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Referee #1

Evidence, reproducibility and clarity

Summary: The authors have previously published Mass-spectrometry data that demonstrates a physical interaction between Sall4 and the BAF chromatin complex in iPSC derived neurectodermal cells that are a precursor cell state to neural crest cells. The authors sought to understand the basis of this interaction and investigate the role of Sall4 and the BAF chromatin remodelling complex during neural crest cell specification. The authors first validate this interaction with a co-IP between ARID1B subunit and Sall4 confirming the mass spec data. The authors then utilise in silico modelling to identify the specific interaction between the BAF complex and Sall4, suggesting that this contact is mediated through the BAF complex member DPF2. To functionally validate the role of Sall4 during neural crest specification, the authors utilsie CRISPR-Cas9 to introduce a premature stop codon on one allele of Sall4 to generate iPSCs that are haploinsufficient for Sall4. Due to the reports of Sall4's role in pluripotency, the authors confirm that this model doesn't disrupt pluripotent stem cells and is viable to model the role of Sall4 during neural crest induction. The authors expand this assessment of Sall4 function further during their differentiation model to cranial neural crest cells, assessing Sall4 binding with Cut+Run sequencing, revealing that Sall4 binds to motifs that correspond to key genes in neural crest differentiation. Moreover, reduction in Sall4 expression also reduces the binding of the BAF complex, through Cut and Run for BRG1. Overall, the authors then propose a model by which Sall4 and BRG1 bind to and open enhancer regions in neurectodermal cells that enable complete differentiation to cranial neural crest cells.

Overall, the data is clear and reproducible and offers a unique insight into the role of chromatin remodellers during cell fate specification.

However, I have some minor comments.

1. Using AlphaFold in silico modelling, he authors propose the interaction between the BAF complex with Sall4 is mediated by DPF2, but don't test it. Does a knockout, or knockdown of DPF2 prevent the interaction?
2. OPTIONAL: Does knockout of DPF2 phenocopy the Sall4 ko? This would be very interesting to include in the manuscript, but it would perhaps be a larger body of work.
3. Figure 1, the day of IP is not clearly described until later in the test. please outline during in the figure

3- What is the expression of Sal1 (and other Sall paralogs) during differentiation. The same with the protein levels of Sall4, does this remain at the below 50%, or is this just during pluripotency? 4. The authors hypothesise that Sall4 binds to enhancers- with the criteria for an enhancer being that these peaks > 1KB from the TSS are enhancers. Can this be reinforced by overlaying with other ChIP tracks that would give more confidence in this? There are several datasets from Joanna Wysocka's lab that also utilise this protocol which can give you more evidence to reinforce the claim and provide further detail as to the role of Sall4 5. The authors state that cells fail to become cranial neural crest cells, however they do not propose what the cells do instead. do they become neural? Or they stay at pluriopotent, which is one option given the higher expression of Nanog, OCT4 and OTX2 that are all expressed in pluripotent stem cells. 6. In general, I would like to see the gating strategy and controls for the flow cytometry in a supplemental figure. 7. For supplementary figure 1- please include the gene names in the main image panels rather than just the germ layer.

Significance

The strength of this study lies in its well-designed and clearly presented experiments and datasets. In particular, identifying the specific SALL4 isoform that interacts with the BAF complex-and further exploring the implications of this interaction-is a major highlight. The authors also make effective use of in silico modelling with AlphaFold, offering valuable mechanistic insight into how this interaction is mediated.

The topic should have appeal to researchers in developmental biology and epigenetics. This study represents a significant step forward in validating the interaction between SALL4 and the BAF complex, and it highlights the requirement of SALL4 for BAF-mediated chromatin remodelling during neural crest specification. These findings are likely to be of interest to those studying the gene regulatory mechanisms underlying craniofacial development.

However, while the authors outline the roles of SALL4 and the BAF complex in chromatin remodeling during neural crest development, the downstream effects on cell fate specification could be more thoroughly examined. Currently, Gene Ontology analysis is the primary method used to interpret these consequences, and additional functional validation would strengthen the conclusions.

Intended audience: Basic research, epigenetics in pluripotency and neural crest development.

I like the language that he uses in this piece of texts because it shows that he is conscious of who his target audience is, proving it to be people who are mostly younger than him that he is trying to appeal to.

eLife Assessment

How secretion is regulated during cell division and how membrane trafficking factors cooperate with the cytoskeleton during cell division remain poorly understood. In this work the authors find protein-protein interactions and localization dependencies between the polymeric septin cytoskeleton and the exocyst complex, using fission yeast as a model organism and using alphafold 3 based structural predictions. The work provides a valuable body of new information that will be of great interest to the cell biology community. The evidence is solid and provides the authors and the community a framework to test if the identified interfaces reflect bona fide interaction sites in vivo and in vitro in future.

Reviewer #1 (Public review):

Summary

In this manuscript, Singh, Wu and colleagues explore functional links between septins and the exocyst complex. The exocyst in a conserved octameric complex that mediates the tethering of secretory vesicles for exocytosis in eukaryotes. In fission yeast cells, the exocyst is necessary for cell division, where it localizes mostly at the rim of the division plane, but septins, which localize in a similar manner, are non-essential. The main findings of the work are that septins are required for the specific localization of the exocyst to the rim of the division plane, and the likely consequent localization of the glucanase Eng1 at this same location, where it is known to promote cell separation. In absence of septins, the exocyst still localizes to the division plane, but is not restricted to the rim. They also show some defect in the localization of secretory vesicles and glucan synthase cargo. They further show interactions between septins and exocyst subunits through coIP experiments.

Strengths

The septin, exocyst and Eng1 localization data are well supported, showing that the septin rim recruits the exocyst and (likely consequently) the Eng1 glucanase at this location. One important finding of the manuscript is that of a physical interaction between septins and exocyst subunits in co-immunoprecipitation experiments.

Weaknesses

While interactions are supported by coIP experiments, the AlphaFold-predicted septin-exocyst interactions are not very convincing and the predicted binding interfaces are not supported by mutation analysis. A further open question is whether septins interact with the intact exocyst complex or whether the interactions occur only with individual subunits. The two-hybrid and coIP data only show weak interactions with individual subunits, and some coIPs (for instance Sec3 and Exo70 with Spn1 and Spn4) are negative, suggesting that the exocyst complex may not remain intact in these experiments.

Reviewer #2 (Public review):

Summary:

This interesting study implicates the direct interaction between two multi-subunit complexes, known as the exocyst and septin complexes, in the function of both complexes during cytokinesis in fission yeast. While previous work from several labs had implicated roles for the exocyst and septin complexes in cytokinesis and cell separation, this study describes the importance of protein:protein interaction between these complexes in mediating the functions of these complexes in cytokinesis. Previous studies in neurons had suggested interactions between septins and exocyst complexes occur but the functional importance of such interactions was not known. Moreover, in baker's yeast where both of these complexes have been extensively studied - no evidence of such an interaction has been uncovered despite numerous studies which should have detected it. Therefore while exocyst:septin interactions appear to be conserved in several systems, it appears likely that budding yeast are the exception--having lost this conserved interaction.

Strengths:

The strengths of this work include the rigorous analysis of the interaction using multiple methods including Co-IP of tagged but endogenously expressed proteins, 2 hybrid interaction, and Alphafold Multimer. Careful quantitative analysis of the effects of loss of function in each complex and the effects on localization and dynamics of each complex was also a strength. Taken together this work convincingly describes that these two complexes do interact and that this interaction plays an important role in post Golgi vesicle targeting during cytokinesis.

Comments on revisions:

The authors have added substantial work to the revised manuscript, and it is much improved. In particular, the figures portraying the AlphaFold Multimer model of the exocyst:septin interactions are much clearer. I also appreciate the effort that went into modeling the fission yeast exocyst complex based on the yeast CryoEM structure in order to determine if the predicted interfaces with septins were likely to be surface accessible in the intact exocyst complex.

Author response:

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

Reviewer #1 (Public Review): 

Summary: 

In this manuscript, Singh, Wu and colleagues explore functional links between septins and the exocyst complex. The exocyst in a conserved octameric complex that mediates the tethering of secretory vesicles for exocytosis in eukaryotes. In fission yeast cells, the exocyst is necessary for cell division, where it localizes mostly at the rim of the division plane, but septins, which localize in a similar manner, are non-essential. The main findings of the work are that septins are required for the specific localization of the exocyst to the rim of the division plane, and the likely consequent localization of the glucanase Eng1 at this same location, where it is known to promote cell separation. In the absence of septins, the exocyst still localizes to the division plane but is not restricted to the rim. They also show some defects in the localization of secretory vesicles and glucan synthase cargo. They further propose that interactions between septins and exocysts are direct, as shown through Alphafold2 predictions (of unclear strength) and clean coIP experiments. 

Strengths: 

The septin, exocyst and Eng1 localization data are well supported, showing that the septin rim recruits the exocyst and (likely consequently) the Eng1 glucanase at this location. One major finding of the manuscript is that of a physical interaction between septins and exocyst subunits. Indeed, many of the coIPs supporting this discovery are very clear. 

Weaknesses: 

I am less convinced by the strength of the physical interaction of septins with the exocyst complex. Notably, one important open question is whether septins interact with the intact exocyst complex, as claimed in the text, or whether the interactions occur only with individual subunits. The two-hybrid and coIP data only show weak interactions with individual subunits, and some coIPs (for instance Sec3 and Exo70 with Spn1 and Spn4) are negative, suggesting that the exocyst complex does not remain intact in these experiments.

Given the known structure of the full exocyst complex and septin filaments (at least in S. cerevisiae), the Alphafold2 predicted structure could be used to probe whether the proposed interaction sites are compatible with full complex formation.  

We thank the reviewer for these important and insightful comments. We agree that our current data, particularly the data from yeast two-hybrid and co-immunoprecipitation (coIP) assays, primarily reveal interactions between individual septin and exocyst subunits, and do not conclusively demonstrate binding of septins to the fully assembled exocyst complex. We realize this as a key limitation and have revised the manuscript text accordingly to clarify this point.

We also appreciate the reviewer’s suggestion to use structural prediction to further assess their interaction plausibility. We have now employed the full Saccharomyces cerevisiae exocyst complex (with 4.4 Å resolution) published by the Guo group (Mei et al., 2018) to examine the interfaces of septin and the exocyst interactions, assuming that the S. pombe exocyst has the similar structure. We focused on checking all the interacting residues on the exocyst complex and septins from our AlphaFold modeling to determine whether these predicted interactions are structurally compatible. Our analysis reveals that majority subunit interactions are sterically feasible, while a few would likely require partial disassembly or flexible conformations. These new insights have been added to the revised Results and Discussion sections (Figure Supplement S4, S5 and Videos 4-7).

While we cannot fully resolve whether septins engage with the whole exocyst complex versus selected subunits, our combined data support a model that septins scaffold or spatially regulate the exocyst localization at the division site, potentially through dynamic and multivalent interactions. We now explicitly state this more cautious interpretation in the revised manuscript.

Mei, K., Li, Y., Wang, S., Shao, G., Wang, J., Ding, Y., Luo, G., Yue, P., Liu, J.-J., Wang, X. and Dong, M.-Q., Wang, H-W, Guo W. 2018. Cryo-EM structure of the exocyst complex. Nature Struct & Mol. Biol, 25(2), pp.139-146.

The effect of spn1∆ on Eng1 localization is very clear, but the effect on secretory vesicles (Ypt3, Syb1) and glucan synthase Bgs1 is less convincing. The effect is small, and it is not clear how the cells are matched for the stage of cytokinesis. 

For localizations and quantifications of Eng1, Ypt3, Syb1, and Bgs1 shown in Figures 6 and 7, cells with a closed septum (at or after the end of contractile-ring constriction) were quantified or highlighted. To quantify their fluorescence intensity at the division site using line scan, the line width used was 3 pixels. For Syb1 (Figure 6D), we quantified cells at the end of ring constriction (when Rlc1-tdTomato constricted to a dot) in the middle focal plane. The exact same lines were drawn in both Rlc1 and Syb1 channels. The center of line scan was defined as the pixel with the brightest Rlc1 value. All data were aligned by the center and plotted. For Bgs1 (Figure 7A), we quantified the cells that Rlc1 signal had disappeared from the division site. The line was drawn in the Bgs1 channel in the middle focal plane. The center of line scan was defined as the pixel with the brightest Bgs1 value.

All data were aligned by the center and plotted. These details were added to the Materials and Methods.

Reviewer #2 (Public Review): 

Summary: 

This interesting study implicates the direct interaction between two multi-subunit complexes, known as the exocyst and septin complexes, in the function of both complexes during cytokinesis in fission yeast. While previous work from several labs had implicated roles for the exocyst and septin complexes in cytokinesis and cell separation, this study describes the importance of protein:protein interaction between these complexes in mediating the functions of these complexes in cytokinesis. Previous studies in neurons had suggested interactions between septins and exocyst complexes occur but the functional importance of such interactions was not known. Moreover, in baker's yeast where both of these complexes have been extensively studied - no evidence of such an interaction has been uncovered despite numerous studies which should have detected it. Therefore while exocyst:septin interactions appear to be conserved in several systems, it appears likely that budding yeast are the exception--having lost this conserved interaction. 

Strengths: 

The strengths of this work include the rigorous analysis of the interaction using multiple methods including Co-IP of tagged but endogenously expressed proteins, 2 hybrid interaction, and Alphafold Multimer. Careful quantitative analysis of the effects of loss of function in each complex and the effects on localization and dynamics of each complex was also a strength. Taken together this work convincingly describes that these two complexes do interact and that this interaction plays an important role in post Golgi vesicle targeting during cytokinesis. 

Weaknesses: 

The authors used Alphafold Multimer to predict (largely successfully) which subunits were most likely to be involved in direct interactions between the complexes. It would be very interesting to compare this to a parallel analysis on the budding yeast septin and exocyst complexes where it is quite clear that detectable interactions between the exocyst and septins (using the same methods) do not exist. Presumably the resulting pLDDT scores will be significantly lower. These are in silico experiments and should not be difficult to carry out. 

We thank the reviewer for this insightful suggestion. To assess the specificity of the predicted interactions between septins and the exocyst complex in S. pombe, we performed a comparative AlphaFold2 analysis using some of the homologous subunits from Saccharomyces cerevisiae. We modeled two interactions between Cdc10-Sec5 and Cdc10-Sec15 (Cdc10 is the Spn2 homolog) using the same pipeline and parameters at the time when we did the modeling for S. pombe. We did not find interactions between them using the criteria we used for the fission yeast proteins in this study. These results support the notion that the predicted septin–exocyst interactions in S. pombe are not generalizable to budding yeast. Unfortunately, we did not test all other combinations at that time and the AlphaFold2 platform is not available to us now (showing system error messages when we tried recently). We thank the reviewer again for this helpful suggestion, which should strengthen the evolutionary interpretation of the septin-exocyst interactions once it is able to be systematically carried out.

Reviewer #3 (Public Review): 

Septins in several systems are thought to guide the location of exocytosis, and they have been found to interact with the exocyst vesicle-tethering complex in some cells. However, it is not known whether such interactions are direct or indirect. Moreover, septin-exocyst physical associations were not detected in several other systems, including yeasts, making it unclear whether such interactions reflect a conserved septin-exocytosis link or whether they may missed if they depend on septin polymerization or association into higher-order structures. Singh et. al., set out to define whether and how septins influence the exocyst during S. pombe cytokinesis. Based on three lines of evidence, the authors conclude that septins directly bind to exocyst subunits to regulate localization of the exocyst and vesicle secretion during cytokinesis. The conclusions are consistent with the data presented, but some interpretations need to be clarified and extended: 

(1) The first line of evidence examines septin and exocyst localization during cytokinesis in wild-type and septin-mutant or exocyst-mutant yeast. Quantitative imaging convincingly shows that the detailed localization of the exocyst at the division site is perturbed in septin mutants, and that this is accompanied by modest accumulation of vesicles and vesicle cargos. Whether that is sufficient to explain the increased thickness of the division septum in septin mutants remains unclear.

The modest accumulation of vesicles and vesicle cargos at the division site is one of the reasons for the increased thickness of the division septum in septin mutants. It is more likely that the misplaced exocyst can still tether vesicles along the division plane (less likely at the rim) without septins. Due to the lack of the glucanase Eng1 at the rim of the division plane in septin mutants, daughter-cell separation is delayed and then cells continue to thicken the septum. We have added these points to the Discussion.

(2) The second line of evidence involves a comprehensive Alphafold2 analysis of potential pair-wise interactions between septin and exocyst subunits. This identifies several putative interactions in silico, but it is unclear whether the identified interaction surfaces would be available in the full septin or exocyst complexes.  

We thank the reviewer for raising this important point. We fully agree that a key limitation of pairwise AlphaFold predictions is that they do not account for the higher-order structural context of multimeric protein complexes, such as septin hetero-oligomers or the assembled exocyst complex. As a result, some of the predicted interfaces could indeed be conformationally restricted in the native state.

