JUN and ATF4 here) following perturbation. Since the AP-1 complex is well known to be important in t(8;21) AML, we hypothesize that these other genes are also important. We apologize for the previous lack of interpretation here and have amended the text to clarify this point. 

(11) Abstract: Please detail: how many of the 159 AML patients are t(8;21)? 

We have now amended the abstract to include this. 

(12) Figures: Increase font size where possible, eg age in 1B or risk group in 1G is super small and hard to read. 

Extra attention has been given to improving the figure readability and resolution throughout the whole manuscript.  

(13) Color codes in Figures 2B and 2C are all over the place and misleading: Sort 2C along age, indicate what is adult and adolescent, sort the x axis in 2B along age. 

We have changed this figure accordingly.  

(14) I suggest not coloring dendrograms, in my opinion this is highly irritating. 

The dendrogram colors correspond to clusters which are referenced in the text, this coloring provides informative context and aids interpretation, making it a useful addition to the figure.

(15) The resolution in Figure 4B is bad, I can't read the labels. 

This visualization has been revised, to make presentation of this data clearer.  

(16) In addition to selecting bulk RNA samples matching the two regulon signatures, some effort should have been put into investigating the samples not aligned with those, or assessing how unique these GRN signatures are to the specific cell type and disease of interest, excluding the influence of cell type composition and random noise. The lateonset signatures should also be excluded from being present in an external pre-natal cohort in a more statistically rigorous manner. 

Our use of the bulk RNA-Seq data is solely intended for the validation of predefined regulon signatures, for which we already have a working hypothesis.  While we agree that further investigation of the samples that do not align with these signatures could yield interesting insights, we believe that such an analysis would extend beyond the scope of the current manuscript.

(17) The specific bulk RNA samples used should be specified, along with the tissue of origin. The same goes for the Lambo dataset. 

We have clarified this point in the text and provided a supplementary table detailing all samples used for validation, alongside the sample list from AML scAtlas.

(18) In Supplementary Figure 5 B, the axes should be define. 

We have updated this figure to include axis legends.

(19) Supplementary Figure 4A. There is a mistake in the sex assignment for sample AML14D. Since chrY-genes are expressed, this sample is likely male, while the Xist expression is mostly zero. 

We thank the reviewer for pointing out this error, which has now been corrected.  

(20) Wording suggestions: 

(a) Line 54: not compelling phrasing. 

(b) Line 83: "allows to decipher". 

(c) Line 88: repetition from line 85. 

(d) Line 90: the expression "clean GRN" is not clear. 

These wording suggestions have all been incorporated in the revised manuscript.

(21) Supplementary Figure 3D is not interpretable, I suggest a different visualization. 

We agree that the original figure was not the most informative and have replaced it with UMAPs displaying LSC6 and LSC17 scores.

Author response:

Reviewer #1 (Public review):

Fombellida-Lopez and colleagues describe the results of an ART intensification trial in people with HIV infection (PWH) on suppressive ART to determine the effect of increasing the dose of one ART drug, dolutegravir, on viral reservoirs, immune activation, exhaustion, and circulating inflammatory markers. The authors hypothesize that ART intensification will provide clues about the degree to which low-level viral replication is occurring in circulation and in tissues despite ongoing ART, which could be identified if reservoirs decrease and/or if immune biomarkers change. The trial design is straightforward and well-described, and the intervention appears to have been well tolerated. The investigators observed an increase in dolutegravir concentrations in circulation, and to a lesser degree in tissues, in the intervention group, indicating that the intervention has functioned as expected (ART has been intensified in vivo). Several outcome measures changed during the trial period in the intervention group, leading the investigators to conclude that their results provide strong evidence of ongoing replication on standard ART. The results of this small trial are intriguing, and a few observations in particular are hypothesis-generating and potentially justify further clinical trials to explore them in depth. However, I am concerned about over-interpretation of results that do not fully justify the authors' conclusions.

We thank Reviewer #1 for their thoughtful and constructive comments, which helped us clarify and improve the manuscript. Below, we address each of the reviewer’s points and describe the changes that we implemented in the revised version. We acknowledge the reviewer’s concern regarding potential overinterpretation of certain findings, and in the revised version we took particular care to ensure that all conclusions are supported by the data and framed within the exploratory nature of the study.

(1) Trial objectives: What was the primary objective of the trial? This is not clearly stated. The authors describe changes in some reservoir parameters and no changes in others. Which of these was the primary outcome? No a priori hypothesis / primary objective is stated, nor is there explicit justification (power calculations, prior in vivo evidence) for the small n, unblinded design, and lack of placebo control. In the abstract (line 36, "significant decreases in total HIV DNA") and conclusion (lines 244-246), the authors state that total proviral DNA decreased as a result of ART intensification. However, in Figures 2A and 2E (and in line 251), the authors indicate that total proviral DNA did not change. These statements are confusing and appear to be contradictory. Regarding the decrease in total proviral DNA, I believe the authors may mean that they observed transient decrease in total proviral DNA during the intensification period (day 28 in particular, Figure 2A), however this level increases at Day 56 and then returns to baseline at Day 84, which is the source of the negative observation. Stating that total proviral DNA decreased as a result of the intervention when it ultimately did not is misleading, unless the investigators intended the day 28 timepoint as a primary endpoint for reservoir reduction - if so, this is never stated, and it is unclear why the intervention would then be continued until day 84? If, instead, reservoir reduction at the end of the intervention was the primary endpoint (again, unstated by the authors), then it is not appropriate to state that the total proviral reservoir decreased significantly when it did not.

We agree with the reviewer that the primary objective of the study was not explicitly stated in the submitted manuscript. We clarified this in the revised manuscript (lines 361-364). As registered on ClinicalTrials.gov (NCT05351684), the primary outcome was defined as “To evaluate the impact of treatment intensification at the level of total and replication-competent reservoir (RCR) in blood and in tissues”, with a time frame of 3 months. Accordingly, our aim was to explore whether any measurable reduction in the HIV reservoir (total or replication-competent) occurred during the intensification period, including at day 28, 56, or 84. The protocol did not prespecify a single time point for this effect to occur, and the exploratory design allowed for detection of transient or sustained changes within the intensification window.

We recognize that this scope was not clearly articulated in the original text and may have led to confusion in interpreting the transient drop in total HIV DNA observed at day 28. While total DNA ultimately returned to baseline by the end of intensification, the presence of a transient reduction during this 3-month window still fits within the framework of the study’s registered objective. Moreover, although the change in total HIV DNA was transient, it aligns with the consistent direction of changes observed across the multiple independent measures, including CA HIV RNA, RNA/DNA ratio and intact HIV DNA, collectively supporting a biological effect of intensification.

We would also like to stress that this is the first clinical trial ever, in which an ART intensification is performed not by adding an extra drug but by increasing the dosage of an existing drug. Therefore, we were more interested in the overall, cumulative, effect of intensification throughout the entire trial period, than in differences between groups at individual time points. We clarified in the revised manuscript that this was a proof-of-concept phase 2 study, designed to reveal biological effects of ART intensification rather than confirm efficacy in a powered comparison. The absence of a prespecified statistical endpoint or sample size calculation reflects the exploratory nature of the trial.

(2) Intervention safety and tolerability: The results section lacks a specific heading for participant safety and tolerability of the intervention. I was wondering about clinically detectable viremia in the study. Were there any viral blips? Was the increased DTG well tolerated? This drug is known to cause myositis, headache, CPK elevation, hepatotoxicity, and headache. Were any of these observed? What is the authors' interpretation of the CD4:8 ratio change (line 198)? Is this a significant safety concern for a longer duration of intensification? Was there also a change in CD4% or only in absolute counts? Was there relative CD4 depletion observed in the rectal biopsy samples between days 0 and 84? Interestingly, T cells dropped at the same timepoints that reservoirs declined... how do the authors rule out that reservoir decline reflects transient T cell decline that is non-specific (not due to additional blockade of replication)?

We improved the Methods section to clarify how safety and tolerability were assessed during the study (lines 389-396). Safety evaluations were conducted on day 28 and day 84 and included a clinical examination and routine laboratory testing (liver function tests, kidney function, and complete blood count). Medication adherence was also monitored through pill counts performed by the study nurses.

No virological blips above 50 copies/mL were observed and no adverse events were reported by participants during the 3-month intensification period. Although CPK levels were not included in the routine biological monitoring, no participant reported muscle pain or other symptoms suggestive of muscle toxicity.

The CD4:CD8 ratio decrease noted during intensification was not associated with significant changes in absolute CD4 or CD8 counts, as shown in Figure 5. We interpret this ratio change as a transient redistribution rather than an immunological risk, therefore we do not consider it to represent a safety concern.

We would like to clarify that CD4⁺ T-cell counts did not significantly decrease in any of the treatment groups, as shown in Figure 5. The apparent decline observed concerns the CD4/CD8 ratio, which transiently dropped, but not the absolute number of CD4⁺ T cells. Moreover, although the dynamics of total HIV DNA is indeed similar to that of CD4/CD8 ratio (both declined transiently and then returned to baseline by day 84), the dynamics of unspliced RNA and unspliced RNA/total DNA ratio are clearly different, as these markers demonstrated a sustained decrease that was maintained throughout the trial period, even when the CD4/CD8 ratio already returned to baseline. Also, we observed a significant decrease in intact HIV DNA at day 84 compared to day 0. These effects cannot be easily explained by a transient decline in CD4+ cells.

(3) The investigators describe a decrease in intact proviral DNA after 84 days of ART intensification in circulating cells (Figure 2D), but no changes to total proviral DNA in blood or tissue (Figures 2A and 2E; IPDA does not appear to have been done on tissue samples). It is not clear why ART intensification would result in a selective decrease in intact proviruses and not in total proviruses if the source of these reservoir cells is due to ongoing replication. These reservoir results have multiple interpretations, including (but not limited to) the investigators' contention that this provides strong evidence of ongoing replication. However, ongoing replication results in the production of both intact and mutated/defective proviruses that both contribute to reservoir size (with defective proviruses vastly outnumbering intact proviruses). The small sample size and well-described heterogeneity of the HIV reservoir (with regard to overall size and composition) raise the possibility that the study was underpowered to detect differences over the 84-day intervention period. No power calculations or prior studies were described to justify the trial size or the duration of the intervention. Readers would benefit from a more nuanced discussion of reservoir changes observed here.

We sincerely thank the reviewer for this insightful comment. We fully agree that the reservoir dynamics observed in our study might raise several possible interpretations, and that its complexity, resulting from continuous cycles of expansion and contraction, reflects the heterogeneity of the latent reservoir. 

Total HIV DNA in PBMCs showed a transient decline during intensification (notably at day 28), ultimately returning to baseline by day 84. This biphasic pattern likely reflects the combined effects of suppression of ongoing low-level replication by an increased DTG dosage, followed by the expansion of infected cell clones (mostly harbouring defective proviruses). In other words, the transient decrease in total (intact + defective) DNA at day 28 may be due to an initial decrease in newly infected cells upon ART intensification, however at the subsequent time points this effect was masked by proliferation (clonal expansion) of infected cells with defective proviruses. Recent studies suggest that intact and defective proviruses are subjected to different selection pressures by the immune system on ART (PMID: 38337034) and their decay on therapy is different (intact proviruses are cleared much more rapidly than defectives). In addition, defective proviruses can be preferentially expanded as they can reprogram the host cell proliferation machinery (https://doi.org/10.1101/2025.09.22.676989). This explains why in our study the intact proviruses decreased, but the total proviruses did not change, between days 0 and 84, in the intensification group. Interestingly, in the control group, we observed a significant increase in total DNA at day 84 compared to day 0, with no difference for the intact DNA, which is also in line with the clonal expansion of defective proviruses.

Importantly, we observed a significant decrease in intact proviral DNA between day 0 and day 84 in the intensification group (Figure 2D). This result directly addresses the study’s primary objective: assessing the impact of intensification on the replication-competent reservoir. In comparison, as the reviewer rightly points out, total HIV DNA includes over 90% defective genomes, which limits its interpretability as a biomarker of biologically relevant reservoir changes. In addition, other reservoir markers, such as cell-associated unspliced RNA and RNA/DNA ratios, also showed consistent trends supporting a biologically relevant effect of intensification. Even in the absence of sustained changes in total HIV DNA, the coherence across the different independent measures of the reservoir (intact DNA, unspliced RNA), suggests an effect indicative of ongoing replication pre-intensification.

Regarding tissue reservoirs, the lack of substantial change in total HIV DNA between days 0 and 84 is also in line with the predominance of defective sequences in these compartments. Moreover, the limited increase in rectal tissue dolutegravir levels during intensification (from 16.7% to 20% of plasma concentrations) may have limited the efficacy of the intervention in this site.

As for the IPDA on rectal biopsies, we attempted the assay using two independent DNA extraction methods (Promega Reliaprep and Qiagen Puregene), but both yielded high DNA shearing index values, and intact proviral detection was successful in only 3 of 40 samples. Given the poor DNA integrity, these results were not interpretable.

That said, we fully acknowledge the limitations of our study, especially the small sample size, and we agree with the reviewer that caution is needed when interpreting these findings. In the revised manuscript, we adopted a more measured tone in the discussion (lines 340-346), stating that these observations are exploratory and hypothesis-generating, and require confirmation in larger, more powered studies. Nonetheless, we believe that the convergence of multiple reservoir markers pointing in the same direction constitutes a meaningful biological effect that deserves further investigation.

(4) While a few statistically significant changes occurred in immune activation markers, it is not clear that these are biologically significant. Lines 175-186 and Figure 3: The change in CD4 cells + for TIGIT looks as though it declined by only 1-2%, and at day 84, the confidence interval appears to widen significantly at this timepoint, spanning an interquartile range of 4%. The only other immune activation/exhaustion marker change that reached statistical significance appears to be CD8 cells + for CD38 and HLA-DR, however, the decline appears to be a fraction of a percent, with the control group trending in the same direction. Despite marginal statistical significance, it is not clear there is any biological significance to these findings; Figure S6 supports the contention that there is no significant change in these parameters over time or between groups. With most markers showing no change and these two showing very small changes (and the latter moving in the same direction as the control group), these results do not justify the statement that intensifying DTG decreases immune activation and exhaustion (lines 38-40 in the abstract and elsewhere).