To address this concern, we predicted the S. pombe exocyst and septin structures using AlphaFold3. We mapped predicted contact residues onto the predicted structure. Most predicted interfaces (86% for the exocyst and 86-96% for septins) appear to be located on accessible surfaces in the assembled complexes (Figure supplement S4, S5, videos 4 - video 7), suggesting that these interactions are sterically plausible. We have added this important caveat to the text of the revised manuscript highlighting the interface accessibility within the assembled complexes. We appreciate the reviewer’s insight, which helped us strengthen the interpretation and limitations of the AlphaFold-based analysis.

(3) The third line of evidence uses co-immunoprecipitation and yeast two hybrid assays to show that several physical interactions predicted by Alphafold2 can be detected, leading the authors to conclude that they have identified direct interactions. However, both methods leave open the possibility that the interactions are indirect and mediated by other proteins in the fission yeast extract (co-IP) or budding yeast cell (two-hybrid). 

We thank the reviewer for this important clarification. We agree that coimmunoprecipitation (co-IP) and yeast two-hybrid (Y2H) assays cannot conclusively distinguish between direct and indirect interactions. As the reviewer points out, co-IPs may reflect associations mediated by bridging proteins within the fission yeast extract, and Y2H readouts can be influenced by fusion context or endogenous host proteins. In our manuscript, we have now revised the relevant statements in the Results and Discussion sections to clarify that the observed associations are consistent with direct interactions predicted by AlphaFold2, but cannot alone establish direct binding. We have also tempered our terminology—substituting phrases such as “direct interaction” with “physical association consistent with direct binding,” where appropriate.

(4) Based on prior studies it would be expected that the large majority of both septins and exocyst subunits are present in cells and extracts as stoichiometric complexes. Thus, one would expect any septin-exocyst interaction to yield associations detectable with multiple subunits, yet co-IPs were not detected in some combinations. It is therefore unclear whether the interactions reflect associations between fully-formed functional complexes or perhaps between transient folding intermediates. 

We thank the reviewer for this thoughtful observation. We agree that both septins and exocyst subunits are generally understood to exist in cells as stable, stoichiometric complexes, and that interactions between fully assembled complexes might be expected to yield co-immunoprecipitation signals involving multiple subunits from each complex. However, it was also found that >50% of septins Spn1 and Spn4 are in the cytoplasm even during cytokinesis when the septin double rings are formed (Table 1 of Wu and Pollard, Science 2005, PMID: 16224022). Thus, it is possible that there are pools of free septin and exocyst subunits in the cytoplasm, which were detected in our Co-IP assays. 

In our experiments, we observed selective co-IP signals between certain septin and exocyst subunits, while other combinations did not yield detectable interactions. We believe these findings could reflect several other possibilities besides the possible interactions among the free subunits in the cytoplasm:

(1) Some interactions may only be strong enough between specific subunits at exposed interfaces under the Co-IP conditions, rather than through wholesome complex–complex interactions;

(2) The detergent and/or salt conditions used in our co-IPs may disrupt labile complex interfaces or partially dissociate multimeric assemblies.

To address this concern, we now include in the Discussion a paragraph highlighting the possibility that some of the observed interactions may not reflect binding between fully assembled, functional complexes. Notably, most detected interactions pairs are consistent with the AlphaFold predictions, which suggest specific subunit interfaces may be responsible for mediating contact. While we cannot fully resolve whether septins engage with the whole exocyst complex versus selected subunits, our combined data supports a model that septins scaffold or spatially regulate the exocyst localization at the division site, potentially through dynamic and multivalent interactions. We now explicitly state this more cautious interpretation in the revised manuscript. Future biochemical studies using native complex purifications, cross-linking mass spectrometry, or in vitro reconstitution with fully assembled septin and exocyst complexes, or in vivo FRET assays will be essential to clarify whether the interactions we observe occur between intact assemblies or intermediate forms.

Reviewer #1 (Recommendations for the Authors): 

A major finding from the manuscript is the description of physical interaction of septin subunits with exocyst subunits. The analysis starts from Alphafold2 predictions, shown in Figures 3 and S3. However, some of the most useful metrics of Alphafold, the PAE plot and the pTM and ipTM values, are not provided. It is thus very difficult to estimate the value of the predicted structures (which are also obscured by all side chains). The power of a predicted structure is that it suggests binding interfaces, which is not explored here. At the very least, it would not be difficult to examine whether the proposed binding interfaces are free in the septin filaments and octameric exocyst complex. 

Please also see response to reviewer #1 (Public Review).

We thank the reviewer for these very helpful suggestions. We agree that inclusion of AlphaFold2 model confidence metrics—specifically the Predicted Aligned Error (PAE) plots, as well as pTM and ipTM values—is essential for evaluating the reliability of the predicted septin–exocyst interfaces.

In the revised manuscript, we have now included the PAE plots (Figure 3 and Supplementary S3) and summarizes the pTM scores for each predicted septin–exocyst subunit pair. We also provide a short description of these metrics in the figure legend to help guide interpretation. The old Alphafold2 version (alphafold2advanced) that we used doesn’t give iPTM score, so are not included. However, according to our methodology, we only counted the interacting residues which have pLDDT scores >50%, predicting the resulting iPTM score should not be very weak.

In addition, we have updated Figures 3 and S3 to show simplified ribbon diagrams of the interface regions, with side chains hidden by default and selectively displayed only at predicted interaction hotspots. This improves structural clarity and makes the interface regions easier to interpret. We mentioned in the Discussion that the preliminary studies show that the predicted interacting interfaces of Sec15 and Sec5 with septin subunits are accessible for interaction in the whole exocyst complex. The new Figure Supplement S4 and S5 and Videos 4-7 now show the interface residues of both the exocyst and septins that are involved in the interactions.

Two further points on the interaction: 

The 2H interaction data is not very convincing. The insets showing beta-gal assays do not look very different from the negative control (compare for instance in panel 4E the Sec15BD alone, last column, with the Sec15-BD in combination with Spn4-AD, third column: roughly same color), which suggests it is mostly driven by autoactivation of Sec15-BD. Providing growth information in addition to beta-gal may be helpful. 

We appreciate the reviewer’s close evaluation of the yeast two-hybrid (Y2H) assay data, and we agree that the signals observed in the Spn4–Sec15 combination is indeed weak. Unfortunately, we did not perform growth assays. However, we would like to clarify that this is consistent with the nature of the interactions that we are investigating. The interaction between individual septin and exocyst subunits is not strong and/or transient as supported by the weak interactions by Co-IP experiments. Given the exocyst only tethers/docks vesicles on the plasma membrane for tens of seconds before vesicle fusion, the multivalent interactions between septins and the exocyst should be very dynamic and not be too strong. 

As evidenced by our Co-IP experiments and multivalent interactions predicted by Alphafold2, the interaction between Spn4 and Sec15 is detectable but weak, suggesting that this may be a low-affinity or transient interaction. Given that Y2H assays have known limitations in detecting such low-affinity interactions—especially those that depend on conformational context or are not optimal in the yeast nucleus—it is perhaps not surprising that the X-gal color development is subtle. These limitations of the Y2H system have been well-documented (e.g., Braun et al., 2009; Vidal & Fields, 2014), particularly for interactions with affinities in the micromolar range or those requiring conformational specificity. Therefore, the weak signal observed is in line with expectations for a lowaffinity, transient interaction such as between Spn4 and Sec15.

Vidal, M. and Fields, S., 2014. The yeast two-hybrid assay: still finding connections after 25 years. Nature methods, 11(12), pp.1203-1206.

Braun, P., Tasan, M., Dreze, M., Barrios-Rodiles, M., Lemmens, I., Yu, H., Sahalie, J.M., Murray, R.R., Roncari, L., De Smet, A.S. and Venkatesan, K., 2009. An experimentally derived confidence score for binary protein-protein interactions. Nature methods, 6(1), pp.91-97.

In the coIP experiments, I am confused by the presence of tubulin signal in some of the IPs. For instance, in Fig 4B, but not 4D, where the same Sec15-GFP is immunoprecipitated. There is also a signal in 4C but not 4A. This needs to be clarified. 

The presence of tubulin in some immunoprecipitates is not unexpected, particularly in experiments involving cytoskeleton-associated proteins such as septins and exocyst subunits. The occasional presence of tubulin in our co-IP samples is consistent with well-documented reports showing tubulin as a frequent non-specific co-purifying protein, particularly under native lysis conditions used to preserve large complexes (Vega and Hsu, 2003; Gavin et al., 2006; Mellacheruvu et al., 2013; Hein et al., 2015). The CRAPome database and quantitative interactomics studies highlight tubulin as one of the most common background proteins in affinity-based workflows. Importantly, tubulin was used as a loading control but not as a marker for interaction in our study, and its variable presence does not reflect a specific interaction with Sec15-GFP or other bait proteins, and we have clarified this point in the revised figure legend.

Gavin, A.C., Aloy, P., Grandi, P., Krause, R., Boesche, M., Marzioch, M., Rau, C., Jensen, L.J., Bastuck, S., Dümpelfeld, B. and Edelmann, A., 2006. Proteome survey reveals modularity of the yeast cell machinery. Nature, 440(7084), pp.631-636.

Mellacheruvu, D., Wright, Z., Couzens, A.L., Lambert, J.P., St-Denis, N.A., Li, T., Miteva, Y.V., Hauri, S., Sardiu, M.E., Low, T.Y. and Halim, V.A., 2013. The CRAPome: a contaminant repository for affinity purification–mass spectrometry data. Nature methods, 10(8), pp.730736.

Hein, M.Y., Hubner, N.C., Poser, I., Cox, J., Nagaraj, N., Toyoda, Y., Gak, I.A., Weisswange, I., Mansfeld, J., Buchholz, F. and Hyman, A.A., 2015. A human interactome in three quantitative dimensions organized by stoichiometries and abundances. Cell, 163(3), pp.712-723.

Vega, I.E., Hsu, S.C. 2003. The septin protein Nedd5 associates with both the exocyst complex and microtubules and disruption of its GTPase activity promotes aberrant neurite sprouting in PC12 cells. Neuroreport, 14, pp.31-37.

Regarding the localization of Ypt3 and Syb1 in WT and spn1∆ in Figure 6C-D and Bgs1 in Figure 7A, it would help to add a contractile ring marker to be able to match the timing of cytokinesis between WT and mutants and ensure that cells of same stage are compared (and add some quantification for Ypt3). In fact, in Figure 7A, next to the cells being pointed at, there are very similar localizations of Bgs1 in WT and spn1∆ at the rim of the ingressing septum, which makes me wonder how the quantified cells were chosen. 

For localizations and quantifications of Eng1, Ypt3, Syb1, and Bgs1 shown in Figures 6 and 7, cells with a closed septum (at or after the end of contractile-ring constriction) were quantified or highlighted. To quantify their fluorescence intensity at the division site using line scan, the line width used was 3 pixels. For Syb1 (Figure 6D), we quantified cells at the end of ring constriction (when Rlc1-tdTomato constricted to a dot) in the middle focal plane. The exact same lines were drawn in both Rlc1 and Syb1 channels. The center of line scan was defined as the pixel with the brightest Rlc1 value. All data were aligned by the center and plotted. For Bgs1 (Figure 7A), we quantified the cells that Rlc1 signal had disappeared from the division site. The line was drawn in the Bgs1 channel in the middle focal plane. The center of line scan was defined as the pixel with the brightest Bgs1 value. All data were aligned by the center and plotted. These details were added to the Materials and Methods.

Finally, the manuscript would benefit from some figure reorganization/compaction. Unless work on the binding interfaces is added, Figure 3 and S3 could be removed and summarized by providing the pTM and ipTM values of the predicted interactions. Figure 5 could be combined with Figure 2, as it is essentially a repeat with additional exocyst subunits. 

Because the binding interfaces are added, we keep the original Figures 3 and S3. The experiments in Figure 5 could not be performed before the interaction tests between septins and the exocyst. Thus, to aid the flow of the story, we keep Figures 2 and 5 separated.

Minor comments: 

The last sentence of the first paragraph of the results does not make much sense at this point of the paper. After the first paragraph, there is no evidence that colocalization would be required for proper function.  

We agree that the sentence in question may have overstated the functional implications of colocalization too early in the Results section, before presenting supporting evidence. Our intention was to introduce the hypothesis that spatial proximity between septins and exocyst subunits may be relevant for their coordination during cytokinesis, which we examine in later figures. We have revised the sentence to more accurately reflect the observational nature of the data at this stage in the manuscript as below:

"These observations suggest the spatial proximity between septins and the exocyst during certain stage of cytokinesis, raising the possibility of their functional coordination, which we would further investigate below."

What is the indicated n in Figure 6B? Number of cells? 

Yes, the n in Figure 6B refers to the thin sections of electron microscopy quantified in the analysis. We have now updated the figure legend to explicitly state this for clarity.

The causal inference made between the alteration of Exocyst localization in septin mutants and the thicker septum is possible, but by no means certain. It should be phrased more cautiously. 

We agree that our original phrasing may have overstated the causal relationship between altered exocyst localization in septin mutants and septum thickening. Our data supports a correlation between these phenotypes, but additional experiments would be required to establish direct causality.

To reflect this, we have revised the relevant sentence in the Discussion to read:

“The modest accumulation of vesicles and vesicle cargos at the division site is one of the reasons for the increased thickness of the division septum in septin mutants. It is more likely that the misplaced exocyst can still tether vesicles along the division plane without septins. Due to the lack of the glucanase Eng1 at the rim of the division plane in septin mutants, daughter-cell separation is delayed and then cells continue to thicken the septum.”

Reviewer #2 (Recommendations for the Authors): 

(1) In the display of the AlphaFold Model for the interactions (Figure 3 and Supplemental Figure 3) it is difficult to identify which subunits are where. Residue numbers and subunits should be labeled and only side chains important for the interactions should be present in the model. 

We appreciate this valuable suggestion. We agree that clearer visual labeling is essential for interpreting the predicted interactions and have revised Figures 3 and S3 accordingly to improve readability and emphasize key structural features.

Specifically, we have:

• Labeled each subunit with its name and color-coded consistently across panels.

•  Annotated key interface residues with residue numbers directly in the figure.

• Removed non-interacting side chains to declutter the model and highlight only those involved in predicted interactions as well as expanded the figure legend for explanation.

(2) In Table 1 the column label "Genetic Interaction at 25C" is confusing when synthetic growth defects are shown with a "plus". Rather this column could be labeled "Growth of double mutants at 25C" and then designate the relative growth rate observed at 25C as in Table 2. Designating a negative effect on growth with a plus is confusing. 

Thanks for the thoughtful suggestions. We have made the suggested changes by deleting the last column so that Tables 1 and 2 are consistent.

(3) In Figure 4, why is tubulin being co-immunoprecipitated in two of the four anti-GFP IPs? Are the IPs dirty and if so why does it vary between the four experiments? If they are dirty can the non-specific tubulin be removed by additional washes with IP buffer or conversely is it necessary to do minimal washes in order to detect the exocyst-septin interaction by coIP? A comment on this would be helpful. 

The presence of tubulin in some immunoprecipitates is not unexpected, particularly in experiments involving cytoskeleton-associated proteins such as septins and exocyst subunits. The occasional presence of tubulin in our co-IP samples is consistent with welldocumented reports showing tubulin as a frequent non-specific co-purifying protein, particularly under native lysis conditions used to preserve large complexes (Vega and Hsu, 2003; Gavin et al., 2006; Mellacheruvu et al., 2013; Hein et al., 2015). The CRAPome database and quantitative interactomics studies highlight tubulin as one of the most common background proteins in affinity-based workflows. Importantly, tubulin was used as a loading control but not marker for interaction in our study, and its variable presence does not reflect a specific interaction with Sec15-GFP or other bait proteins, and we have clarified this point in the revised figure legend.

Gavin, A.C., Aloy, P., Grandi, P., Krause, R., Boesche, M., Marzioch, M., Rau, C., Jensen, L.J., Bastuck, S., Dümpelfeld, B. and Edelmann, A., 2006. Proteome survey reveals modularity of the yeast cell machinery. Nature, 440(7084), pp.631-636.

Mellacheruvu, D., Wright, Z., Couzens, A.L., Lambert, J.P., St-Denis, N.A., Li, T., Miteva, Y.V., Hauri, S., Sardiu, M.E., Low, T.Y. and Halim, V.A., 2013. The CRAPome: a contaminant repository for affinity purification–mass spectrometry data. Nature methods, 10(8), pp.730736.

Hein, M.Y., Hubner, N.C., Poser, I., Cox, J., Nagaraj, N., Toyoda, Y., Gak, I.A., Weisswange, I., Mansfeld, J., Buchholz, F. and Hyman, A.A., 2015. A human interactome in three quantitative dimensions organized by stoichiometries and abundances. Cell, 163(3), pp.712-723.

Vega, I.E., Hsu, S.C. 2003. The septin protein Nedd5 associates with both the exocyst complex and microtubules and disruption of its GTPase activity promotes aberrant neurite sprouting in PC12 cells. Neuroreport, 14, pp.31-37. 

In response to the second part of reviewer’s comment, we washed the pulldown product for 5 times each time with 1 ml IP buffer at 4ºC. We used this standard protocol for all the Co-IP experiments to detect the interaction between different septin-exocyst subunits. So, we are not sure if and how more washes or more stringent buffer conditions can interfere with detection of the interactions.

Reviewer #3 (Recommendations for the Authors): 

In addition to the issues noted in the public review, there were some confusing findings and references to previous literature that merit further consideration or discussion: 

• The current gold standard for validating Alphafold predictions involves making targeted mutants suggested by the structural predictions. The absence of any such validation weakens the conclusions significantly. 

We agree that the targeted mutagenesis based on AlphaFold2-predicted interaction interfaces represents a powerful approach to experimentally validate the in silico models. While we did not pursue structure-guided mutagenesis in this study, our goal was to identify putative interactions between septin and exocyst subunits as a foundation for future functional work. Our current conclusions are intentionally limited to proposing putative interfaces, supported by co-immunoprecipitation and genetic interaction data.

We recognize that direct validation of specific contact residues would significantly strengthen the model. Accordingly, we have revised the Discussion to explicitly state this limitation and to note that structure-based mutagenesis will be an important next step to test the functional relevance of predicted interactions. We have added the following statement:

“Future studies are needed to refine the residues involved in the interactions because the predicted interacting residues from AlphaFold are too numerous. However, it is encouraging that most of the predicted interacting residues are clustered in several surface patches. Experimental validation through targeted mutagenesis is an important next step.”

• Much of the writing appears to imply that differences in mutant phenotypes indicate differences in septin (or exocyst) subunit behaviors/functions. However, my reading of the work in budding yeast is that such differences reflect the partial functionality that can be conferred by aberrant partial septin complexes that assemble and may polymerize in mutants lacking different subunits. In this view, which is supported by data showing that essentially all septins are in stoichiometric octameric complexes in cells, the wild-type functions are all mediated by the full complex. Similarly, the separate exocyst subunit localizations based on tagged Sec3 (Finger et al) were not supported by later work from the Brennwald lab with untagged Sec3, and the idea that different exocyst subunits may function separately from the full complex has very limited support in yeast. I would suggest that the text be edited to better reflect the literature, or that different views be better justified. 

Thanks for the suggestions. We have revised the text accordingly.

• The comprehensive set of Alphafold2 predictions is a major strength of the paper, but it is unclear to this reader whether the multiple predicted interactions truly reflect multivalent multimode interactions or whether many (most?) predictions would not be consistent with interactions between full complexes and may not indicate physiological interactions. Better discussion of these issues is needed to interpret the findings. 

We appreciate the reviewer’s suggestion to use structural prediction to further assess interaction plausibility. We have now employed the full Saccharomyces cerevisiae exocyst complex (with 4.4 Å resolution) published by the Guo group to examine the interfaces of septins and the exocyst interactions, assuming that the S. pombe exocyst has the similar structure. We mapped predicted contact residues onto the predicted structure. Most predicted interfaces (86% for the exocyst and 86-96% for septins) appear to be located on accessible surfaces in the assembled complexes (Figure supplement S4, S5, videos 4 - video 7), suggesting that these interactions are sterically plausible. We have added this important caveat to the text of the revised manuscript highlighting the interface accessibility within the assembled complexes. We appreciate the reviewer’s insight, which helped us strengthen the interpretation and limitations of the AlphaFold-based analysis.

• Some but not all co-IP blots appear to show tubulin (negative control) coming down with the GFP pull-downs. Why is that, and what does it imply for the reliability of the co-IP protocol? 

The presence of tubulin in some immunoprecipitates is not unexpected, particularly in experiments involving cytoskeleton-associated proteins such as septins and exocyst subunits. The occasional presence of tubulin in our co-IP samples is consistent with welldocumented reports showing tubulin as a frequent non-specific co-purifying protein, particularly under native lysis conditions used to preserve large complexes (Vega and Hsu, 2003; Gavin et al., 2006; Mellacheruvu et al., 2013; Hein et al., 2015). The CRAPome database and quantitative interactomics studies highlight tubulin as one of the most common background proteins in affinity-based workflows. Importantly, tubulin was used as a loading control but not a marker for interaction in our study, and its variable presence does not reflect a specific interaction with Sec15-GFP or other bait proteins, and we have clarified this point in the revised figure legend.

Gavin, A.C., Aloy, P., Grandi, P., Krause, R., Boesche, M., Marzioch, M., Rau, C., Jensen, L.J., Bastuck, S., Dümpelfeld, B. and Edelmann, A., 2006. Proteome survey reveals modularity of the yeast cell machinery. Nature, 440(7084), pp.631-636.

Mellacheruvu, D., Wright, Z., Couzens, A.L., Lambert, J.P., St-Denis, N.A., Li, T., Miteva, Y.V., Hauri, S., Sardiu, M.E., Low, T.Y. and Halim, V.A., 2013. The CRAPome: a contaminant repository for affinity purification–mass spectrometry data. Nature methods, 10(8), pp.730736.

Hein, M.Y., Hubner, N.C., Poser, I., Cox, J., Nagaraj, N., Toyoda, Y., Gak, I.A., Weisswange, I., Mansfeld, J., Buchholz, F. and Hyman, A.A., 2015. A human interactome in three quantitative dimensions organized by stoichiometries and abundances. Cell, 163(3), pp.712-723.

Vega, I.E., Hsu, S.C. 2003. The septin protein Nedd5 associates with both the exocyst complex and microtubules and disruption of its GTPase activity promotes aberrant neurite sprouting in PC12 cells. Neuroreport, 14, pp.31-37.

• Why were two different protocols used for different yeast-two-hybrid analyses? 

The purpose of using two protocols was to test which protocol is more reliable and sensitive.

• The different genetic interactions between septin and exocyst mutants when combined with TRAPP-II mutants merits further discussion: might the difference reflect relocation of exocyst from rim to center in septin mutants versus inactivation of exocyst in exocyst mutants? 

We appreciate this insightful comment and agree that this distinction is likely meaningful. The reviewer correctly notes that septin mutants may not abolish exocyst function but rather cause its spatial mislocalization: from the rim to the center of the division site, whereas the exocyst mutants likely result in partial or complete loss of vesicle tethering activity at the plasma membrane.

To address this important nuance, we have expanded the Discussion as follows:

“The genetic interactions between mutations in the exocyst and septins when combined with TRAPP-II mutants may reflect fundamentally different consequences for compromising the exocyst function (Tables 1 and 2). In septin mutants, the exocyst complex still localizes to the division site but is mispositioned from the rim to the center of the division plane. This mislocalization allows partial retention of exocyst function, leading to very mild synthetic or additive defects when combined with compromised TRAPP-II trafficking and tethering. In contrast, in exocyst subunit mutants, the exocyst becomes partial or non-functional, resulting in a more severe loss of exocyst activity. These differing consequences could explain the qualitative differences in genetic interactions observed with TRAPP-II mutants (Tables 1 and 2). Thus, septins and the exocyst also work in different genetic pathways for certain functions in fission yeast cytokinesis.”

• The vesicle accumulation in septin mutants was quite modest. Does that imply that most vesicles are still fusing in the septum? Further discussion would be beneficial to understand what the authors think this means. 

We thank the reviewer for this important point. We agree that the modest vesicle accumulation observed in septin mutants suggests that a significant proportion of vesicles continue to successfully fuse at the division site, even in the absence of fully functional septin structures.

We now discuss this in greater detail in the revised manuscript:

“The relatively modest vesicle accumulation in septin mutants suggests that septins are not absolutely required for vesicle tethering or fusion per se at the division site. Instead, septins primarily function to spatially organize the targeting sites of exocyst-directed vesicles by stabilizing the localization of the exocyst at the rim of the cleavage furrow. In septin mutants, mislocalization of the exocyst reduces the spatial precision of membrane insertion but still permits vesicle tethering and fusion, albeit in a less controlled manner. Thus, septins likely play a modulatory rather than essential role in exocytic vesicle delivery during cytokinesis. This interpretation aligns with our localization and genetic interaction data, which indicates that septins act as scaffolds to optimize secretion geometry, rather than as core components of the fusion machinery.”

• It was unclear to this reader why relocation of some exocyst complexes from the rim to the center of the septal region would lead to dramatic thickening of the septum. Further discussion would be beneficial to understand what the authors think this means. 

The modest accumulation of vesicles and vesicle cargos at the division site is one of the reasons for the increased thickness of the division septum in septin mutants. It is more likely that the misplaced exocyst can still tether vesicles along the division plane without septins. Because of the lack of glucanase Eng1 at the rim of the division plane in septin mutants, daughter-cell separation is delayed and then cells continue to thicken the septum. We have added these points to the Discussion.

how slowly? Do we need to consider the evaporation?

This is their main argument and point to a notable gap in conservation's use of the fossil record and its use as extra data/analogue

Probably one of the more important aspects considering how influential climate state is on microfauna

A notable exclusion as I thought using more spatially relative species in the past would create a better analogue

I'm glad to see a mention of how humans pose a significant chance of alteration to the model because as mentioned its impossible to predict how we move forward in technology and its subsequent impact on the environment.

Read the other person's body language

Walking away from the person before getting any type of response

availability heuristic = “ease of recall” rule. Tversky & Kahneman (1974) showed it drives errors like overestimating accident deaths. it’s conceptually parallel to face-dominance in Media 1: both are cognitive shortcuts where one salient channel overwhelms the full dataset.

availability heuristic = “ease of recall” rule. Tversky & Kahneman (1974) showed it drives errors like overestimating accident deaths. it’s conceptually parallel to face-dominance: both are cognitive shortcuts where one salient channel overwhelms the full dataset.

the horoscope/personality test effect mirrors universality of expression: we map vague, generic input onto our own representative cases. the trick is that representativeness feels diagnostic even when it’s just broad enough to fit anyone.

the $10 divide-the-money experiment is basically a goal-priming test. fits with Stroop: whichever schema/goal is most activated “wins” automatically unless controlled thought intervenes.

Masuda & Nisbett’s airport scene study shows cultural tool preferences: westerners lock on objects (analytic), east asians scan context (holistic). Ekman’s universality work adds nuance. Core emotions look the same, but interpretation rules (display vs context) diverge by culture.

the Bantu cattle memory vs. stock market schemas is a culture-driven accessibility effect. schemas reflect what’s chronically primed by daily life, just like how goal primes bias the $10 money-split task (Bargh)

Rosenthal & Jacobson’s bloomers study is a textbook case: expectation - subtle changes in treatment - performance shifts. notice it was automatic, not conscious favoritism (Chen & Bargh, 1997). Demonstrating deception; where unconscious micro-signals leak through despite intentions.

priming is a clean example of automaticity. Higgins et al. (1977) showed that just memorizing words like “reckless” or “adventurous” shaped impressions of Donald. this matches Ekman’s (1964) point that static snapshots miss how quickly preceding context primes interpretation.

the passage on Korsakov’s patients inventing coherence links nicely to how redundancy across channels strengthens impressions (Media 1). without memory continuity, the mind “confabulates,” which is basically a desperate attempt to build a schema on the fly.

schemas aren’t just useful. They can actually override direct evidence. Mehrabian & Ferris (1967) found that when faces and voices conflict, people weight the face 1.5× more. it’s the same logic as Kelley’s warm/cold professor study: a single cue can set the frame for interpreting everything else.

Rodin’s Thinker is a great visual for controlled, effortful cognition. but the irony is that most of our judgments are automatic; fast, unconscious, and effortless. this is the same split shown in the Stroop task: automatic reading overrides our deliberate control unless we slow down.

eLife Assessment

This work presents important findings suggesting that a combination of transcranial stimulation approaches applied for a short period could improve memory performance. Solid methods and evidence, in line with current standards for non-invasive stimulation and recording, are included to broadly support the main findings. The results potentially have implications for non-invasive enhancement of cognitive functions.

Reviewer #1 (Public review):

Summary:

The authors make a bold claim that a combination of repetitive transcranial magnetic stimulation (intermittent theta burst-iTBS) and transcranial alternating current stimulation (gamma tACS) causes slight improvements in memory in a face/name/profession task.

Strengths:

The idea of stimulating the human brain non-invasively is very attractive because, if it worked, it could lead to a host of interesting applications. The current study aims to evaluate one such exciting application.

Reviewer #2 (Public review):

Summary:

The manuscript by Borghi and colleagues provides evidence that the combination of intermittent theta burst TMS stimulation and gamma transcranial alternating current stimulation (γtACS) targeting the precuneus increases long-term associative memory in healthy subjects compared to iTBS alone and sham conditions. Using a rich dataset of TMS-EEG and resting-state functional connectivity (rs-FC) maps and structural MRI data, the authors also provide evidence that dual stimulation increased gamma oscillations and functional connectivity between the precuneus and hippocampus. Enhanced memory performance was linked to increased gamma oscillatory activity and connectivity through white matter tracts.

Strengths:

The combination of personalized repetitive TMS (iTBS) and gamma tACS is a novel approach to targeting the precuneus, and thereby, connected memory-related regions to enhance long-term associative memory. The authors leverage an existing neural mechanism engaged in memory binding, theta-gamma coupling, by applying TMS at theta burst patterns and tACS at gamma frequencies to enhance gamma oscillations. The authors conducted a thorough study that suggests that simultaneous iTBS and gamma tACS could be a powerful approach for enhancing long-term associative memory. The paper was well-written, clear, and concise.

Reviewer #3 (Public review):

Summary:

Borghi and colleagues present results from 4 experiments aimed at investigating the effects of dual γtACS and iTBS stimulation of the precuneus on behavioral and neural markers of memory formation. In their first experiment (n = 20), they find that a 3-minute offline (i.e., prior to task completion) stimulation that combines both techniques leads to superior memory recall performance in an associative memory task immediately after learning associations between pictures of faces, names, and occupation, as well as after a 15-minute delay, compared to iTBS alone (+ tACS sham) or no stimulation (sham for both iTBS and tACS). Performance in a second task probing short-term memory was unaffected by the stimulation condition. In a second experiment (n = 10), they show that these effects persist over 24 hours and up to a full week after initial stimulation. A third (n = 14) and fourth (n = 16) experiment were conducted to investigate neural effects of the stimulation protocol. The authors report that, once again, only combined iTBS and γtACS increases gamma oscillatory activity and neural excitability (as measured by concurrent TMS-EEG) specific to the stimulated area at the precuneus compared to a control region, as well as precuneus-hippocampus functional connectivity (measured by resting state MRI), which seemed to be associated with structural white matter integrity of the bilateral middle longitudinal fasciculus (measured by DTI).

Strengths:

Combining non-invasive brain stimulation techniques is a novel, potentially very powerful method to maximize the effects of these kinds of interventions that are usually well-tolerated and thus accepted by patients and healthy participants. It is also very impressive that the stimulation-induced improvements in memory performance resulted from a short (3 min) intervention protocol. If the effects reported here turn out to be as clinically meaningful and generalizable across populations as implied, this approach could represent a promising avenue for treatment of impaired memory functions in many conditions.

Methodologically, this study is expertly done! I don't see any serious issues with the technical setup in any of the experiments. It is also very commendable that the authors conceptually replicated the behavioral effects of experiment 1 in experiment 2 and then conducted two additional experiments to probe the neural mechanisms associated with these effects. This certainly increases the value of the study and the confidence in the results considerably.

The authors used a within-subject approach in their experiments, which increases statistical power and allows for stronger inferences about the tested effects. They also used to individualize stimulation locations and intensities, which should further optimize the signal-to-noise ratio.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Public review):

Summary:

The authors make a bold claim that a combination of repetitive transcranial magnetic stimulation (intermittent theta burst-iTBS) and transcranial alternating current stimulation (gamma tACS) causes slight improvements in memory in a face/name/profession task.

Strengths:

The idea of stimulating the human brain non-invasively is very attractive because, if it worked, it could lead to a host of interesting applications. The current study aims to evaluate one such exciting application.

Weaknesses:

(1) The title refers to the "precuneus-hippocampus" network. A clear definition of what is meant by this terminology is lacking. More importantly, mechanistic evidence that the precuneus and the hippocampus are involved in the potential effects of stimulation remains unconvincing.

Thank you for the observation. We believe that the evidence collected supports our state relative to the stimulation of the precuneus and the involvement of the hippocampus. In particular, given the existing evidence on TMS methodology and precuneus non-invasive stimulation (see Koch et al., Brain, 2022, Koch et al., Alzheimer's research & therapy, 2025), the computation of the biophysical model with the E-field we produced (see Biophysical modeling and E-field calculation section in the supplementary information), together with the individual identification of the precuneus through the RM (see iTBS+γtACS neuromodulation protocol and MRI data acquisition in the main text), we can reasonably assume that the individually identified PC was stimulated.