We agree with the reviewer that the observed changes in immune activation and exhaustion markers were modest. We revised the abstract and the manuscript text (including a section header) to reflect this more accurately (lines 39, 175, 185, 253). We noted that these differences, while statistically significant (e.g., in TIGIT+ CD4+ T cells and CD38+HLA-DR+ CD8+ T cells), were limited in magnitude. We explicitly acknowledged these limitations and interpreted the findings with appropriate caution.

(5) There are several limitations of the study design that deserve consideration beyond those discussed at line 327. The study was open-label and not placebo-controlled, which may have led to some medication adherence changes that confound results (authors describe one observation that may be evidence of this; lines 146-148). Randomized/blinded / cross-over design would be more robust and help determine signal from noise, given relatively small changes observed in the intervention arm.There does not seem to be a measurement of key outcome variables after treatment intensification ceased - evidence of an effect on replication through ART intensification would be enhanced by observing changes once intensification was stopped. Why was intensification maintained for 84 days? More information about the study duration would be helpful. Table 1 indicates that participants were 95% male. Sex is known to be a biological variable, particularly with regard to HIV reservoir size and chronic immune activation in PWH. Worldwide, 50% of PWH are women. Research into improving management/understanding of disease should reflect this, and equal participation should be sought in trials. Table 1 shows differing baseline reservoir sizes between the control and intervention groups. This may have important implications, particularly for outcomes where reservoir size is used as the denominator.

We expanded the limitations section to address several key aspects raised by the reviewer: the absence of blinding and placebo control, the predominantly male study population, and the lack of postintervention follow-up. While we acknowledge that open-label designs can introduce behavioural biases, including potential changes in adherence, we now explicitly state that placebo-controlled, blinded trials would provide a more robust assessment and are warranted in future research (lines 340346). 

The 84-day duration of intensification was chosen based on previous studies and provided sufficient time for observing potential changes in viral transcription and reservoir dynamics. However, we agree that including post-intervention follow-up would have strengthened the conclusions, and we highlighted this limitation and future direction in the revised manuscript (lines 340-346). 

The sex imbalance is now clearly acknowledged as a limitation in the revised manuscript, and we fully support ongoing efforts to promote equitable recruitment in HIV research. We would like to add that, in our study, rectal biopsies were coupled with anal cancer screening through HPV testing. This screening is specifically recommended for younger men who have sex with men (MSM), as outlined in the current EACS guidelines (see: https://eacs.sanfordguide.com/eacs part2/cancer/cancerscreening-methods). As a result, MSM participants had both a clinical incentive and medical interest to undergo this procedure, which likely contributed to the higher proportion of male participants in the study.

Lastly, although baseline total HIV DNA was higher in the intensified group, our statistical approach is based on a within-subject (repeated-measures) design, in which the longitudinal change of a parameter within the same participant during the study was the main outcome. In other words, we are not comparing absolute values of any marker between the groups, we are looking at changes of parameters from baseline within participants, and these are not expected to be affected by baseline imbalances.

(6) Figure 1: the increase in DTG levels is interesting - it is not uniform across participants. Several participants had lower levels of DTG at the end of the intervention. Though unlikely to be statistically significant, it would be interesting to evaluate if there is a correlation between change in DTG concentrations and virologic / reservoir / inflammatory parameters. A positive relationship between increasing DTG concentration and decreased cell-associated RNA, for example, would help support the hypothesis that ongoing replication is occurring.

We agree with the reviewer that assessing correlations between DTG concentrations and virological, immunological, or inflammatory markers would be highly informative. In fact, we initially explored this question in a preliminary way by examining whether individuals who showed a marked increase in DTG levels after intensification also demonstrated stronger changes in the viral reservoir. While this exploratory analysis did not reveal any clear associations, we would like to emphasize that correlating biological effects with DTG concentrations measured at a single timepoint may have limited interpretability. A more comprehensive understanding of the relationship between drug exposure and reservoir dynamics would ideally require multiple pharmacokinetic measurements over time, including pre-intensification baselines. This is particularly important given that DTG concentrations vary across individuals and over time, depending on adherence, metabolism, and other individual factors.

(7) Figure 2: IPDA in tissue- was this done? scRNA in blood (single copy assay) - would this be expected to correlate with usCaRNA? The most unambiguous result is the decrease in cell-associated RNA - accompanying results using single-copy assay in plasma would be helpful to bolster this result.

As mentioned in our response to point 3, we attempted IPDA on tissue samples, but technical limitations prevented reliable detection of intact proviruses. Regarding residual viremia, we did perform ultra-sensitive plasma HIV RNA quantification but due to a technical issue (an inadvertent PBMC contamination during plasma separation) that affected the reliability of the results we felt uncomfortable including these data in the manuscript.

The use of the US RNA / Total DNA ratio is not helpful/difficult to interpret since the control and intervention arms were unmatched for total DNA reservoir size at study entry.

We respectfully disagree with this comment. The US RNA/total DNA ratio is commonly used to assess the relative transcriptional activity of the viral reservoir, rather than its absolute size. While we acknowledge that the total HIV-1 DNA levels differed at baseline between the two groups, the US RNA/total DNA ratio specifically reflects the relationship between transcriptional activity and reservoir size within each individual, and is therefore not directly confounded by baseline differences in total DNA alone.

Moreover, our analyses focus on within-subject longitudinal changes from baseline, not on direct between-group comparisons of absolute marker values. As such, the observed changes in the US RNA/total DNA ratio over time are interpreted relative to each participant's baseline, mitigating concerns related to baseline imbalances between groups.

Reviewer #2 (Public review):

Summary:

An intensification study with a double dose of 2nd generation integrase inhibitor with a background of nucleoside analog inhibitors of the HIV retrotranscriptase in 2, and inflammation is associated with the development of co-morbidities in 20 individuals randomized with controls, with an impact on the levels of viral reservoirs and inflammation markers. Viral reservoirs in HIV are the main impediment to an HIV cure, and inflammation is associated with co-morbidities.

Strengths:

The intervention that leads to a decrease of viral reservoirs and inflammation is quite straightforward forward as a doubling of the INSTI is used in some individuals with INSTI resistance, with good tolerability.

This is a very well documented study, both in blood and tissues, which is a great achievement due to the difficulty of body sampling in well-controlled individuals on antiretroviral therapy. The laboratory assays are performed by specialists in the field with state-of-the art quantification assays. Both the introduction and the discussion are remarkably well presented and documented.

The findings also have a potential impact on the management of chronic HIV infection.

Weaknesses:

I do not think that the size of the study can be considered a weakness, nor the fact that it is open-label either.

We thank Reviewer #2 for their constructive and supportive comments. We appreciate their positive assessment of the study design, the translational relevance of the intervention, and the technical quality of the assays. We also take note of their perspective regarding sample size and study design, which supports our positioning of this trial as an exploratory, hypothesis-generating phase 2 study.