As we acknowledged in the Limitations section, we cannot entirely rule out the possibility that our results might also reflect stimulation of more superficial parietal regions adjacent to the precuneus. Nor do we provide direct evidence of microscopic changes in the precuneus following stimulation. However, the results we provide in terms of changes in precuneus oscillatory activity and precuneus-hippocampi connectivity sustain both our thesis of the precuneus stimulation and of hippocampi involvement in the stimulation effects.

Despite this consideration, we agree on the fact that a clear definition of what is meant by the terminology “precuneus-hippocampus network” is lacking. Moreover, since our data and previous evidence sustain the notion of PC stimulation, while this study does not produce direct evidence of the hippocampi stimulation - but only of the effect of the neuromodulation protocol on its connection with the precuneus, we soften the claim in the title. We remove the mention of the precuneus-hippocampus network so that the modified title will be as follows: “Dual transcranial electromagnetic stimulation of the precuneus boosts human long-term memory.”

(2) The question of the extent to which the stimulation approach and the stimulation parameters used in these experiments causes specific and functionally relevant neural effects remains open. Invasive recordings that could address this question remain out of the scope of this non-invasive study. The authors conducted scalp EEG experiments in an attempt to address this question using non-invasive methods. However, the results shown in Fig. 3 are unclear. The results are inconsistently reported in units of microvolts squared in some panels (3A, 3B) and in units of microvolts in other panels (3C). Also, there is insufficient consideration of potential contamination by signal components reflecting eye movements, other muscle artifacts, or another volume-conducted signal reflecting aggregate activity inside the brain.

As you correctly noted, Figure 3 presents results obtained from the TMS–EEG recordings. However, there is no inconsistency regarding the measurement units, as we are referring to two distinct indices: one in the frequency domain—oscillatory power shown in Figures 3A and 3B, expressed in microvolts squared (μV^²)—and one in the time domain—the TMS-evoked potential shown in Figure 3C, expressed in microvolts (μV).

Regarding the concern about artifacts, this is an important issue on which our group has a strong expertise, having published well-established, highly cited procedures on how to record and clean TMS-EEG signals (e.g., Casula et al., Clinical Neurophysiology, 2017; Rocchi et al., Brain Stimulation, 2021). In the current study, we adopted a well-established and rigorous approach for both data acquisition and preprocessing. This ensured that the recorded TMS–EEG signals were not contaminated by physiological or electrical artifacts.

As regards the recording procedure, all participants were instructed to fixate on a black cross to minimize eye movements. To avoid auditory-related components caused by the TMS click, we adopted an ad-hoc procedure optimized for TMS-EEG recordings (Rocchi et al., Brain Stimulation, 2021). First, participants were given earphones that continuously played an ad-hoc masking noise composed of white noise mixed with specific time-varying frequencies of the TMS click (Rocchi et al., Brain Stimulation, 2021). The masking noise volume was adjusted to ensure that participants could not detect the TMS click, or as much as tolerated (always below 90 dB). To further reduce the impact of the TMS click on the EEG signal, we placed ear defenders (SNR=30) on top of the earphones. Please see TMS–EEG data acquisition section in the main text.

As regards the offline cleaning process, we applied Independent Component Analysis (INFOMAX-ICA) to the EEG data to identify and remove components associated with muscle activity, eye movements, blinking, and residual TMS-related artifacts, in line with the most recent guidelines on TMS–EEG preprocessing (Hernandez-Pavon et al., Brain Stimulation, 2023). Specifically, for TMS-related muscle artefacts, we strictly followed the criteria based on their scalp topography, spectral content, timing, and amplitude, which we published in a paper focused on this topic (Casula et al., Clinical Neurophysiology, 2017). We add this detail in the TMS–EEG preprocessing and analysis section in the supplementary information (lines 119-120).

(3) Figure 3 indicates "Precuneus oscillatory activity ...", but evidence that the activity presented reflects precuneus activity is lacking. The maps shown at the bottom of Figure 3C suggest that the EEG signals recorded with scalp EEG reflect activity generated across a wide spatial range, with a peak encompassing at least tens of centimeters. Thus, evidence that effects specifically reflect precuneus activity, as the paper's title and text throughout the manuscript suggest, is lacking.

We believe there may have been a misunderstanding. As indicated in the figure caption, panels A and B represent oscillatory activity, whereas panel C displays the TMS-evoked potentials (TEPs). Therefore, the topographical maps mentioned (i.e., those in panel C) did not refer to oscillatory activity, but to differences in TEP amplitude. Specifically, the topographies shown in Figure 3C illustrate statistically significant differences in TEP amplitudes between post-stimulation time points (T1—immediately after stimulation, and T2—20 minutes after stimulation) and the pre-stimulation baseline (T0).

In this figure, we focused our analysis on a cluster of electrodes overlying the individually identified precuneus, capturing EEG responses to single TMS pulses delivered to that target. This approach, widely used in previous literature (e.g., Koch et al., NeuroImage, 2018; Casula et al., Annals of Neurology, 2022; Koch et al., Brain, 2022; Maiella et al., Clinical Neurophysiology, 2024; Koch et al., Alzheimer’s Research & Therapy, 2025), supports the interpretation that the observed responses reflect precuneus-related activity. Furthermore, the wide spatial range change you mention proved to be statistically different only when conducting the TMS-EEG over the precuneus (i.e., administering the TMS single pulse over the precuneus) and not when performing it over the left parietal cortex. We modified the discussion section in the main text to make it more clear (lines 196-199).

“Moreover, we observed specific cortical changes in the posteromedial parietal areas, as evidenced by the whole-brain analysis conducted on TMS-EEG data when performed over the precuneus and the absence of effect when TMS-EEG was performed on the lateral posterior parietal cortex used as a control condition.”

That said, we do not state that the effects observed specifically reflect the precuneus activity; indeed, we think the effect of the stimulation is broader, as discussed in the Discussion section. We rather sustain, in line with the literature (Koch et al., Neuroimage 2018; Koch et al., Brain, 2022; Koch et al., Alzheimer's research & therapy, 2025), the idea that the effects observed are a consequence of the precuneus stimulation by the dual stimulation.

(4) The paper as currently presented (e.g., Figure 3) also lacks rigorous evidence of relevant oscillatory activity. Prior to filtering EEG signals in a particular frequency band, clear evidence of oscillations in the frequency band of interest should be shown (e.g., demonstration of a clear peak that emerges naturally in the frequency range of interest when spectral analysis is applied to "raw" signals). The authors claim that gamma oscillations change because of the stimulation, but a clear peak in the gamma range prior to stimulation is not apparent in the data as currently presented. Thus, the extent to which spectral measurements during stimulation reflect physiological gamma oscillations remains unclear.

If we understand correctly, your concern relates to the lack of a clear gamma peak before neuromodulation, which may suggest uncertainty about the observed changes in gamma oscillatory activity. Is that correct?

First, it is important to underline that the natural frequency typically observed in the precuneus falls within the beta range, not the gamma range (see Rosanova et al., Journal of Neuroscience, 2009; Casula et al., Annals of Neurology, 2022). This explains why a prominent gamma peak is not expected at baseline (T0).

Differently, our neuromodulatory protocol was specifically aimed at boosting gamma oscillatory activity given its well-established role in learning and memory processes (Griffiths & Jensen, Trends in Neurosciences, 2023). Thus, to assess the effect of the neuromodulatory protocol, we compared the oscillatory activity before (T0) and after stimulation (T1 and T2), which showed a clear increase in the gamma band. This effect is visible in the raw oscillatory power plot and is most clearly represented in Figure 3B, where the gamma band emerged as the only frequency range showing significant changes across time points.

(5) Concerns remain regarding the rigor of statistical analyses in the revised manuscript (see also point 8 below). Figure 3B shows an undefined statistical test with p<0.05. The statistical test that was used is not explained. Also, a description of how corrections for multiple comparisons were made is missing. Figures 3A and 3C are not accompanied by statistics, making the results difficult to interpret. For Figure 4C, a claim was made based on a significant p-value for one statistical test and a non-significant p-value in another test. This is a common statistical mistake (see Figure 1 and accompanying discussion in Makin and Orban de Xivry (2019) Science Forum: Ten common statistical mistakes to watch out for when writing or reviewing a manuscript. eLife 8:e48175).

All statistical tests are described in the Statistical Analysis section of the main text. Specifically, to assess cortical oscillation changes in Experiment 3, we conducted repeated-measures ANOVAs with stimulation condition (iTBS+γtACS vs. iTBS+sham-tACS) and time (ΔT1 = T1–T0; ΔT2 = T2–T0) as within-subject factors, for each frequency band. To further explore the effects of stimulation at each time point, we performed paired t-tests with Bonferroni correction for multiple comparisons. A one-tailed hypothesis was adopted, based on our a priori prediction of gamma-band increase derived from previous work (Maiella et al., 2022).

Please note that Figures 3A and 3C are purely descriptive and are therefore not accompanied by statistical tests. Figure 3A shows the full spectral profile across frequencies and conditions, while statistical significance for these data is reported in Figure 3B. Similarly, the upper part of Figure 3C displays the TMS-evoked potential (TEP) in the precuneus, while the statistical comparison of TEP amplitudes across time points is shown in the lower part of Figure 3C.

Regarding Figure 4C and the article you cited, are you referring to the error described as “Interpreting comparisons between two effects without directly comparing them”? If we understand correctly, this refers to the mistake of inferring an effect by observing that a significant result occurs in one condition or group, while the corresponding result in another condition or group is not significant, without directly testing the difference between them.

In the case of Experiment 4, which investigates fMRI effects and is illustrated in Figure 4, we employed a general linear model that explicitly modeled both conditions and time points, allowing for a direct statistical comparison. Therefore, the connectivity effect reported does not fall into the category of the error you mentioned.

Importantly, Figure 4C does not depict the effect of the neuromodulatory protocol itself. Rather, its purpose is to show that, within the real stimulation condition, there is a correlation between the observed effect and the integrity of the bilateral Middle Longitudinal Fasciculus. No conclusions or assumptions were made based on the absence of a significant correlation in the sham condition. However, since it was an exploratory analysis, we decided to soften our claims relative to the neural mechanism in the discussion section of the main text (lines 241-246).

(6) In the second question posed in the original review, I highlighted that it was unclear how such stimulation would produce memory enhancement. The authors replied that, in the absence of mechanisms, there are many other studies that suffer from the same problem. This raises the question of placebo effects. The paper does not sufficiently address or discuss the possibility that any potential stimulation effects may reflect placebo effects.

We agree with the reviewer on the potential role of a placebo effect in our study. For this reason, our experimental study had several stimulation conditions, including a placebo condition, which corresponded to the sham iTBS-sham tACS condition, which did not produce any effect.

(7) The third major concern in the original review was the lack of evidence for a mechanism that is specific to the precuneus. Evidence for specific involvement of the precuneus remains lacking in the revised manuscript. The authors state: "the non-invasive stimulation protocol was applied to an individually identified precuneus for each participant". However, the meaning of this statement is unclear. Specifically, it is unclear how the authors know that they are specifically targeting the precuneus. Without directly recording from the precuneus and directly demonstrating effects, which is outside of the scope of the study, specific involvement of the precuneus seems speculative. Also, it does not seem as though a figure was included in the paper to show how the stimulation protocol specifically targets the precuneus. In their response to the original reviews, the authors state that posterior medial parietal areas are the only regions that show significant differences following the stimulation, but they did not cite a specific figure, or statistics reported in the text, that show this. In any event, posterior medial parietal areas encompass a wide area of the brain, so this would still not provide evidence for an effect specifically involving the precuneus.

We respectfully disagree with the claim that targeting the precuneus in our study is speculative. The statement that “without directly recording from the precuneus and directly demonstrating effects, which is outside the scope of the study, specific involvement of the precuneus seems speculative” would, by that logic, implicitly call into question a large body of cognitive neuroscience research employing non-invasive techniques such as EEG and fMRI.

Our methodological approach—combining MRI-guided stimulation, biophysical modeling, and TMS–EEG—is well established and widely used for targeting and studying the role of specific cortical regions, including the precuneus (e.g., Wang et al., Science, 2014; Koch et al., NeuroImage, 2018; Casula et al., Annals of Neurology, 2022, 2023; Koch et al., Brain, 2022; Maiella et al., Clinical Neurophysiology, 2024; Koch et al., Alzheimer’s Research & Therapy, 2025).

In line with previously published protocols (Santarnecchi et al., Human Brain Mapping, 2018; Özdemir et al., PNAS, 2020; Mantovani et al., Journal of Psychiatric Research, 2021), we identified individual targets (i.e., the precuneus) for each participant based on structural and resting-state functional MRI data (see MRI Data Acquisition and Preprocessing section in the main text). This target was then accurately localized using MRI-guided stereotaxic neuronavigation, ensuring reproducible and anatomically precise stimulation across subjects.

Finally, concerning the last comment about the lack of figures/statistics showing how the stimulation protocol targets the precuneus and the specificity of the effect observed, we would like to let the focus go over:

Figure 3 in the main text, where we show the results of the TME-EEG over the posterior medial parietal areas;

Figure S1 in the supplementary information, which shows with the e-fied simulation how the stimulation protocol targets the brain;

the Precuneus iTBS+γtACS increases gamma oscillatory activity section in the main text results, where we report the results of the statistical analysis of the TMS-EEG conducted over the precuneus and the left posterior parietal cortex, used as a control condition to test for the specificity of the neuromodulation protocol.

(8) Regarding chance levels, it is unfortunate that the authors cannot quantify what chance levels are in the immediate and delayed recall conditions. This makes interpretation of the results challenging. In the immediate and delayed conditions, the authors state that the chance level is 33%. It would be useful to mark this in the figures. If I understand correctly, chance is 33% in Fig. 2A. If this is the case and if I am interpreting the figure correctly:

Gray bars for the sham condition appear to be below chance (~20-25%). Why is this condition associated with an accuracy level that is lower than chance?

Cyan bars and red bars do not appear to be significantly different from chance (i.e., 33%), with red slightly higher than cyan. What statistic was performed to obtain the level of significance indicated in the figure? The highest average value for the red condition appears to be around 35%. More details are needed to fully explain this figure and to support the claims associated with this figure.

The immediate and recall conditions you mention correspond to a free recall task. In this case, the notion of a fixed "chance level" is not straightforward as it would be in recognition or forced-choice paradigms, which is why we did not quantify it at first. I will now try to explain this extensively.

Unlike multiple-choice tasks, where participants select the answer from a limited set of alternatives and the probability of a correct response by chance can be precisely quantified (e.g., 33% in a 3-alternative forced choice), free recall involves the spontaneous retrieval of items from memory without external cues or predefined options. As such, the response range in free recall is essentially unconstrained, encompassing the entire vocabulary of the participant.

Because of this open-ended nature, the probability of correctly recalling a studied item purely by chance is exceedingly low and could be approximated to zero. Also, in our task, participants had to correctly recollect both name and occupation, doubling the possibility of the answers.

This assumption is further supported by the fact that random guesses in free recall are unlikely to match any of the studied items, given the vast number of possible alternatives. As a result, performance above zero can be reasonably interpreted as reflecting genuine memory retrieval, rather than random guessing.

As regards statistics, repeated-measures ANOVAs with stimulation condition as a within-subject factor (i.e., iTBS+γtACS; iTBS+sham-tACS; sham-iTBS+sham-tACS) for each dependent variable (see statistical analysis section in main text).

(9) In the revised version of the paper, the authors did not address concerns associated with the block design (please see question 4d in the original review).

We are sorry for the misunderstanding. We did not address your concerns related to block design since it does not apply to our study. As reported in the paper you mentioned in the original review, block design involves data collection performed in response to different stimuli of a given class presented in succession. If this is the case, it does not correspond to our experimental design since both TMS-EEG and fMRI were conducted in the resting state (i.e., without the presentation of stimuli) on different days according to the different randomized stimulation conditions.  

In sum, this study presents an admirable aspirational goal, the notion that a non-invasive stimulation protocol could modulate activity in specific brain regions to enhance memory. However, the evidence presented at the behavioral level and at the mechanistic level (e.g. the putative involvement of specific brain regions) remains unconvincing.

We hope our response will be carefully considered, fostering a constructive exchange and leading to a reassessment of your evaluation.

Reviewer #2 (Public review):

Summary:

The manuscript by Borghi and colleagues provides evidence that the combination of intermittent theta burst TMS stimulation and gamma transcranial alternating current stimulation (γtACS) targeting the precuneus increases long-term associative memory in healthy subjects compared to iTBS alone and sham conditions. Using a rich dataset of TMS-EEG and resting-state functional connectivity (rs-FC) maps and structural MRI data, the authors also provide evidence that dual stimulation increased gamma oscillations and functional connectivity between the precuneus and hippocampus. Enhanced memory performance was linked to increased gamma oscillatory activity and connectivity through white matter tracts.

Strengths:

The combination of personalized repetitive TMS (iTBS) and gamma tACS is a novel approach to targeting the precuneus, and thereby, connected memory-related regions to enhance long-term associative memory. The authors leverage an existing neural mechanism engaged in memory binding, theta-gamma coupling, by applying TMS at theta burst patterns and tACS at gamma frequencies to enhance gamma oscillations. The authors conducted a thorough study that suggests that simultaneous iTBS and gamma tACS could be a powerful approach for enhancing long-term associative memory. The paper was well-written, clear, and concise.

Comments on Revision:

I thank the authors for their thoughtful responses to my first review and their inclusion of more detailed methodological discussion of their rationale for the stimulation protocol conditions and timing. Regarding the apparent difference in connectivity at baseline between conditions, the explanation that this is due to intrinsic dynamics, state, or noise implies the baseline is reflecting transient changes in dynamics rather than a true or stable baseline. Based on this, it looks like iTBS solely is significantly greater than the baseline before the iTBS and γtACS condition but maybe not that much lower than post-stimulation period for iTBS and γtACS. A longer baseline period should be used to ensure transient states are not driving baseline levels such that these endogenous fluctuations would average out. This also raises questions about whether the effect of iTBS and γtACS or iTBS alone are dependent on the intrinsic state at the time when stimulation begins. Their additional clarification of memory scoring is helpful but also reveals that the effect of dual iTBS+γtACS specifically on the association between faces and names is just significant. This modest increase in associative memory should be taken into consideration when interpreting these findings.

We thank the reviewer for the feedback. We fully agree that considering baseline dynamics is critical when assessing the neurophysiological and connectivity effects of stimulation protocols.

In Experiments 3 and 4, baseline measurements were specifically included in our design to account for the possibility that intrinsic dynamics, state, or noise could influence the observed effects of neuromodulation. Indeed, if we had compared only post-stimulation connectivity between the real and sham conditions, the effects might have appeared larger. The inclusion of baseline measurements allows us to contextualize and better isolate the neuromodulatory impact by controlling such endogenous fluctuations. Importantly, the fMRI connectivity measurements, which comprise the baseline, are derived from 10-minute BOLD signal acquisitions, which help mitigate the influence of transient fluctuations and provide a quite stable estimate of intrinsic connectivity.

Moreover, regarding the possibility that stimulation effects may depend on the intrinsic state at stimulation onset, we hypothesize that gamma-frequency entrainment induced by tACS could reduce the variability of intrinsic dynamics, promoting a more stable neural state that is favorable for the induction of long-term plasticity.

As regards the memory scoring, we would like to clarify that the significant improvement observed in the dual iTBS+γtACS condition does not pertain solely to the face–name association. Rather, it concerns the more demanding task of recalling the association between face, name, and occupation. While we agree that the observed effect could be considered modest, it is worth noting that it follows from only 3 minutes of stimulation.

Reviewer #3 (Public review):

Summary:

Borghi and colleagues present results from 4 experiments aimed at investigating the effects of dual γtACS and iTBS stimulation of the precuneus on behavioral and neural markers of memory formation. In their first experiment (n = 20), they find that a 3-minute offline (i.e., prior to task completion) stimulation that combines both techniques leads to superior memory recall performance in an associative memory task immediately after learning associations between pictures of faces, names, and occupation, as well as after a 15-minute delay, compared to iTBS alone (+ tACS sham) or no stimulation (sham for both iTBS and tACS). Performance in a second task probing short-term memory was unaffected by the stimulation condition. In a second experiment (n = 10), they show that these effects persist over 24 hours and up to a full week after initial stimulation. A third (n = 14) and fourth (n = 16) experiment were conducted to investigate neural effects of the stimulation protocol. The authors report that, once again, only combined iTBS and γtACS increases gamma oscillatory activity and neural excitability (as measured by concurrent TMS-EEG) specific to the stimulated area at the precuneus compared to a control region, as well as precuneus-hippocampus functional connectivity (measured by resting state MRI), which seemed to be associated with structural white matter integrity of the bilateral middle longitudinal fasciculus (measured by DTI).

Strengths:

Combining non-invasive brain stimulation techniques is a novel, potentially very powerful method to maximize the effects of these kinds of interventions that are usually well-tolerated and thus accepted by patients and healthy participants. It is also very impressive that the stimulation-induced improvements in memory performance resulted from a short (3 min) intervention protocol. If the effects reported here turn out to be as clinically meaningful and generalizable across populations as implied, this approach could represent a promising avenue for treatment of impaired memory functions in many conditions.

Methodologically, this study is expertly done! I don't see any serious issues with the technical setup in any of the experiments. It is also very commendable that the authors conceptually replicated the behavioral effects of experiment 1 in experiment 2 and then conducted two additional experiments to probe the neural mechanisms associated with these effects. This certainly increases the value of the study and the confidence in the results considerably.

The authors used a within-subject approach in their experiments, which increases statistical power and allows for stronger inferences about the tested effects. They also used to individualize stimulation locations and intensities, which should further optimize the signal-to-noise ratio.

Weaknesses:

I think one of the major weaknesses of this study is the overall low sample size in all of the experiments (between n = 10 and n = 20). This is, as I mentioned when discussing the strengths of the study, partly mitigated by the within-subject design and individualized stimulation parameters. The authors mention that they performed a power analysis but this analysis seemed to be based on electrophysiological readouts similar to those obtained in experiment 3. It is thus unclear whether the other experiments were sufficiently powered to reliably detect the behavioral effects of interest. In the revised manuscript, the authors provide post-hoc sensitivity analyses that help contextualize the strength of the findings.

While the authors went to great lengths trying to probe the neural changes likely associated with the memory improvement after stimulation, it is impossible from their data to causally relate the findings from experiments 3 and 4 to the behavioral effects in experiments 1 and 2. This is acknowledged by the authors and there are good methodological reasons for why TMS-EEG and fMRI had to be collected in separate experiments, but readers should keep in mind that this limits inferences about how exactly dual iTBS and γtACS of the precuneus modulate learning and memory.

We thank the reviewer for the feedback.

Reviewer #1 (Recommendations for the authors):

I suggest:

(1) Removing all mechanistic claims about the precuneus and hippocampus.

We soften our claims about the precuneus-hippocampus network.

(2) Repeating and focusing on the behavioral experiments with a much larger number of images and stronger statistical power to try to demonstrate a compelling behavioral correlate of the proposed stimulation protocol.

We clarified the misunderstanding relative to the chance level of the behavioral experiments raised by the reviewer.

Reviewer #2 (Recommendations for the authors):

Use longer baseline to establish stable gamma level for comparisons in Figure 3

If we understand correctly, you propose to increase the baseline to establish the gamma oscillatory activity as expressed in Figure 3 (showing the results of experiment 3). Is that right? In the figure, you see a baseline of -100; 0ms, which we use for a merely graphical reason, since no activity is usually observable before the TMS pulse. However, to establish the level of gamma, we used a larger baseline correction ranging from -700 ms to -300 ms (i.e., 400ms). We added this important information in the cortical oscillation section of the supplementary information (lines 134-135).

Reviewer #3 (Recommendations for the authors):

I think that the authors did a great job responding to the concerns raised by the reviewers. All of my own comments have been satisfactorily addressed. I will update my public review to be more concise, so that it only includes the overall assessment of the manuscript, including the strengths and weaknesses, but without the requests for clarification. Strengths and weaknesses remain largely the same, as the authors did not conduct additional experiments.

Thank you.

eLife Assessment

This study presents a valuable finding that KDM5 inhibitors may enable a wide therapeutic window as compared to STING agonists or Type I Interferons. The evidence supporting the claims of the authors is convincing. The work will be of broad interest to scientists working in the field of breast cancer research.

Reviewer #1 (Public review):

In this manuscript, Lau et al reported that KDM5 inhibition in luminal breast cancer cells results in R-loop-mediated DNA damage, reduced cell fitness and an increase in ISG and AP signatures as well as cell surface Major Histocompatibility Complex (MHC) class I, mediated by RNA:DNA hybrid activation of the CGAS/STING pathway.

Their studies have shown that KDM5 inhibition/loss mediates a viral mimicry and DNA damage response through the generation of R-loops in genomic repeats. This is a different mechanism from the more well studied double-stranded RNA-induced "viral mimicry" response.

More importantly, they have shown that KDM5 inhibition does not result in DNA damage or activation of the CGAS/STING pathway in normal breast epithelial cells, suggesting that KDM5 inhibitors may enable a wide therapeutic window in this setting, as compared to STING agonists or Type I Interferons.

Their findings provide new insights into the interplay between epigenetic regulation of genomic repeats, R-loop formation, innate immunity, and cell fitness in the context of cancer evolution and therapeutic vulnerability.

Comments on revised version:

The authors have satisfactorily addressed my comments and revised the manuscript accordingly.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors investigated how the type-I interferon response (ISG) and antigen presentation (AP) pathways are repressed in luminal breast cancer cells and how this repression can be overcome. They found that a STING agonist can reactivate these pathways in breast cancer cells, but it also does so in normal cells, suggesting that this is not a good way to create a therapeutic window. Depletion of ADAR and inhibition of KDM5 also activate ISG and AP genes. The activation of ISG and AP genes is dependent on cGAS/STING and the JAK kinase. Interestingly, although both ADAR depletion and KDM5 inhibition activate ISG and AP genes, their effects on cell fitness are different. Furthermore, KDM5 inhibitor selectively activates ISG and AP genes in tumor cells but not normal cells, arguing that it may create a larger therapeutic window than the STING agonist. These results also suggest that KDM5 inhibition may activate ISG and AP genes in a way different from ADAR loss, and this process may affect tumor cell fitness independently of the activation of ISG and AP genes.

The authors further showed that KDM5 inhibition increases R-loops and DNA damage in tumor cells, and XPF, a nuclease that cuts R-loops, is required for the activation of ISG and AP genes. Using H3K4me3 CUT&RUN, they found that KMD5 inhibition results in increased H3K4me3 not only at genes, but also at repetitive elements including SINE, LINE, LTR, telomeres, and centromeres. Using S9.6 CUT&TAG, they confirmed that R-loops are increased at SINE, LINE, and LTR repeated with increased H3K4me3. Together, the results of this study suggest that KMD5 inhibition leads to H3K4me3 and R-loop accumulation in repetitive elements, which induces DNA damage and cGAS/STING activation and subsequently activates AP genes. This provides an exciting approach to stimulate the anti-tumor immunity against breast tumors.

KDM5 inhibition activates interferon and antigen presentation genes through R-loops.

Strengths:

A new approach to make breast tumors "hot" for anti-tumor immunity.

Weaknesses:

Future in vivo studies are needed to show the effects of KDM5 inhibitors on the immunotherapy responses of breast tumors.

Comments on revised version:

The authors have adequately addressed my comments.

Author response:

The following is the authors’ response to the original reviews

We thank the reviewers for their careful and positive assessment of our manuscript. Maybe our findings are best summarized in the model below, showing that KDM5 inhibition/loss mediates a viral mimicry and DNA damage response through the generation of R-loops in genomic repeats. This is a different mechanism from the more well studied double-stranded RNA-induced “viral mimicry” response. Our studies also suggest that KDM5 inhibition may have a larger therapeutic window than STING agonists, since KDM5 inhibition seemingly does not induce “viral mimicry” in normal breast epithelial cells. 

Author response image 1.

Model of viral mimicry activation. De-repression of repetitive elements may trigger dsRNA formation, which activates the RIG-1/MDA5 pathway, as well as PKR. Alternatively, derepression of these elements may induce transcription replication conflicts (TRCs), resulting in R-loop formation. R-loops can lead to DNA damage, and/or activate the cGAS/STING pathway. Both the MAVS pathway and the cGAS/STING pathway converge to activate type I interferon (IFN) responses, resulting in decreased cell fitness and/or increased immunogenicity.

We do agree with the assessment that the study would be strengthened by in vivo studies. However, there are 4 different isoforms of KDM5 (3 in females), and existing KDM5specific inhibitors do not have adequate PK/PD properties for in vivo studies. We would also like to note that most mouse studies have not been proven to accurately predict immunotherapy responses in patients. Future studies in ex vivo tumor models would strengthen the clinical relevance of these studies. In the interim, we have added some normal macrophage studies in Figure S5 and an example of studies in normal T-cells below. Such studies will also be important to ensure that future KDM5 inhibitors do not have adverse effects on the immune system. Here, we observe that KDM5 inhibition appears to have neutral or slightly reduced T cell viability with KDM5 inhibition (Author response image 2a). However, KDM5 inhibition also results in increased CD107a expression in T-cells, indicative of a more cytotoxic phenotype (Author response image 2b). These studies suggest that KDM5 inhibitors do not have significant adverse effects on T cells or macrophages (figure S5) in the normal immune environment.

Author response image 2.

KDM5 inhibition does not have significant adverse effects on T-cells. a) Fold change proliferation of T-cells from 2 different human donors (left and right panels on graph) activated with 0.25ug/ml CD3 and treated with the indicated concentrations of C48 or a positive control (CBLB) compared to vehicle controls. b. FACS plots and histograms of CD107a surface expression (x-axis) versus forward scatter (FSC, y-axis) of T-cells from 2 different humans donors activated with 0.25ug/ml or 0.5mug/ml CD3 and treated with the indicated concentrations of C48.

Specific comments and answers to Reviewer #1:

We have added some additional analysis of data from other breast cancer cell lines to strengthen our points (Figure S2f, Figure S3e, Figure S4g-h, k.) We have also uploaded all the data to Geo with the following accession numbers :

GSE296387: H3K4me3 CUT-and-Tag data

GSE296584: S9.6 CUT-and-Tag data

GSE296974: RNA-sequencing data

Responses to Reviewer #1 (Recommendations for the authors):

(1) We have not conducted genomic studies comparing KDM5 expression to retroelement activation status in the tumor data sets but recognize that this is important for future studies. Again, there are several KDM5 isoforms and looking at repeat expression in these larger data sets is complex. We have added some data correlating KDM5 expression with ISG signatures in Figure S3j-l as well as in the graph below (Author response image 3). The correlation with ISG and AP signatures is modest, but strongest for KDM5B and C in breast cancer data sets, consistent with our disruption data for these 2 isoforms. As mentioned above, we do agree that future studies of KDM5s along with a broader analysis of other epigenetic modifying enzymes over repeats in various cancer types will shed light on the role of histone modifying enzymes in suppressing “viral mimicry” in tumors.

Author response image 3.

Correlation between gene expression and IFN gene set GSVA scores in breast cancer cell lines. a) Pearson correlation score between gene expression and IFN signature (ISG) gene set variation analysis (GSVA) scores in breast cancer cell lines as reported in DepMap. Higher ranks indicate an inverse correlation between expression of the individual gene and the expression of the ISG gene set. Correlation ranks for KDM5A, B and C are highlighted. b) as in a), but comparing gene expression to antigen presentation (AP) GSVA scores.

(2) We apologize for the mislabeling in figure 2B – has been corrected in the revised version.

(3) We agree that blocking the cGAS/STING pathway, only partially rescues the ISREGFP and HLA-A, B, C phenotype in HCC1428 cells. We have added data (Figure S2f) showing that this rescue is stronger in MCF7 cells. It is possible that the MDA5/MAVS pathway may also contribute to activation of the Type I interferon response. However, we have data that MAVS plays a minor (if any) role in this context, as MAVS KO minimally decreases C48-induced ISRE-GFP activity and HLA-A, B, C surface expression in HCC1428 cells (added Figure S2g).

Furthermore, there is no significant increase in dsRNA observed (using J2 antibody as a readout in immunofluorescence experiments) with C48 treatment as compared to 5’-azacytidine treatment or ADAR K/O (data not included). However, we have not performed MAVS/PKR K/O experiments to completely rule out the involvement of the dsRNA sensing pathways.

(4) These experiments were performed in the operetta imaging system, rather than confocal imaging, and therefore we do not have such images. Quantification of RNaseH1-GFP in the whole cell is reported in the figure, as RNaseH1-GFP signal is increased in both the nucleus and the cytoplasm with C48 treatment. This is not unexpected, as our data suggest that R-loop formation occurs in repetitive regions of the genome that are de-repressed by KDM5 inhibition in the nucleus, and the RNA/DNA hybrids, generated from R-loops, may activate cGAS/STING pathway in the cytoplasm.

(5) Disruption of siXPF and siXPG is relatively toxic in itself. Complete knockouts in breast cancer cells were not viable and we partially knocked down XPF using siRNA instead. We do agree that these kinds of rescue studies need to be expanded upon in future studies, but they served as further proof of the conclusions presented here.

(6) We have provided all the data in Geo and alternative representations can be made.

(7) Unfortunately, CUT-and-Tag experiments were not performed in cells expressing siXPF and therefore we cannot provide this data. However, XPF has been previously shown to be responsible for excising R-loops from the genome, rendering them detectable by cGAS/STING in the cytoplasm (Crossley et al, 2022, referenced in the current MS). Therefore, while we demonstrate that XPF knockdown attenuates type I IFN pathway activation upon KDM5 inhibition, it may not necessarily reduce R-loop formation in retroelements; it may just prevent their excision and downstream cGAS/STING activation. We do agree that CUT-and-Tag experiments in cells treated with siXPF versus siControl will have to be performed in the future to test this hypothesis.