Reviewer #3 (Public review):

The introduction does a very good job of discussing the issue around whether there is ongoing replication in people with HIV on antiretroviral therapy. Sporadic, non-sustained replication likely occurs in many PWH on ART related to adherence, drug-drug interactions and possibly penetration of antivirals into sanctuary areas of replication and as the authors point out proving it does not occur is likely not possible and proving it does occur is likely very dependent on the population studied and the design of the intervention. Whether the consequences of this replication in the absence of evolution toward resistance have clinical significance challenging question to address.

It is important to note that INSTI-based therapy may have a different impact on HIV replication events that results in differences in virus release for specific cell type (those responsible for "second phase" decay) by blocking integration in cells that have completed reverse transcription prior to ART initiation but have yet to be fully activated. In a PI or NNRTI-based regimen, those cells will release virus, whereas with an INSTI-based regimen, they will not.

Given the very small sample size, there is a substantial risk of imbalance between the groups in important baseline measures. Unfortunately, with the small sample size, a non-significant P value is not helpful when comparing baseline measures between groups. One suggestion would be to provide the full range as opposed to the inter-quartile range (essentially only 5 or 6 values). The authors could also report the proportion of participants with baseline HIV RNA target not detected in the two groups.

We thank Reviewer #3 for their thoughtful and balanced review. We are grateful for the recognition of the strength of the Introduction, the complexity of evaluating residual replication, and the technical execution of the assays. We also appreciate the insightful suggestions for improving the clarity and transparency of our results and discussion.

We revised the manuscript to address several of the reviewer’s key concerns. We agree that the small sample size increases the risk of baseline imbalances. We acknowledged these limitations in the manuscript (lines 327-330). For transparency, we now provide both the full range and the IQR for all parameters in Table 1. However, we would like to stress that our statistical approach is based on a within-subject (repeated-measures) design, in which the longitudinal change of a parameter within the same participant during the study was the main outcome. In other words, we are not comparing absolute values of any marker between the groups, we are looking at changes of parameters from baseline within participants, and these are not expected to be affected by baseline imbalances.

A suggestion that there is a critical imbalance between groups is that the control group has significantly lower total HIV DNA in PBMC, despite the small sample size. The control group also has numerically longer time of continuous suppression, lower unspliced RNA, and lower intact proviral DNA. These differences may have biased the ability to see changes in DNA and US RNA in the control group.

We acknowledge the significant baseline difference in total HIV DNA between groups, which we have clearly reported. However, the other variables mentioned, such as duration of continuous viral suppression, unspliced RNA levels, and intact proviral DNA, did not differ significantly between groups at baseline, despite differences in the median values (that are always present). These numerical differences do not necessarily indicate a critical imbalance.

Notably, there was no significant difference in the change in US RNA/DNA between groups (Figure 2C).

The nonsignificant difference in the change in US RNA/total DNA between groups is not unexpected, given the significant between-group differences for both US RNA and total DNA changes. Since the ratio combines both markers, it is likely to show attenuated between-group differences compared to the individual components. However, while the difference did not reach statistical significance (p = 0.09), we still observed a trend towards a greater reduction in the US RNA/total DNA ratio in the intervention group.

The fact that the median relative change appears very similar in Figure 2C, yet there is a substantial difference in P values, is also a comment on the limits of the current sample size. 

Although we surely agree that in general, the limited sample size impacts statistical power, we would like to point out that in Figure 2C, while the medians may appear similar, the ranges do differ between groups. At days 56 and 84, the median fold changes from baseline are indeed close but the full interquartile range in the DTG group stays below 1, while in the control group, the interquartile range is wider and covers approximately equal distance above and below 1. This explains the difference in p values between the groups.

The text should report the median change in US RNA and US RNA/DNA when describing Figures 2A-2C.

These data are already reported in the Results section (lines 164–166): "By day 84, US RNA and US RNA/total DNA ratio had decreased from day 0 by medians (IQRs) of 5.1 (3.3–6.4) and 4.6 (3.1–5.3) fold, respectively (p = 0.016 for both markers)."

This statistical comparison of changes in IPDA results between groups should be reported. The presentation of the absolute values of all the comparisons in the supplemental figures is a strength of the manuscript.

In the assessment of ART intensification on immune activation and exhaustion, the fact that none of the comparisons between randomized groups were significant should be noted and discussed.

We would like to point out that a statistically significant difference between the randomized groups was observed for the frequency of CD4⁺ T cells expressing TIGIT, as shown in Figure 3A and reported in the Results section (p = 0.048).

The changes in CD4:CD8 ratio and sCD14 levels appear counterintuitive to the hypothesis and are commented on in the discussion.

Overall, the discussion highlights the significant changes in the intensified group, which are suggestive. There is limited discussion of the comparisons between groups where the results are less convincing.

We observed statistically significant differences between the randomized groups for total DNA (p<0.001) and US RNA (p=0.01), as well as for the frequency of CD4⁺ T cells expressing TIGIT (p=0.048). We would like to stress that US RNA is a key marker of residual replication as it is very sensitive to de novo infection events. As discussed in the manuscript (lines 291-294), a newly infected CD4+ T lymphocyte can contain hundreds to thousands of US HIV RNA copies at the peak of infection. Therefore, a change in the US RNA level upon ART intensification is a very sensitive indicator of new infections. The fact that for US RNA we observed both a significant reduction in the intensified group and a significant difference between the groups is a strong indicator that some new infections had been occurring prior to intensification.

The limitations of the study should be more clearly discussed. The small sample size raises the possibility of imbalance at baseline. The supplemental figures (S3-S5) are helpful in showing the differences between groups at baseline, and the variability of measurements is more apparent. The lack of blinding is also a weakness, though the PK assessments do help (note 3TC levels rise substantially in both groups for most of the time on study (Figure S2).

The many assays and comparisons are listed as a strength. The many comparisons raise the possibility of finding significance by chance. In addition, if there is an imbalance at baseline outcomes, measuring related parameters will move in the same direction.

We agree that the multiple comparisons raise the possibility of chance findings but would like to stress that in an exploratory study like this it is very important to avoid a type II error. In addition, the consistent directionality of the most relevant outcomes (US RNA and intact DNA) lends biological plausibility to the observed effects.

The limited impact on activation and inflammation should be addressed in the discussion, as they are highlighted as a potentially important consequence of intermittent, not sustained replication in the introduction.

The study is provocative and well executed, with the limitations listed above. Pharmacokinetic analyses help mitigate the lack of blinding. The major impact of this work is if it leads to a much larger randomized, controlled, blinded study of a longer duration, as the authors point out.

Finally, we fully endorse the reviewer’s suggestion that the primary contribution of this study lies in its value as a proof-of-concept and foundation for future randomized, blinded trials of greater scale and duration. We highlighted this more clearly in the revised Discussion (lines 340-346).

Reviewer #1 (Recommendations for the authors):

(1) Lines 84-87: How would chronic immune activation/inflammation be expected to differ if viral antigen is being released from stable reservoirs rather than low-level replication?