Responses to Reviewer #2 (Recommendations for the authors):

(1) We have modified the text as well as the figure legend to state that this is a simplistic representation of the pathway in normal cells. As stated in the introduction, these pathways can be modified in tumors. The data presented suggest that the dsRNA pathway can be activated in all breast cancer cell lines tested, whereas more variation is observed in the activation of the STING pathway.  

(2) The ADAR guides target ADAR 110 and p150 but not ADAR2. This has been clarified in the text.  

(3) The guides have been renamed in the figure as the reviewer suggests.  

(4) It has been shown by others that KDM5 can occupy the STING promoter (https://pubmed.ncbi.nlm.nih.gov/30080846/); which supports the reviewer’s suggestion that STING upregulation in HMECs may be due to increased H3K4me3 at the STING gene. However, we argue that STING upregulation is not sufficient to activate “viral mimicry” due to the absence of “tumor-specific R-loops” (due to an increase in TRC in tumor cells) in normal cells. It is interesting to note that the S9.6 signal in subtelomeric regions is increased in HMECS similar to what is observed in tumor cells. However, the S9.6 signal over other repeats is not (Author response image 4), suggesting that C48-induced increases over non-telomeric repeats are tumor specific. This suggests that the tumor-specific increases in R-loop formation, which lead to “viral mimicry” activation, are not driven by those formed in subtelomeric regions. Future studies will have to expand on these findings.

Author response image 4.

Percent of S9.6 reads that align to repetitive genome in HMEC cells. (a) % of total aligned S9.6 reads that map to subtelomeric region in HMEC cells treated with DMSO or 2.5 μM C48. (b) % of total aligned S9.6 reads that map to repetitive elements in general in HMEC cells treated as in a).

(5) Clarity on R-loop quantification has been added to the figure legend as well as in the Materials and Methods section. Mean fluorescence intensity in the whole cell (this includes both nuclear and cytoplasmic signals) was quantified together and normalized to the number of DAPI-stained nuclei per well. As mentioned above all quantified in the Operetta imaging system.

(6) We have added some data that shows that increases in H3K4me3 is observed in and around ISGs upon KDM5 inhibition (Figure S4f). However, without time course experiments it is difficult to assess whether these are direct effects of the KDM5 inhibitor or indirect effects from activation of Type I IFN (similarly to what has previously been reported with 5’-azacytidine induction of “viral mimicry”, https://pubmed.ncbi.nlm.nih.gov/26317465/).

(7) We have previously included data showing that S9.6 reads in repeats that do not display C48-mediated increases in H3K4me3 also do not increase with C48 treatment (this is now Figure S4o). In addition, we have added some data showing that repeats with increased H3K4me3 and repeats with increased transcription upon C48 treatment also have increased S9.6 reads. Repeats that display both increases in H3K4me3 and mRNA expression have even greater increases in S9.6 signal compared to repeats that have increases in either one (Figure S4m-n). Taken together, this data suggest that KDM5 inhibition increases H3K4me3 in repeats, thereby allowing for their transcription, which can increase the probability of Transcription replication conflicts (TRC) and R-loop formation at such loci.

(8) As mentioned earlier in this response, while we observe increased S9.6 reads in subtelomeric regions of HCC1428 cells upon KDM5 inhibition, we also observe this in normal HMEC cells. Since KDM5 inhibition does not induce viral mimicry in HMEC cells, this suggests that R-loops formed in subtelomeric regions do not dictate the response observed with C48 treatment in breast cancer cells.

We hope that these answers to the reviewers comments as well as the additional data provided strengthens our findings.

Again the fact that they lost the word winner suggests that they still haven't "won" against the catastrophe and are living through it.

Initially we're made to think that not being able to die isn't a terrible thing but the narrator then uses the word "robbed" which basically implies it was a possession that was stolen from her.

This seems very dystopian to have an organization running the country especially in the way that they're doing it (very authoritarian).

It's interesting that after the catastrophes people feared what they could say perhaps because they feared repercussions. The author does highlight that the words they chose to use now wouldn't have been the same as the ones chosen in "normal circumstances".

In the media that we have examined so far in this class, there's always a character that doesn't quite understand or yet grasp that a catastrophe is on the way. Initially, it's Will Smith's character in Independence Day and it's Martha in Dark.

This could perhaps relate again to some sort of tragedy that the narrator went through and is still coping with.

It's unclear whether the narrator was forced to relocate due to a catastrophic event, but regardless she struggles with letting go her old nationality/identity.

The use of italics suggests an event that happens that we have yet to discover. Based on how the narrator is framing it, we can assume it's something bad and it clearly affects the way people view her.

We can assume that some sort of catastrophe happened as these two lines very similarly matches the descriptions of catastrophes we came up with in class. It's this idea of losing everything and then some more.

eLife Assessment

The study showcases a significant and important enhancement of the MAGIC transgenesis method, by extending it genome-wide to all chromosomes. The authors convincingly demonstrate that the MAGIC mosaic clones can be generated for genes from all, including the 4th chromosome. With this toolkit extension, the method is now most likely set to strongly rival the classical FRT/Flp recombination system for gene manipulation in flies.

Reviewer #1 (Public review):

Summary:

In this manuscript, Shen et al. have improved upon the mitotic clone analysis tool MAGIC that their lab previously developed. MAGIC uses CRISPR/Cas9-mediated double-stranded breaks to induce mitotic recombination. The authors have replaced the sgRNA scaffold with a more effective scaffold to increase clone frequency. They also introduced modifications to positive and negative clonal markers to improve signal-to-noise and mark the cytoplasm of the cells instead of the nuclei. The changes result in increase in clonal frequencies and marker brightness. The authors also generated the MAGIC transgenics to target all chromosome arms and tested the clone induction efficacy.

Strengths:

MAGIC is a mitotic clone generation tool that works without prior recombination to special chromosomes (e.g., FRT). It can also generate mutant clones for genes for which the existing FRT lines could not be used (e.g., the genes that are between the FRT transgene and the centromere).

This manuscript does a thorough job in describing the method and provides compelling data that support improvement over the existing method.

Weaknesses:

It would be beneficial to have a greater variety of clonal markers for nMAGIC. Currently, the only marker is BFP, which may clash with other genetic tools (e.g., some FRET probes) depending on the application. It would be nice to have far-red clonal markers.

Reviewer #2 (Public review):

Summary:

In this study, the authors present the latest improvement of their previously published methods, pMAGIC and nMAGIC, which can be used to engineer mosaic gene expression in wild-type animals and in a tissue-specific manner. They address the main limitation of MAGIC, the lack of gRNA-marker transgenes, which has hampered the broader adoption of MAGIC in the fly community. To do so, they create an entire toolkit of gRNA markers for every Drosophila chromosome and test them across a range of different tissues and in the context of making Drosophila species hybrid mosaic animals. The study provides a significant and broadly useful improvement compared to earlier versions, as it broadens the use-cases for transgenic manipulation with MAGIC to virtually any subfield of Drosophila cell biology.

Strengths:

Major improvements to MAGIC were made in terms of clone induction efficiency and usability across the Drosophila model system, including wild-type genotypes and the use in non-melanogaster species.

Notably, mosaic mutants can now be created for genes residing on the 4th chromosome, which is exciting and possibly long-awaited by 4th chromosome gene enthusiasts.

Selection of the standard set of gRNA markers was done thoughtfully, using non-repetitive conserved and unique sequences.

The authors demonstrate that MAGIC can be used easily in the context of interspecific hybrids. I believe this is a great advancement for the Drosophila community, especially for evolutionary biologists, because this may allow for easy access to mechanistic, tissue-specific insight into the process of a range of hybrid incompatibilities, an important speciation process that is normally difficult to study at the level of molecular and cell biology.

In the same way, because it is not limited to usage in any particular genetic background, genome-wide MAGIC can be potentially used in wild-type genotypes relatively easily. This is exciting, especially because natural genetic diversity is rarely investigated more mechanistically and at the scale/resolution of cells or specific tissues. Now, one can ask how a particular naturally occurring allele influences cell physiology compared to another (control) while keeping the global physiological context of the particular genetic background largely intact.

Weaknesses:

It is not entirely clear how functionally non-critical regions were evaluated, besides that they are selected based on conservation of sequence between species. It may be useful to directly test the difference in viability or other functionally relevant phenotype for flies carrying different markers. Similarly, the frequency of off-targets could be investigated or documented in a bit more detail, especially if one of the major use-cases is meant for naturally derived, diverse genetic backgrounds. It is, at the moment, unclear how consistently the clones are induced for each new gRNA marker across different WT genetic backgrounds, for example, a set of DGRP genotypes, which could be highly useful information for future users.

Reviewer #3 (Public review):

Summary:

In the manuscript by Shen, Yeung, and colleagues, the authors generate an improved and expanded Mosaic analysis by gRNA-induced crossing-over (MAGIC) toolkit for use in making mosaic clones in Drosophila. This is a clever method by which mitotic clones can be induced in dividing cells by using CRISPR/Cas9 to generate double-strand breaks at specific locations that induce crossing over at those locations. This is conceptually similar to previous mosaic methods in flies that utilized FRT sites that had been inserted near centromeres along with heat-shock inducible FLPase. The advantage of the MAGIC system is that it can be used along with chromosomes lacking FRT sites already introduced, such as those found in many deficiency collections or in EMS mutant lines. It may also be simpler to implement than FRT-based mosaic systems. There are two flavors of the MAGIC system: nMAGIC and pMAGIC. In nMAGIC, the main constituents are a transgene insertion that contains gRNAs that target DNA near the centromere, along with a fluorescent marker. In pMAGIC, the main constituents are a transgenic insertion that contains gRNAs that target DNA near the centromere, along with ubiquitous expression of GAL80. As such, nMAGIC can be used to generate clones that are not labelled, whereas pMAGIC (along with a GAL4 line and UAS-marker) can be used much like MARCM to positively label a clone of cells. This manuscript introduces MAGIC transgenic reagents that allow all 4 chromosomes to be targeted. They demonstrate its use in a variety of tissues, including with mutants not compatible with current FLP/FRT methods, and also show it works well in tissues that prove challenging for FLP/FRT mosaic analyses (such as motor neurons). They further demonstrate that it can be used to generate mosaic clones in non-melanogaster hybrid tissues. Overall, this work represents a valuable improvement to the MAGIC method that should promote even more widespread adoption of this powerful genetic technique.

Strengths:

(1) Improves the design of the gRNA-marker by updating the gRNA backbone and also the markers used. GAL80 now includes a DE region that reduces the perdurance of the protein and thus better labeling of pMAGIC clones. The data presented to demonstrate these improvements is rigorous and of high quality.

(2) Introduces a toolkit that now covers all chromosome arms in Drosophila. In addition, the efficiency of 3 target different sites is characterized for each chromosome arm (e.g., 3 different gRNA-Marker combinations), which demonstrate differences in efficiency. This could be useful to titrate how many clones an experimenter might want (e.g., lower efficiency combinations might prove advantageous).

(3) The manuscript is well written and easy to follow. The authors achieved their aims of creating and demonstrating MAGIC reagents suitable for mosaic analysis of any Drosophila chromosome arm.

(4) The MAGIC method is a valuable addition to the Drosophila genetics toolkit, and the new reagents described in this manuscript should allow it to become more widely adopted.

Weaknesses:

(1) The MAGIC method might not be well known to most readers, and the manuscript could have benefited from schematics introducing the technique.

(2) Traditional mosaic analyses using the FLP/FRT system have strongly utilized heat-shock FLPase for inducible temporal control over mitotic clones, as well as a way to titrate how many clones are induced (e.g., shorter heat shocks will induce fewer clones). This has proven highly valuable, especially for developmental studies. A heat-shock Cas9 is available, and it would have been beneficial to determine the efficiency of inducing MAGIC clones using this Cas9 source.

Author response:

Reviewing Editor Comments:

The following are some consolidated review remarks after discussions amongst all three reviewers:

The reviewers feel the evidence level could be raised from 'convincing' to 'compelling' if the following key (and partially shared) suggestions by the reviewers are followed adequately:

(1) Expand labeling options for nMAGIC, which is currently just a BFP marker. This would increase the utility of the method. A far-red marker would be very helpful. Could the authors just do this for one chromosome arm and make the reagent available for others to generate other chromosome arms?

This is a great suggestion. We will make an nMAGIC vector containing a far-red fluorescent marker and generate a 40D2 version of this nMAGIC gRNA-maker to demonstrate its utility. This vector will be available for others to make additional nMAGIC gRNA-markers.

(2) Verify that destabilized GAL80 is potent enough to suppress GAL4. Repeat Figure 1C-E with tub-GAL80-DE-SV40.

We will use a tub-GAL80-DE-SV40 gRNA-marker to test suppression of pxn-Gal4.

(3) Concern about the health of the induced mitotic clones. This is an important consideration, but the reviewers were not sure what the necessary experiments would be. To gauge twin-spot clone sizes? Please address.

We will assess the health of induced mitotic clones in wing imaginal discs. We will do this by generating twin spots with a nMAGIC gRNA-marker in wing discs and compare the sizes of the two cell populations (BFP^+/+ and BFP^-/-) in twin spots.

(4) Include a schematic of the MAGIC method as Figure 1 or add it to Figure 1. Many may not be familiar with the method, so to promote its adoption, the authors should clearly introduce the MAGIC method in this paper (and not rely on readers to go to previous publications). For this paper to become a MAGIC reference paper, it should be self-contained.

We will add a diagram of the MAGIC method in the revised manuscript.

(5) Determine the utility of using a hs-Cas9 line for temporal induction of MAGIC clones. This is a traditional method for mitotic clone induction (with hsFLP/FRTs), and its use with the MAGIC system (especially pMAGIC) could also make it more attractive, especially to label small populations of neurons born at known times. To this point, the authors could generate pMAGIC clones using hs-Cas9 for commonly used adult target neurons, such as projection neurons, central complex neurons, or mushroom body neurons. The method to label small numbers of these adult neurons is well worked out with known GAL4 lines, and demonstrating that pMAGIC could have similar results would capture the attention of many not familiar with the pMAGIC method.

We thank the reviewers for this suggestion. We will test hs-Cas9 in inducing pMAGIC clones in one of the neuronal populations in the adult brain, as suggested by the reviewers.

In addition, we will address all other minor concerns of the reviewers.

Author response:

Public Reviews:

Reviewer #1 (Public Review):

Summary:

Plasmodium vivax can persist in the liver of infected individuals in the form of dormant hypnozoites, which cause malaria relapses and are resistant to most current antimalarial drugs. This highlights the need to develop new drugs active against hypnozoites that could be used for radical cure. Here, the authors capitalize on an in vitro culture system based on primary human hepatocytes infected with P. vivax sporozoites to screen libraries of repurposed molecules and compounds acting on epigenetic pathways. They identified a number of hits, including hydrazinophthalazine analogs. They propose that some of these compounds may act on epigenetic pathways potentially involved in parasite quiescence. To provide some support to this hypothesis, they document DNA methylation of parasite DNA based on 5-methylcytosine immunostaining, mass spectrometry, and bisulfite sequencing.

Strengths:

-The drug screen itself represents a huge amount of work and, given the complexity of the experimental model, is a tour de force.

-The screening was performed in two different laboratories, with a third laboratory being involved in the confirmation of some of the hits, providing strong support that the results were reproducible.

-The screening of repurposing libraries is highly relevant to accelerate the development of new radical cure strategies.

We thank the reviewer for pointing out the strengths of our report.

Weaknesses:

The manuscript is composed of two main parts, the drug screening itself and the description of DNA methylation in Plasmodium pre-erythrocytic stages. Unfortunately, these two parts are loosely connected. First, there is no evidence that the identified hits kill hypnozoites via epigenetic mechanisms. The hit compounds almost all act on schizonts in addition to hypnozoites, therefore it is unlikely that they target quiescence-specific pathways. At least one compound, colforsin, seems to selectively act on hypnozoites, but this observation still requires confirmation. Second, while the description of DNA methylation is per se interesting, its role in quiescence is not directly addressed here. Again, this is clearly not a specific feature of hypnozoites as it is also observed in P. vivax and P. cynomolgi hepatic schizonts and in P. falciparum blood stages. Therefore, the link between DNA methylation and hypnozoite formation is unclear. In addition, DNA methylation in sporozoites may not reflect epigenetic regulation occurring in the subsequent liver stages.

We agree our report lacks direct evidence that hydrazinophthalazines are interacting with parasite epigenetic mechanisms. We spent significant resources attempting several novel approaches to establish a direct connection, but technological advances are needed to enable such studies, which we mention in the introduction and discussion. We disagree that schizonticidal activity automatically excludes the possibility a hypnozonticidal hit is acting on quiescence-specific pathways because both hypnozoites and schizonts are under epigenetic control and these pathways are likely performing different functions in different stages. Also important is the use of the word ‘specific’ as this term could be used to indicate parasite versus host (a drug that clears a parasite infection with a safety margin), parasite-directed effect versus host-directed effect (a drug acting via an agonistic or antagonistic effect on parasite or host pathway(s), but leading to parasite death in either case), hypnozoite versus schizont, or P. vivax versus other Plasmodium species. We were careful to indicate the usage of ‘specific’ throughout the text. Given the almost-nonexistent hit rate when screening diverse small molecule libraries screening against P. vivax hypnozoites, and remarkable increase in hits when screening epigenetic inhibitors as described in this report, our data suggests epigenetic pathways are important to the regulation of hypnozoite dormancy in addition to regulation of other parasite stages, but those effects are outside the scope of this report.

-The mode of action of the hit compounds remains unknown. In particular, it is not clear whether the drugs act on the parasite or on the host cell. Merely counting host cell nuclei to evaluate the toxicity of the compounds is probably acceptable for the screen but may not be sufficient to rule out an effect on the host cell. A more thorough characterization of the toxicity of the selected hit compounds is required.

We agree, and mention in the results and discussion, that the effect could be mediated through host pathways. This is not unlike the 8-aminoquinolones, which are activated by host cytochromes and kill via ROS, which is a nonspecific mechanism (that is, the compound is not directly interacting with a parasite target) leading to a parasite-specific effect (the parasite cannot tolerate the ROS produced, but the host can). During screening, it is generally the case that detecting hits with direct effects on the target organism are more desirable, so hits are counterscreened for general cytotoxicity. In this report, we show an effect on the parasite in direct comparison to the effect on host primary hepatocytes in the P. vivax assay itself, and follow up on hits with general counterscreens using two mammalian cell lines using CellTiter Glo, which does not rely on nuclei counts. Some compounds did show general cytotoxic effects, but with selectivity (more potency) against P. vivax liver stages, while other hits like the hydrazinophthalazines did not show an effect against primary hepatocytes and show only weak toxicity against mammalian cells at the highest dose tested. Further studies are needed to determine if the effect is indeed host- or parasite-directed and, if hydrazinophthalazines are to be developed into marketed antimalarials, extensive safety testing would be part of the development process.

-There is no convincing explanation for the differences observed between P. vivax and P. cynomolgi. The authors question the relevance of the simian model but the discrepancy could also be due to the P. vivax in vitro platform they used.

Fully characterizing the chemo-sensitivity of P. vivax and P. cynomolgi liver stages is outside the scope of this report. Rather, we report tool compounds which could be used in future studies to further characterize these sister species. We also make the point that P. cynomolgi is the gold standard for in vivo antirelapse activity, but it is still a model species, not a target species, and so few experimental hypnozonticidal compounds have been reported that the predictive value of P. cynomolgi is not fully understood. We found that several of our hits were species-specific using our in vitro platforms, thus future studies are needed to ensure this predictive value.

-Many experiments were performed only once, not only during the screen (where most compounds were apparently tested in a single well) but also in other experiments. The quality of the data would be increased with more replication.

Due to their size, compound library screens are typically performed once, with confirmation in dose-response assays, which were repeated several times. Rhesus PK studies was performed once on three animals, which is typical. All other studies were performed at least twice and most were performed three times or more. We provide a data table showing readers the source material for all replication as well as other source data tables showing the raw data for dose-response and other assays.

-While the extended assay (12 days versus 8 days) represents an improvement of the screen, the relevance of adding inhibitors of core cytochrome activity is less clear, as under these conditions the culture system deviates from physiological conditions.

We agree that cytochrome inhibitors render the platform less physiologically relevant, but the goal of screening is to detect hits which could be improved upon using medicinal chemistry, including metabolic stability. Metabolic stability is better assessed using standard assays such as liver microsomes, thus our goal was to characterize the effects of test compounds on the parasite without the confounding effect of hepatic metabolism.

Reviewer #2 (Public Review):

Summary:

In this manuscript, inhibitors of the P. vivax liver stages are identified from the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library as well as a 773-member collection of epigenetic inhibitors. This study led to the discovery that epigenetics pathway inhibitors are selectively active against P. vivax and P. cynomolgi hypnozoites. Several inhibitors of histone post-translational modifications were found among the hits and genomic DNA methylation mapping revealed the modification on most genes. Experiments were completed to show that the level of methylation upstream of the gene (promoter or first exon) may impact gene expression. With the limited number of small molecules that act against hypnozoites, this work is critically important for future drug leads. Additionally, the authors gleaned biological insights from their molecules to advance the current understanding of essential molecular processes during this elusive parasite stage.

Strengths:

-This is a tremendously impactful study that assesses molecules for the ability to inhibit Plasmodium hypnozoites. The comparison of various species is especially relevant for probing biological processes and advancing drug leads.

-The SI is wonderfully organized and includes relevant data/details. These results will inspire numerous studies beyond the current work.

We thank the reviewer for pointing out the strengths of our report.

Reviewer #3 (Public Review):

Although this work represents a massive screening effort to find new drugs targeting P. vivax hypnozoites, the authors should balance their statement that they identified targetable epigenetic pathways in hypnozoites.

-They should emphasize the potential role of the host cell in the presentation of the results and the discussion, as it is known that other pathogens modify the epigenome of the host cell (i.e. toxoplasma, HIV) to prevent cell division. Also, hydrazinophtalazines target multiple pathways (notably modulation of calcium flux) and have been shown to inhibit DNA-methyl transferase 1 which is lacking in Plasmodium.

-In a drug repurposing approach, the parasite target might also be different than the human target.

-The authors state that host-cell apoptotic pathways are downregulated in P. vivax infected cells (p. 5 line 162). Maybe the HDAC inhibitors and DNA-methyltransferase inhibitors are reactivating these pathways, leading to parasite death, rather than targeting parasites directly.

We agree caution must be taken as we did not directly confirm the mechanism of our hits. Many follow up studies will be needed to do so. We do point out in the discussion that the mechanism of hits could be host-directed. We agree with the notion that some of these hits could be affecting parasitized host cell pathways, which lead to death of the parasitized cell, with the parasite being collateral damage, yet such a mechanism could lead to a safe and effective novel antimalarial.

It would make the interpretation of the results easier if the authors used EC50 in µM rather than pEC50 in tables and main text. It is easy to calculate when it is a single-digit number but more complicated with multiple digits.

We apologize for the atypical presentation of potency data. However, there is growing concern in drug discovery when Standard Deviation is applied to Potency data because Standard Deviation is a linear calculation and Potency is a log effect, making the math incompatible. We understand thousands of papers are reported every year using this mathematically incorrect method, making our presentation of these data less familiar. However, we define pEC50 in its use in the text and table legends and hope to increase its use in the broader scientific community.

Authors mention hypnozoite-specific effects but in most cases, compounds are as potent on hypnozoite and schizonts. They should rather use "liver stage specific" to refer to increased activity against hypnozoites and schizonts compared to the host cell. The same comment applies to line 351 when referring to MMV019721. Following the same idea, it is a bit far-fetched to call MMV019721 "specific" when the highest concentration tested for cytotoxicity is less than twice the EC50 obtained against hypnozoites and schizonts.

We have reviewed and revised statements in the manuscript to ensure the effect we are describing is accurate in terms of parasite versus parasite form.

Page 5 lines 187-189, the authors state "...hydrazinophtalazines were inactive when tested against P. berghei liver schizonts and P. falciparum asexual blood stages, suggesting that hypnozoite quiescence may be biologically distinct from developing schizonts". The data provided in Figure 1B show that these hydrazinophtalazines are as potent in P. vivax schizonts than in P. vivax hypnozoites, so the distinct activity seems to be Plasmodium species specific and/or host-cell specific (primary human hepatocytes rather than cell lines for P. berghei) rather than hypnozoite vs schizont specific.

We agree the effect of hydrazinophtalazine could be more species specific than stage specific, but the context of our comment has to do with current methods in antimalarial discovery and development. Given the biological uniqueness of the various Plasmodium species and stages, any hypnozonticidal hit may or may not have pan-species or pan-stage activity; our goal was to characterize this. Regardless of the mechanism, we found it interesting that the hydrazinophtalazines kill P. vivax hypnozoites, but not P. cynomolgi hypnozoites nor other species and stages used in antimalarial drug development. This result makes the point that hypnozoite-focused assays may be required to detect and develop hypnozonticidal hits, regardless of what other species or stages they may or may not act on.

Why choose to focus on cadralazine if abandoned due to side effects? Also, why test the pharmacokinetics in monkeys? As it was a marketed drug, were no data available in humans?

Cadralazine was found more potent than hydralazine and PK data was available from humans, thus dose prediction calculations showed an efficacious dose was more achievable with cadralazine than hydralazine. Side effects are often dependent on dose and regimen, which are very likely to be much different for treating malaria versus hypertension. Thus, the potential side effects of cadralazine if it was to be used as an antimalarial are simply unknown and are not disqualifying at this step. The PK study was done in Rhesus macaques so we could calculate the dose needed to achieve coverage of EC90 during a planned follow up in a Rhesus-P. cynomolgi relapse model. However, this planned in vivo efficacy study was not justified once we concurrently discovered cadralazine was inactive on P. cynomolgi in vitro.

In the counterscreen mentioned on page 6, the authors should mention that the activity of poziotinib in P. berghei and P. cynomolgi is equivalent to cell toxicity, so likely not due to parasite specificity.

Poziotinib shows activity against mammalian cell lines but not against the primary hepatocyte cultures supporting dose-response assays against P. vivax liver forms, which do not replicate. Thus, poziotinib appears selective in the liver stage assay but also may have a much more potent effect in continuously replicating cell lines.

To improve the clarity and flow of the manuscript, could the authors make a recapitulative table/figure for all the data obtained for poziotinib and hydrazinophtalazines in the different assays (8-days vs 12-days) and laboratory settings rather than separate tables in main and supplementary figures. Maybe also reorder the results section notably moving the 12-day assay before the DNA methylation part.

We apologize for the large amount of data presented but believe we are presenting it in the clearest way possible. All raw data is available if readers wish to re-analyze or re-organize our findings.

The isobologram plot shows an additive effect rather than a synergistic effect between cadralazine and 5-azacytidine, please modify the paragraph title accordingly. Please put the same axis scale for both fractional EC50 in the isobologram graph (Figure 2A).

The isobologram shows the effect approaching synergy at some combinations. The isobologram was rendered using standard methods. The raw data is available if readers wish to re-analyze it.

Concerning the immunofluorescence detection of 5mC and 5hmC, the authors should be careful with their conclusions. The Hoechst signal of the parasites is indistinguishable because of the high signal given by the hepatocyte nuclei. The signal obtained with the anti-5hmC in hepatocyte nuclei is higher than with the anti-5mC, thus if a low signal is obtained in hypnozoites and schizonts, it might be difficult to dissociate from the background. In blood stages (Figure S18), the best to obtain a good signal is to lyse the red blood cell using saponin, before fixation and HCl treatment.

We spent many hours using high resolution imaging of hundreds of parasites trying to detect clear 5hmC signal in both hypnozoites and schizonts but never saw a clearly positive signal. Indeed, the host signal can be confounding, thus we felt the most clear and unbiased way to quantify and present these data was using HCI. We appreciate the suggestion to lyse cells first for detecting in the blood stage.

To conclude that 5mC marks are the predominate DNA methylation mark in both P. falciparum and P. vivax, authors should also mention that they compare different stages of the life cycle, that might have different methylation levels.

We do mention at the start of this section our reasoning that quantifying marks in sporozoites was technically achievable, but not in a mixed culture of parasites and hepatocytes. We agree they could have different marks at these different stages.

Also, the authors conclude that "[...] 5mC is present at low level in P. vivax and P. cynomolgi sporozoites and could control liver stage development and hypnozoite quiescence". Based on the data shown here, nothing, except presence the of 5mC marks, supports that DNA methylation could be implicated in liver stage development or hypnozoite quiescence.

We clearly show sporozoite and liver stage DNA is methylated, which implicates this fundamental cell function exists in P. vivax liver stages, and that compounds with characterized activity against DNMT are active on liver stages. We acknowledge we were unable to show a direct effect and use the qualifier ‘could’ for this very reason.

How many DNA-methyltransferase inhibitors were present in the epigenetic library? Out of those, none were identified as hits, maybe the hydrazinophtalazines effect is not linked to DNMT inhibition but another target pathway of these molecules like calcium transport?

We supply the complete list of inhibitors in the epigenetic library as a supplemental file, the library contained 773 compounds. Hydrazinophtalazines were not included in the library, but several other DNA methyltransferase inhibitors were inactive. It is possible that hydrazinophtalazine activity is linked to other mechanisms but the inactivity of other DNMT inhibitors does not preclude the possibility hydrazinophtalazines are acting through DNMT.

The authors state (line 344): "These results corroborate our hypothesis that epigenetic pathways regulate hypnozoites". This conclusion should be changed to "[...] that epigenetic pathways are involved in P. vivax liver stage survival" because:

-The epigenetic inhibitors described here are as active on hypnozoite than liver schizonts.

-Again, we cannot rule out that the host cell plays a role in this effect and that the compound may not act directly on the parasite.

The same comment applies to the quote in lines 394 to 396. There is no proof in the results presented here that DNA methylation plays any role in the effect of hydrazinophtalazines in the anti-plasmodial activity obtained in the assay.

We maintain that we use words throughout the text that express uncertainty about the mechanisms involved. It is important to point out that, prior to this paper, the number of hypnozonticidal hits was incredibly low and this field is just emerging. The fundamental role of epigenetic mechanisms is regulation of gene expression. Finding several hypnozonticial hits when screening epigenetic libraries implies epigenetic pathways are important for hypnozoite survival. We intentionally do not specify exact mechanisms or if they are host or parasite pathways. Host-parasite interactions in the liver stage are incredibly difficult to resolve and are outside the scope of this report. Furthermore, this statement is not exclusive to schizonts, but since screens of diversity sets against schizonts result in a much higher hit rate, the focus of this comment is unearthing rare hypnozonticidal hits.

eLife Assessment

This study provides valuable insights into human valve development by integrating snRNA-seq and spatial transcriptomics to characterize cell populations and regulatory programs in the embryonic and fetal outflow tract. The methods, data, and analyses are solid overall, but with some weaknesses that can be strengthened. The findings will be of interest to those who work in the field of heart development and congenital heart disease.

Reviewer #1 (Public review):

Summary:

The study by Bobola et al reports single-nucleus expression analysis with some supporting spatial expression data of human embryonic and fetal cardiac outflow tracts compared to adult aortic valves. The transcription factor GATA6 is identified as a top regulator of one of the mesenchymal subpopulations, and potential interacting factors and downstream target genes are identified bioinformatically. Additional bioinformatic tools are used to describe cell lineage relationships and trajectories for developmental and adult cardiac cell types.

Strengths:

The studies of human tissue and extensive gene expression data will be valuable to the field.

Weaknesses:

(1) The expression data are largely confirmatory of previous studies in humans and mice. Thus, it is not clear what novel biological insights are being reported. While there is some novelty and impact in using human tissue, there are extensive existing publications and data sets in this area.

(2) Major conclusions regarding spatial localization, differential gene expression, or cell lineage relationships based on bioinformatic data are not validated in the context of intact tissues.

(3) The conclusions regarding lineage relationships are based on common gene expression in the current study and may not reflect cellular origins or lineage relationships that have previously been reported in genetic mouse models.

(4) An additional limitation is the exclusive examination of adult aortic valve leaflets that represent only a subset of outflow tract derivatives in the mature heart. The conclusion, as stated in the title regarding adult derivatives of the outflow tract, is not accurate based on the limited adult tissue evaluated, exclusive bioinformatic approach, and lack of experimental lineage analysis of cell origins.

Reviewer #2 (Public review):

Summary:

The manuscript by Leshem et al. presents a transcriptomic analysis of the developing human outflow tract (OFT) at embryonic and fetal stages using snRNAseq and spatial transcriptomics. Additionally, the authors analyze transcriptomic data from the adult aortic valve to compare embryonic and adult cell populations, aiming to identify persistent embryonic transcriptional signatures in adult cells. A total of 15 clusters were identified from the embryonic and fetal OFT samples, including three mesenchymal and four endothelial clusters. Using SCENIC analysis on the embryonic snRNAseq data, the authors identified GATA6 as a key regulator of valve precursor cells. Spatial transcriptomic analysis of four fetal OFT sections further revealed the spatial distribution of mesenchymal nuclei, smooth muscle cells, and valvular interstitial cells. Trajectory analysis identified two distinct developmental origins of fetal mesenchymal cells: the neural crest and the second heart field. Finally, the authors used snRNAseq data from the adult aortic valve to propose that embryonic transcriptional signatures persist in a subset of adult cells.

Strengths:

(1) The study offers a rich and detailed dataset, combining snRNA-seq and spatial transcriptomics in human embryonic and fetal OFT, which are challenging to obtain.