This is a very insightful question. Although release of viral antigens from stable reservoirs could certainly also trigger immune activation/inflammation, the reservoir cells in PWH on long-term ART are constantly being negatively selected by the immune system (PMID: 38337034; PMID: 36596305) so that after a number of years on therapy, most proviruses are either transcriptionally silent or express only a low amount of viral RNA/antigen. Recent evidence suggests that these selected cells possess specific biological properties that include mechanisms that limit proviral gene expression (PMID: 36599977; PMID: 36599978). In comparison, low-level replication would result in de novo infection of unselected, activated CD4+ cells that are expected to produce much more viral antigen than preselected reservoir cells.

(2) Lines 249-253: There are multiple ways to explain this observation - alternatively, the total proviral DNA declined due to transient CD4 depletion.

As discussed above, CD4⁺ T-cell counts did not significantly decrease in any of the treatment groups, as shown in Figure 5. The apparent decline observed concerns the CD4/CD8 ratio, which transiently dropped, but not the absolute number of CD4⁺ T cells. Moreover, although the dynamics of total HIV DNA is indeed similar to that of CD4/CD8 ratio (both declined transiently and then returned to baseline by day 84), the dynamics of unspliced RNA and unspliced RNA/total DNA ratio is clearly different, as these markers demonstrated a sustained decrease that was maintained throughout the trial period. Also, we observed a significant decrease in intact HIV DNA at day 84 compared to day 0. These effects cannot be easily explained by a transient decline in CD4+ cells.

(3) Lines 301-305: This is a confusing explanation for not seeing an effect in tissue. Overall, there was no change in total proviral DNA in blood between days 0 and 84 either - yet the explanation for this observation is different (249-253). Was IPDA not performed on the tissue? Wouldn't this be the preferred test for reservoir depletion?

We thank the reviewer for bringing this point to our attention. We modified the Discussion to prevent the confusion (lines 303-305). As for the IPDA on tissue, we attempted this assay on the tissue samples using two independent DNA extraction methods (Promega Reliaprep and Qiagen Puregene), but both yielded high DNA shearing index values, and intact proviral detection was successful in only 3 of 40 samples. Given the poor DNA integrity, these results were not interpretable.

Reviewer #2 (Public review):

Summary:

The work set out to better understand the phenomenon of antibiotic persistence in mycobacteria. Three new observations are made using the pathogenic Mycobacterium abscessus as an experimental system: phenotypic tolerance involves suppression of ROS, protein synthesis inhibitors can be lethal for this bacterium, and levofloxacin lethality is unaffected by deletion of catalase, suggesting that this quinolone does not kill via ROS.

Strengths:

The ROS experiments are supported in three ways: measurement of ROS by a fluorescent probe, deletion of catalase increases lethality of selected antibiotics, and a hypoxia model suppresses antibiotic lethality. A variety of antibiotics are examined, and transposon mutagenesis identifies several genes involved in phenotypic tolerance, including one that encodes catalase. The methods are adequate for making these statements.

Weaknesses:

The work can be improved by a more comprehensive treatment of prior work, especially comparison of E. coli work with mycobacterial studies.<br /> Moreover, the work still has some technical issues to fix regarding description of the methods, supplementary material, and reference formating.

Overall impact: Showing that ROS accumulation is suppressed during phenotypic tolerance, while expected, adds to the examples of the protective effects of low ROS levels. Moreover, the work, along with a few others, extends the idea of antibiotic involvement with ROS to mycobacteria. These are field-solidifying observations.

Comments on revisions:

The authors have moved this paper along nicely. I have a few general thoughts.

(1) It would be helpful to have more references to specific figures and panels listed in the text to make reading easier.

(2) I would suggest adding a statement about the importance of the work. From my perspective, the work shows the general nature of many statements derived from work with E. coli. This is important. The abstract says this overall, but a final sentence in the abstract would make it clear to all readers.

(3) The paper describes properties that may be peculiar to mycobacteria. If the authors agree, I would suggest some stress on the differences from E. coli. Also, I would place more stress on novel findings. This might be done in a section called Concluding Remarks. The paper by Shee 2022 AAC could be helpful in phrasing general properties.

(4) Several aspects still need work to be of publication quality. Examples are the materials table and the presentation of supplementary material. Reference formatting also needs attention.

Author response:

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

Reviewer #1 (Public review):

Weaknesses:

Only 1 gene (katG) gave a strong and 1 (Mab_1456c) exhibited a minor defect. Two of the clones did not show any persistence phenotype (blaR and recR) and one (pafA) showed a minor phenotype,

We have now carried out more detailed validation studies on the Tn-Seq, with analysis of timedependent killing over 14 d. This more comprehensive analysis shows that 4 of 5 genes analyzed do indeed have antibiotic tolerance defects under the conditions that Tn-Seq predicted a survival defect (Revised Figure 3). In addition, we found that even before actual cell death, several mutants had delayed resumption of growth after antibiotic removal (Figure 3 Supplemental).

Fig 3 - Why is there such a huge difference in the extent of killing of the control strain in media, when exposed to TIG/LZD, when compared to Fig. 1C and Fig. 4. In Fig. 1C, M. abs grown in media decreases by >1 log by Day 3 and >4 log by Day 6, whereas in Fig. 3, the bacterial load decreases by <1 log by Day 3 and <2 log by Day 6. This needs to be clarified, if the experimental conditions were different, because if comparing to Fig. 1C data then the katG mutant strain phenotype is not very different.

We agree with the reviewer that there is variability in the timing and extent of cell death from experiment to experiment. As noted by the reviewer, in Figure 1C the largest decrement in survival is between day 1 - day 3 (also seen in Figure 6A). As they noted in Figure 4 the largest decrement is between day 3 – day 6 (also seen in Figure 3A, Figure 5F). In each experiment with katG mutants we carefully compare the mutant vs. the control strain within that experiment, which is more accurate than comparing the behavior of mutant in one experiment to a control in another experiment.

Reviewer #2 (Public review):

Weaknesses:

.First, word-choice decisions could better conform to the published literature. Alternatively, novel definitions could be included. In particular, the data support the concept of phenotypic tolerance, not persistence. 

We appreciate the reviewers comments, text modified.

Second, two of the novel observations could be explored more extensively to provide mechanistic explanations for the phenomena. 

We have added several additional experiments, these are detailed below in response to specific comments.

Reviewer #3 (Public review):

Weaknesses:

The findings could not be validated in clinical strains.

We understand the reviewer’s concern that the katG phenotype was only observed in one of the two clinical strains we studied. We feel that our findings are relevant beyond the ATCC 19977 strain for two reasons

(1) We have performed additional analyses of the two clinical isolates and indeed find significant accumulation of ROS following antibiotic exposure in both of these strains (revised Figure 6A).

(2) We do in fact see a role for katG in starvation-induced antibiotic tolerance in Mabs clinical strain-2. It is not surprising that different strains from a particular species may have some different responses to stresses – for example, there is wide strain-specific variability in susceptibility to different phages within a species based on which particular phage defense modules a given strain carries (for example PMID: 37160116). We speculate that different Mabs strains may express varying levels of other antioxidant factors and note that the genes encoding several such factors were identified by our Tn-Seq screen including the peroxidases ahpC, ahpD, and ahpE. Our analysis of the genetic interactions between katG and these other factors is ongoing. 

Comments/Suggestions

(1) In Fig1E, the authors show no difference in killing Mtb with or without adaptation in PBS. These data are contrary to the data presented in Figure 1B. These also do not align with the data of M. smegmatis and M. abscesses. Please discuss these observations in light of the Duncan model of persistence (Mol Microbiol. 2002 Feb;43(3):717-31.).’

The above referenced Duncan laboratory study found tolerance after prolonged starvation but did not actually examine tolerance at early time points. While some of the transcriptional and metabolic changes seen by Duncan and others are slow, other groups have described starvation responses in Mtb that are quite rapid. For example, the stringent response mediator ppGpp accumulates within a few hours after onset of starvation in Mtb (PMID: 30906866). We suspect that a rapid signaling response such as this underlies the phenotype we observe. Regarding the difference between Mtb and other mycobacterial species we also find it surprising that Mtb had a much more rapid starvation response. This is a clear species-specific difference that may reflect an adaptation of Mtb to the nutrient-limited physiologic niche within host macrophages.

(2) Line 151, the authors state that they have used an M. abscesses Tn mutant library of ~ 55,000 mutant strains. The manuscript will benefit from the description of the coverage of total TA sites covered by the mutants.

Text modified to add this detail. There are 91,559 TA sites in the abscessus genome. Thus, our Tn density is ~60%.

(3) Line 155: Please explain how long the cells were kept in an Antibiotic medium.

This technical detail was noted above on line 153 in the original text: “…and then exposed them to TIG/LZD for 6 days”. To clarify the overall conditions, we have also revised the text of the manuscript and added the detail of how long cells were passaged after removal of antibiotics.

(4) Line 201: data not shown. Delayed resumption of growth after removal of antibiotic would be helpful in indicating drug resilience. This data could enhance the manuscript.

Data now provided in Figure 3 Supplemental

(5) Figures 4C and 4F represent the kill curve. It will be good to show the date with CFU against the drug concentration in place of OD600. CFU rather than OD600 best reflects growth inhibition.

Figures 4C and 4F are measuring the minimum inhibitory concentration (MIC) to stop the overall growth of the bacterial population. While we agree that CFU could be analyzed, this would be measuring a different outcome – cell death and the minimum bactericidal concentration (MBC). In these experiments we sought to specifically examine the MIC so as to separate growth inhibition from cell death. For this we used the standard method employed by clinical microbiology laboratories for MIC, which is optical density of the culture (PMID: 10325306).

(6) Figure 5C. The authors shall show the effect of TIG/LZD on M. abscesses ROS production without the PBS adaptation. It is important to conclude that TIG/LZD induces ROS in cells. Authors should utilize ROS scavengers such as Thiourea, DFO, etc., to conclude ROS's contribution to bacterial killing following inhibition of transcription and translation.

New data added (revised Figure 5 and Figure 5 Supplemental)  

(7) Line 303. Remove "note".

Text revised. We thank the reviewer for identifying this typographical error.  

(8) The introduction and Discussion are very similar, and several lines are repeated.

Text revised with overlapping content removed.

Reviewer #1 (Recommendations for the authors):

It appears that the same datasets for PBS adapted cultures were plotted in A-C and D-F. Either this should be specifically mentioned in the legend or it might be better to integrate the non-adapted plots into A-C which would also allow easier comparison.

Appreciate the reviewer’s suggestion; text modified with added clarification to figure legend.

This manuscript is focused on M. abs and the antibiotics TIG/LZD, so the Mtb data or data using the antibiotics INH/RIF/EMB and serves more as a distraction and can be removed

We appreciate the reviewer’s perspective. However, we wish to include these data to show the similarities (and differences) in starvation-induced tolerance between the three organisms.

Fig 3 -As mentioned for Fig. 1, it appears that the same dataset was used for the control in all the figures A-E. This should be explicitly stated in the Figure legend.

Appreciate the reviewer’s suggestion; text modified with added clarification to figure legend.

The divergent results from the clinical strains are extremely interesting. It would be helpful to determine the oxidative stress levels (similar to the cellROX data shown in 5E), to tease out if the difference in katG role is because of lack of ROS induction in these strains or due to expression of alternate anti-oxidative stress defense mechanisms.

We have performed additional cellROX analysis as suggested by the reviewer and found that the ROS induction is indeed present across all three Mabs strains, but that katG is only required in one of the two strains (Strain #2). These data are now included in the revised Figure 6.

Reviewer #2 (Recommendations for the authors):

GENERAL COMMENTS

This is a nice piece of work that uses the pathogen Mabs as a test subject.

The work has findings that likely apply generally to antibiotics and mycobacteria: 1) phenotypic tolerance is associated with suppression of ROS, 2) lethal protein synthesis inhibitors act via accumulation of ROS, and 3) levofloxacin behaves in an unexpected way. Each is a new observation. However, I believe that each topic requires more work to be firmly established to be suitable for eLife.

Phenotypic tolerance: Association with suppression of ROS is important but expected. I would solidify the conclusion by performing several additional experiments. For example, confirm the lethal effect of ROS by reducing it with an iron chelator and a radical scavenger. There is a large literature on effects of iron uptake, levels, etc. on antibiotic lethality that could be applied to this question. In 2013 Imlay argued against the validity of fluorescent probes. Perhaps getting the same results with another probe would strengthen the conclusion.

We have carried out additional experiments with both an iron chelator and small molecule ROS scavengers to further test this idea but note that these experiments have several inherent limitations: 1) These compounds have highly pleiotropic effects. For example while N-acetyl cysteine (NAC) is an antioxidant it also increases mycobacterial respiration and was shown to paradoxically decrease antibiotic tolerance in M. tuberculosis (PMID: 28396391). 2) It has been shown by the Imlay group that small-molecule antioxidants are often ineffective in quenching ROS in bacteria (PMID: 388893820), making negative results difficult to interpret. Nonetheless, we present new experimental data showing that iron chelation does indeed improve the survival of antibiotic-treated Mabs (revised Figure 5).  However,  small molecule antioxidants such as thiourea do not restore antibiotic tolerance and actually increased bacterial cell death, suggesting that they may be affecting respiration in Mabs in a manner similar to that seen for NAC in Mtb. We also note that our genetic analysis, which identified numerous other genes encoding proteins with antioxidant function (Figure 2) is a strong additional argument in support of the importance of ROS in antibiotic-mediated lethality. 

Regarding the concern raised by Imlay about the validity of oxidation-sensitive dyes - this relates to concern bacterial autofluorescence induced by antibiotics that can confound analyses in some species. We have ruled this out in our analyses by using bacteria unstained by cellROX as controls to confirm that there is negligible autofluorescence in Mabs (<0.1%, Figure 5E, Figure 6A).

Protein synthesis inhibitors: At present, this is simply an observation. More work is needed to suggest a mechanism. For example, with E. coli the aminoglycosides are protein synthesis inhibitors that also cause membrane damage. Membrane damage is known to stimulate ROS-mediated killing. Your observation needs to be extended because chloramphenicol, another protein synthesis inhibitor, blocks ROS production. The lethality may be a property of mycobacteria: does it occur with E. coli (note that rifampicin is bacteriostatic with E. coli but lethal to Mtb)?

We agree with the reviewer that the mechanism underlying ROS accumulation following transcription or translational inhibition in Mabs is of significant interest. It is likely to be a mechanism different from E. coli, because in E. coli tetracyclines and rifamycins are both bacteriostatic, whereas in Mabs they are both bactericidal. Determining the mechanism by which translation inhibitors cause ROS accumulation in Mabs is an ongoing effort in our laboratory using proteomics and metabolomics, but is outside the scope of this manuscript.

Levofloxacin: This is also at the observational stage but is unexpected. In other studies, ROS is involved in quinolone-mediated killing of bacteria. Why is this not the case with Mabs? The observation should be solidified by showing the contrast with moxifloxacin, since this compound has been studied with mycobacteria (Shee 2022 AAC). With E. coli, quinolone structure can affect the relative contribution of ROS to killing (Malik 2007 AAC), as is also seen with Mtb (Malik 2006 AAC). What is happening in the present work with levofloxacin, an important anti-tuberculosis drug? Is there a structure explanation (compare with ofloxacin)?

While these are interesting questions, a detailed exploration of the structure-function relationships between different fluoroquinolone antibiotics and their varying activities on Mtb and Mabs is outside the scope of this manuscript.  

The writing is generally easy to follow. However, the concept of persistence should be changed to phenotypic tolerance with text changes throughout. I base this suggestion on the definitions of tolerance and persistence as stated in the consensus review (Balaban 2019 Nat Micro Rev). Experimentally, tolerance is seen as a gradual decline in survival following antibiotic addition; the decline is slower than seen with wild-type cells. The data presented in this paper fit that definition. In contrast, persistence refers to a rapid drop in survival followed by a distinct plateau (Balaban 2019 Nat Micro Rev; for example, see Wu Lewis AAC 2012 ). Moreover, to claim persistence, it would be necessary to demonstrate subpopulation status, which is not done. The Balaban review is an attempt to bring order to the field with respect to persistence and tolerance, since the two are commonly used without regard for a consistent definition.

We appreciate the reviewer’s suggestion; text modified in multiple places to clarify.

Another issue requiring clarification is the relationship between resistance and tolerance. Killing by antibiotics is a two-step process, as most clearly seen with quinolones. First a reversible bacteriostatic event occurs. Resistance blocks that bacteriostatic damage. Then a lethal metabolic response to that damage occurs. Tolerance selectively blocks the second, killing event, a distinct process that often involves the accumulation of ROS. Direct antibiotic-mediated damage is an additional mode of killing that also stems from the reversible, bacteriostatic damage created by antibiotics. The authors recognize the distinction but could make it clearer. Take a look at Zheng (JJ Collins) 2020, 2022.

Text modified to clarify this point

Many readers would also like to see a bit more background on Mabs. For example, does it grow rapidly? Are there features that make it a good model for studying mycobacteria or bacteria in general? The more general, the better.

Text modified, background added

Below I have listed specific comments that I hope are useful in bringing the work to publication and making it highly cited.

SPECIFIC COMMENTS

Line 30 unexpectedly. I would delete this word because the result is expected from the ROS work of Shee et al 2022 with mycobacteria. Moreover, Zeng et al 2022 PNAS showed that ROS participates in antimicrobial tolerance, and persistence is a form of tolerance (Balalban et al, 2019, Nat Micro Rev).

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Line 39 key goal: this is probably untrue in the general sense stated, since bacteriostatic antibiotics are sufficient to clear infection (Wald-Dickler 2019 Clin Infect Dis). However, it is likely to be the goal for Mtb infections.

We agree with the reviewer that bacteriostatic antibiotics are effective in treating most types of infections and do not claim otherwise in the manuscript. However, from a clinical standpoint, eradication of the pathogen causing the infection is indeed the goal of antibiotic therapy in virtually all circumstances (with the exception of specific scenarios such as cystic fibrosis where it is recognized that the infecting organism cannot be fully eliminated). In most cases, the combination of bacteriostatic antibiotics and the host immune response is sufficient to achieve eradication. We have modified the manuscript text to reflect this nuance noted by the reviewer.

Line 62 several: you list three, but hipAB works via ppGpp, so the sentence needs fixing

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Line 70 uncertain: this uncertainty is unreferenced. Since everything is uncertain, this vague phrase does not add to the story.

The reviewer makes an interesting philosophical argument. However, we would submit that some aspects of biology, for example the regulation of glycolysis, are understood in great detail. However, other mechanisms, such as the precise mechanisms of lethality for diverse antibiotics in different bacterial species, are far more uncertain and remain a subject of debate (for example PMID: 39910302). Text not modified.

Line 72 somewhat controversial: I would delete this, because the points in the Science papers by Lewis and Imlay have been clarified and in some cases refuted by prior and subsequent work.

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Line 72 presumed: this suggests that it is wrong and perhaps a different idea has replaced it. Another, and more likely view is that there is an additional mode of killing. I suggest rephrasing to be more in line with the literature.

Text modified for clarity. In this sentence “presume” refers to the historical concept that direct target inhibition was solely responsible for antibiotic lethality. As the reviewer notes, there is now significant literature that ROS (and perhaps other secondary effects) also contribute to bacterial killing.  

Line 73 However and the following might also: this phrasing, plus the presumed, misleads the reader from your intent. I suggest rephrasing.

See above re: line 72

Line 75 citations: these are inappropriate and should be changed to fit the statement. I suggest the initial paper by Collins (Kohanski 2007 Cell) a recent paper by Zhao (Zeng PNAS 2022), and a review Drlica Expert Rev Anti-infect Therapy 2021). The present citations are fine if you want to narrow the statement to mycobacteria, but the history is that the E. coli work came first and was then generalized to mycobacteria. A mycobacterial paper for ROS is Shee 2022 AAC.

We thank the reviewer for noticing that we inadvertently omitted several important E. coli-related references. These have been added.

Line 75 and 76: Conversely ... unresolved. Compelling arguments have been made that show major flaws in the two papers cited, and a large body of evidence has now accumulated showing the validity of the idea promoted by the Collins lab, beginning with Kohanski 2007. In addition to many papers by Collins, see Hong 2019 PNAS and Zeng 2022 PNAS). It is fine if you want to counter the arguments against the Lewis and Imlay papers (summarized in Drlica & Zhao 2021 Expert Rev Anti-infect Therapy), but making a blanket statement suggests that the authors are unfamiliar with the literature.

We agree with the reviewer that the weight of the evidence supports a role for antibiotic-induced ROS as an important mechanism for antibiotic lethality under many (though not all) conditions. We have revised the text to better reflect this nuance.

Line 78. Advantages over what?

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Line 80 exposure: to finish the logic you need to show that E. coli and S. aureus persisters fail to do this.

We thank the reviewer for their suggestion but studying these other organisms is outside the scope of this study. 

Line 82 whereas: this misdirects the reader. It would seem that a simple "and" is better

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Line 89 I think this paragraph is about the need to study Mabs, the subject of the present report. This paragraph could use a more appropriate topic sentence to guide the reader so that no guessing is involved. I suggest rephrasing this paragraph to make the case for studying more compelling.

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Line 96. I suggest citing several references after subinhibitory concentration of antibiotic.

The references are in the following sentence alongside the key observations.

Line 99. Genetic analysis: how does this phrase fit with the idea of persister cells arising stochastically?

There are two issues: 1) We would argue that persister formation is not completely stochastic, but rather a probability that can be modified both genetically and by environment (for example hipA PMID: 6348026). 2) Even if persister formation were totally stochastic, the survival of these cells may depend on specific genes – as we indeed find in our Tn-Seq analysis of Mabs.  

Line 106. In this paragraph you need to define persister. The consensus definition (Balaban 2019 Nat Micro Rev) is a subpopulation of tolerant cells. Tolerance is defined as the slowing or absence of killing while an antibiotic retains its ability to block growth. See Zeng 2022 PNAS for example with rapidly growing cells. Phenotypic tolerance is the absence of killing due to environmental perturbations, most notably nutrient starvation, dormancy, and growth to stationary phase. By extension, phenotypic persistence would be subpopulation status of a phenotypically tolerant cells. If you have a different definition, it is important to state it and emphasize that you disagree with the consensus statement.

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Line 109 unexpectedly. I would delete this word, because the literature leads the reader to expect this result unless you make a clear case for Mabs being fundamentally different from other bacteria with respect to how antibiotics kill bacteria (this is unlikely, see Shee 2022 AAC). Indeed, lines 111-113 state extensions of E. coli work, although suppression of ROS in phenotypic tolerance and genetic persistence have not been demonstrated.

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Line 124 you might add, in parentheses and with references, that a property of persisters is crosspersistence to multiple antibiotic classes. This is also true for tolerance, both genetic and phenotypic. An addition will support your approach.

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Line 128 minimal

Text not modified. We appreciate the reviewer’s preference but both “minimal” and “minimum” are both widely accepted terms. Indeed, the Balaban et al 2019 consensus statement on definitions cited by the author above also uses “minimum” (PMID: 30980069), as do IDSA clinical guidelines (PMID: 39108079).

Line 130 is MIC somehow connected to killing or did you also measure killing? Note that blocking growth and killing cells are mechanistically distinct phenomena, although they are related. By being upstream from killing, blockage of growth will also interfere with killing.

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Line 133 PBS is undefined

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Line 134 increase in persisters ... you need to establish that these are not phenotypically tolerant cells. Do they constitute the entire population (tolerance)? Your data would be more indicative of persisters if you saw a distinct plateau with the PBS samples, as such data are often used to document persistence (retardation of killing is a property of tolerance, Balaban 2019). Fig. 1B is clearly phenotypic tolerance, as the entire population grows. Your data suggest that you are not measuring persistence as defined in the literature (Balaban 2019). Line 139 persister should be tolerance •

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Lines 142, 143, 144. 159, 163, 171, 181, 211, 226, 238, 246, 277, 279,289 persistent should be tolerant

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Line 146 fig 1E Mtb does not show the adaptation phenomenon and it is clearly tolerant, not persistent. This should be pointed out. As stated, you may be misleading the reader.

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*Line 169. Please make it clear whether these genes are affecting antibiotic susceptibility (MIC will affect killing because blocking growth is upstream) or if you are dealing with tolerance (no change in MIC). These measurements are essential and should included as a table. By antibiotic response, do you mean that antibiotics change expression levels?

Regarding MICs, the data for MICs in control and katG mutant are presented in Figure 4C and 4F. Regarding ‘response’ we have clarified the text of this sentence.

Line 174 Interestingly should be as expected

Text not modified; tetracyclines do not induce ROS in E. coli and oxazolidinones have not been studied in this regard.

Line 183 you need to include citations. You can cite the ability of chloramphenicol to block ROS-mediated killing of E. coli. That allows you to use the word unexpected

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Line 199. All of the data in Fig. 3 shows tolerance, not persistence, requiring word changes in this paragraph.

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Line 226. The MIC experiment is important. You can add that this result solidifies the idea that blocking growth and killing cells are distinct phenomena. You can cite Shee 2022 AAC for a mycobacterial paper

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Line 241. The result with levofloxacin is unexpected, because the fluoroquinolones are widely reported to induce ROS, even with mycobacteria (see Shee 2022 AAC). You need to point this out and perhaps redo the experiment to make sure it is correct.

We appreciate the reviewer’s interest in this question. All experiments in this paper were repeated multiple times. This particular experiment was repeated 3 times and in all replicates the katG mutant was sensitized to translation inhibitors but not levofloxacin. Shee et al examined Mtb treated with moxifloxacin and found ROS generation, but did not assess whether a Mtb katG mutant had impaired survival. Thus, in addition to differences in: i) the species studied and ii) the particular fluoroquinolone used, the two sets of experiments were designed to address different questions (ROS accumulation vs protection by katG) . A cell might accumulate ROS without a katG mutant having impaired survival if genetic redundancy exists – a result we indeed see in our clinical Mabs strains under some conditions (new data included in revised Figure 6A).  

Line 269 Additional controls would bolster the conclusion: use of an antioxidant such as thiourea and an iron chelator (dipyridyl) both should reduce ROS effects.

New experiments performed, revised Figure 5.

Line 276 the word no is singular

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Line 284 this suggested ... in fact previous work suggested. This summary paragraph might go better as the first paragraph of the Discussion

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Lines 294-299 Most of this is redundant and should be deleted.

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Line 299 this species is vague

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Line 310 Do you want to discuss spoT?

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Line 313 paragraph is largely redundant

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Line 314 controversial. As above, I would delete this, especially since it is not referenced and is unlikely to be true. If you believe it, you have the obligation to show why the ROS-lethality idea is untrue. If you are referring to Lewis and Imlay, there were almost a dozen supporting papers before 2013 and many after. This statement does not make the present work more important, so deletion costs you nothing.

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Line 314 direct disruption of targets. This is clearly not a general principle, because the quinolones rapidly kill while inhibition of gyrase by temperature-sensitive mutations does not (Kreuzer 1979 J.Bact; Steck 1985). Indeed, formation of drug-gyrase-DNA complexes is reversible: death is not.

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Line 318 as pointed out above, you have not brought this story up to date. The two papers mainly focused on Kohanski 2007, ignoring other available evidence.’’

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Line 326 you need to cite Shee 2022 AAC

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Line 342 the idea of mutants being protective is not novel, as several have been reported with E. coli studies. Thus, there is a general principle involved.

We agree that this suggests a potential general principle

Line 344. It depends on the inhibitor. For example, aminoglycosides are translation inhibitors and they also cause the accumulation of ROS.

We agree that ROS generation depends on the inhibitor, and indeed upon other variables including drug concentration, growth conditions, and bacterial species as well.  

Line 347. You need to point out the considerable data showing that the absence of catalase increases killing

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Line 363 look at Shee 2022 AAC and Jacobs 2021 AAC

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Line 585 I suggest having a colleague provide critical comments on the manuscript and acknowledge that person.

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