(2) The use of SCENIC and trajectory analysis adds mechanistic insight into cell lineage and regulatory programs during valve development.

(3) This study confirms GATA6 as a key regulator of valve precursor cells.

(4) Comparison between embryonic/fetal and adult datasets represents a novel attempt to trace persistence of developmental transcriptional programs.

Weaknesses:

(1) A major limitation is the lack of experimental validation to support key conclusions, particularly the claim of persistent embryonic transcriptional signatures in adult cells.

(2) The manuscript would benefit from a clearer discussion of how these results advance beyond previous studies in human heart and valve development.

(3) The comparison between embryonic and adult data is interesting, but would be more convincing with additional evidence supporting the proposed persistence of embryonic transcriptional signatures in adult cells.

Reviewer #3 (Public review):

Leshem et al have generated a transcriptional cell atlas of the human outflow tract at two developmental timepoints and its adult valvular derivatives. This carefully performed study provides a useful resource for the study of known genes implicated in outflow tract defects and potentially also for discovering new disease genes. The authors reveal neural crest and mesodermal contributions to different outflow tract components and show that GATA6, known to play a role in arterial valve development, controls a set of genes expressed in endocardium-derived cells during valve development. Interestingly, the results suggest lineage persistence of expression of certain genes through to the adult timepoint, a main new finding of this study.

The following points should be addressed to reinforce the conclusions and emphasize the novel features of this study.

(1) It would be helpful to clarify how these new findings confirm or diverge from what is known from analysis of neural crest and mesodermal lineage contributions to different cell populations in the mouse heart. Did the authors identify any human-specific populations of cells, such as the LGR5 population reported by Sahara et al?

(2) The authors should clarify in the introduction and results that they consider the endocardium to be on the SHF trajectory as indicated in Figure S4C. Please add a reference for this point.

(3) The GATA6 results are interesting and support this experimental approach. The paper would be reinforced if the authors could provide any functional validation (in addition to their GATA6 genomic occupancy data) that the designated target genes are regulated by GATA6. This might involve looking at mutant mouse embryos or cultured cells. Do the authors consider that GATA6 may regulate the endocardial to mesenchymal transition during the early stages of valve development? Or the valve interstitial cell versus fibroblast fate choice?

(4) Do the new findings reveal whether human valves have a direct SHF to VIC trajectory (ie, without transiting through endocardium) as has been recently shown in the murine non-coronary valve leaflet? Relevant to this point, Figure 5E appears to show contributions to a single adult aortic valve leaflet - this should be explained, or corrected.

Author response:

We thank the editors and reviewers for the time and effort they have invested in evaluating our manuscript. We appreciate the constructive feedback, which highlights both the strengths of the work and areas for improvement. We will carefully consider all comments and, in the coming months, revise the manuscript to incorporate additional data, address the concerns regarding limited referencing, and provide further clarification on the points raised.

eLife Assessment

This manuscript reports important findings that have theoretical or practical implications beyond a single subfield. However, despite the combination of numerous analytical tools established and applied in the study, the work has substantial experimental limitations leading to incomplete evidence, indicating that the conclusions may be an over-interpretation of the findings.

Reviewer #1 (Public review):

Summary:

In the study by Wang et al. entitled "Dissecting organoid-bacteria interaction highlights decreased contractile force as a key factor for heart infection", a simple cardiac organoid (CO) model was established, by combining a heterologous mixture of patient-specific human induced pluripotent stem cells (hiPSC)-derived cardiomyocytes (CMs) in combination with primary HUVECs (Human Umbilical Vein Endothelial Cells) and human mesenchymal stem cells (MSCs, representing stromal cells). This model was applied for investigating the interplay of COs' bacterial infections in vitro, aiming at revealing pathological mechanisms of bacterial infections of the heart in vivo, which may induce myocarditis and consequently heart failure in affected patients.

Strengths:

The paper is systematic, well written, and easy to follow.

Based on their results, the authors state that: "In this study, by developing quantitative tools for analyzing bacterial-cardiac organoid interactions in a 3D, dynamic, clinically relevant setting, we discovered the significant role of cardiac contractility in preventing bacterial infection."

In principle, the idea of establishing a simple yet functionally and physiologically relevant in vitro model and relevant analytical tools for enabling the study of complex pathological mechanisms of cardiovascular diseases is intriguing.

Weaknesses:

However, despite the combination of numerous analytical tools established and applied in the study, the work has substantial experimental limitations, indicating that the bold conclusions may represent a misinterpretation or overinterpretation of the findings.

Key limitations and questions:

(1) It seems that iPSCs from only one patient ("dilated cardiomyopathy (DCM) cells were derived from a 47-year-old Asian male with an LMNA gene mutation") were used in the study. Moreover, it seems that only one iPSC-line/clone from that DCM patient was used and compared to a single control iPSC line from a "healthy donor". Therefore, despite the different assays and experimental controls used in the study, there is a high risk that the observed phenomena reflect iPSC-line-/ clone-dependent effects, rather than revealing general pathophysiologic mechanisms. Thus, key experiments must be shown by cardiomyocytes/ cardiac organoids derived from additional independent iPSC-lines representing different patients and other non-diseased control lines as well. Moreover, it is established good experimental practice in the iPS cell field to generate and include isogenic iPSC controls i.e. iPSC lines of the same genetic background but with corrections of the hypothesised gene mutation underlying the respective e.g., cardiovascular disease.

(2) In Figure 1 (A) immunohistochemical staining for cardiomyocytes for the cardiac marker Troponin is shown, apparently indicating successful cardiomyogenic differentiation of the applied hiPSC lines. In supplemental Figure S1, a flow cytometry analysis specific to cTnT is shown to reveal the CMs content resulting from the monolayer differentiation of respective iPSC lines. Already, the exemplified plots indicate that the CMs' content/ purity for DCM-CMs was notably lower compared to healthy cardiomyocytes (CM; control). This is an important issue, since the non-CMs ("contaminating bystander cells") may have a substantial effect on the functional (including contractile) properties of the COs.

Interestingly, based on the method description, it seems that COs were generated from cryopreserved iPSC-CMs and iPSC-DCMs, including intermediate seeding and culture on Matrigel before COs formation. However, it remains unclear whether the CMs FACS analysis, which is apparently: "Representative FACS plots for analysis of the cell types in DCM monolayer culture after 33 days of differentiation" shows a CMs purity relevant to CO formation, or something different.

The lineage phenotype of non-CMs in respective differentiations should also be clarified. Moreover, it should be noted in the results that the CMs content in COs is lower than the 6:2:2 (CM:ECs:MSC) ratio indicated by the authors, since the CMs purity is not 100%, and is particularly reduced in the iPSC-DCMs.

Finally, to investigate the important latter questions of the "real CMs content" in COs, systematic technologies should be applied to quantify the lineage composition in COs (e.g. by IF staining for the 3 lineages plus DAPI, followed by COs clearance, confocal microscopy "3D stags" and automated, ImageJ-based quantitative cell counts for total cell number definition (see e.g. doi: 10.1038/s41596-024-00976-2) per CO, and quantification of respective lineage content as well.

These questions are of key importance since the presence of non-CMs and their phenotype has profound consequences on the cardiac organoid model, its contractile/ biophysical properties, and, in general, on models' sensitivity to bacterial infections as well.

(3) Figure 2: (F) Why is this figure (Confocal Observations) showing only healthy cardiac organoids (HCOs) but not DCM-COs?

The overall quality of these pictures is poor and not informative regarding the structural identity and tissue composition of the COs, which actually is an important topic in the frame of the paper, as the 3D structure and tissue composition - and differences between HCOs and DCM-COs - are of key importance to their contractile properties.

Moreover, the expective overlay of the cardiac markers alpha-actinin and MHC is not obvious from Figure 2F (see also comments on Figure 7, below).

In Figure 2E: COs at later stages/days should be shown, in particular at that stage, which was used for the functional assays i.e., bacteria infections and contraction pattern monitoring.

(4) Figure 7 (A) (B) - In the IF sections, it seems that there is no overlay between the expression of the cardiac marker MHC (seems to be expressed in the centre of COs only) and the cardiac markers alpha-actinin (which seems to be unexpectedly expressed in all cells on the sections) and Troponin (which seems to be vocally expressed on the outside, excluding the area of MHC expression).

(F) Quantification of the mean area of gene expression, e.g., for MHC indicates a larger area after MHC expression; this seems to entirely contradict the IF pictures (in Figures 7 A-D) of MHC expression before and after infection. This contraction is deemed very critical to this reviewer as it may indicate that the IF staining, data analysis, and/or data interpretation in this part of the manuscript is poor, misleading, or simply wrong.

(5) Overall, from the perspective of this reviewer, the CO-derived results do not reflect in a meaningful way the contractile and hydrodynamic conditions in the mouse heart or the human heart. Thus, it seems that the conclusions may rather represent a hypothesised outcome bias.

Reviewer #2 (Public review):

Summary:

The authors tried deconvoluting, for the first time, the effect of various components of heart contraction on initial bacterial adhesion, which increases the risk of infective endocarditis. The proposed organoid platform might be used to develop and test novel therapeutic agents for infective endocarditis.

Strengths:

(1) Use of a broad range of methods: finite element methods, -omics, particle tracking, animal experiments to investigate the connections between contractility and infective endocarditis.

(2) Detailed procedure and supportive information, which will allow other groups to replicate the results and extend the application of the proposed organoid platform.

(3) Despite the complexity of the work reported, the manuscript is rather readable and understandable by non-specialists.

Weaknesses:

There is a minor issue with some of the vocabulary (e.g., magnificent amount of bacteria).

eLife Assessment

This fundamental study provides new insights into the plasticity mechanisms underlying the formation of spatial maps in the hippocampus. Supported by a large and comprehensive dataset, the evidence is convincing. This study will be of interest to neuroscientists focusing on spatial navigation, learning, and memory.

Reviewer #1 (Public review):

Summary:

The authors aimed to investigate the cellular mechanisms underlying place field formation (PFF) in hippocampal CA1 pyramidal cells by performing in vivo two-photon calcium imaging in head-restrained mice navigating a virtual environment. Specifically, they sought to determine whether BTSP-like (behavioral time scale synaptic plasticity) events, characterized by large calcium transients, are the primary mechanism driving PFFs or if other mechanisms also play a significant role. Through their extensive imaging dataset, the authors found that while BTSP-like events are prevalent, a substantial fraction of new place fields are formed via non-BTSP-like mechanisms. They further observed that large calcium transients, often associated with BTSP-like events, are not sufficient to induce new place fields, indicating the presence of additional regulatory factors (possibly local dendritic spikes).

Strengths

The study makes use of a robust and extensive dataset collected from 163 imaging sessions across 45 mice, providing a comprehensive examination of CA1 place cell activity during navigation in both familiar and novel virtual environments. The use of two-photon calcium imaging allows the authors to observe the detailed dynamics of neuronal activity and calcium transients, offering insights into the differences between BTSP-like and non-BTSP-like PFF events. The study's ability to distinguish between these two mechanisms and analyze their prevalence under different conditions is a key strength, as it provides a nuanced understanding of how place fields are formed and maintained. The paper supports the idea that BTSP is not the only driving fore behind PFF, and other mechanisms are likely sufficient to drive PFF, and BTSP events may also be insufficient to drive PFF in some cases. The longer-than-usual virtual track used in the experiment allowed place cells to express multiple place fields, adding a valuable dimension to the dataset that is typically lacking in similar studies. Additionally, the authors took a conservative approach in classifying PFF events, ensuring that their findings were not confounded by noise or ambiguous activity.

Weaknesses

The stand out weakness of the paper is the lack of direct measures of BTSP events. Without direct confirmation that large calcium transients correspond to actual BTSP events (including associated complex spikes and calcium plateau potentials), concluding that BTSP is not necessary or sufficient for PFF formation is speculative (although I do believe it).

Reviewer #2 (Public review):

Summary:

The authors of this manuscript aim to investigate the formation of place fields (PFs) in hippocampal CA1 pyramidal cells. They focus on the role of behavioral time scale synaptic plasticity (BTSP), a mechanism proposed to be crucial for the formation of new PFs. Using in vivo two-photon calcium imaging in head-restrained mice navigating virtual environments, employing a classification method based on calcium activity to categorize the formation of place cells' place fields into BTSP, non-BTSP-like, and investigated their properties.

Strengths:

This work shows that place fields formation could induced by both BSTP and non-BSTP events, and it also provided a new and solid method to classify BTSP and non-BTSP place field formation using calcium image to the field. This work offers novel knowledge and new methods and factual evidence for other researchers in the field.

The method enabled the authors to reveal that while many PFs are formed by BTSP-like events, a significant number of PFs emerge with calcium dynamics that do not match BTSP characteristics, suggesting a diversity of mechanisms underlying PF formation. The characteristics of place fields under the first two categories are comprehensively described, including aspects such as formation timing, quantity, and width.

Weaknesses:

The authors have addressed the weaknesses in the revised version.

Reviewer #3 (Public review):

Summary:

In this manuscript, Sumegi et al. use calcium imaging in head-fixed mice to test whether new place fields tend to emerge due to events that resemble behavioral time scale plasticity (BTSP) or other mechanisms. An impressive dataset was amassed (163 sessions from 45 mice with 500-1000 neurons per sample) to study spontaneous emergence of new place fields in area CA1 that had the signature of BTSP. The authors observed that place fields could emerge due to BTSP and non-BTSP-like mechanisms. Interestingly, when non-BTSP mechanisms seemed to generate a place field, this tended to occur on a trial with a spontaneous reset in neural coding (a remapping event). Novelty seemed to upregulate non-BTSP events relative to BTSP events. Finally, large calcium transients (presumed plateau potentials) were not sufficient to generate a place field.

Strengths:

I found this manuscript to be exceptionally well written, well powered, and timely given the outstanding debate and confusion surrounding whether all place fields must arise from BTSP event. Working at the same institute, Albert Lee (e.g. Epszstein et al., 2011 - which should be cited) and Jeff Magee (e.g. Bittner et al., 2017) showed contradictory results for how place fields arise. These accounts have not fully been put toe-to-toe and reconciled in the literature. This manuscript addresses this gap and shows that both accounts are correct - place fields can emerge due to a pre-existing map and due to BTSP.

Weaknesses:

I find only three significant areas for improvement in the present study:

First, can it be concluded that non-BTSP events occur exclusively due to a global remapping event, as stated in the manuscript "these PFF surges included a high fraction of both non-BTSP- and BTSP-like PFF events, and were associated with global remapping of the CA1 representation"? Global remapping has a precise definition that involves quantifying the stability of all place fields recorded. Without a color scale bar in Figure 3D (which should be added), we cannot know whether the overall representations were independent before and after the spontaneous reset. It would be good to know if some neurons are able to maintain place coding (more often than expected by chance), suggestive of a partial-remapping phenomenon.

Second, BTSP has a flip side that involves weakening of existing place fields when a novel field emerges. Was this observed in the present study? Presumably place fields can disappear due to this bidirectional-BTSP or due to global remapping. For a full comparison of the two phenomena, the disappearance of place fields must also be assessed.

Finally, it would be good to know if place fields differ according to how they are born. For example, are there differences in reliability, width, peak rate, out of field firing, etc for those that arise due BTSP vs non-BTSP.

Comments on revisions:

The authors have mostly addressed my feedback. Compelling evidence for a fundamental observation.

Author response:

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

Reviewer #1 (Public review):

Summary:

The authors aimed to investigate the cellular mechanisms underlying place field formation (PFF) in hippocampal CA1 pyramidal cells by performing in vivo two-photon calcium imaging in head-restrained mice navigating a virtual environment. Specifically, they sought to determine whether BTSP-like (behavioral time scale synaptic plasticity) events, characterized by large calcium transients, are the primary mechanism driving PFFs or if other mechanisms also play a significant role. Through their extensive imaging dataset, the authors found that while BTSP-like events are prevalent, a substantial fraction of new place fields are formed via non-BTSP-like mechanisms. They further observed that large calcium transients, often associated with BTSP-like events, are not sufficient to induce new place fields, indicating the presence of additional regulatory factors (possibly local dendritic spikes).

Strengths

The study makes use of a robust and extensive dataset collected from 163 imaging sessions across 45 mice, providing a comprehensive examination of CA1 place-cell activity during navigation in both familiar and novel virtual environments. The use of two-photon calcium imaging allows the authors to observe the detailed dynamics of neuronal activity and calcium transients, offering insights into the differences between BTSP-like and non-BTSP-like PFF events. The study's ability to distinguish between these two mechanisms and analyze their prevalence under different conditions is a key strength, as it provides a nuanced understanding of how place fields are formed and maintained. The paper supports the idea that BTSP is not the only driving force behind PFF, and other mechanisms are likely sufficient to drive PFF, and BTSP events may also be insufficient to drive PFF in some cases. The longer-than-usual virtual track used in the experiment allowed place cells to express multiple place fields, adding a valuable dimension to the dataset that is typically lacking in similar studies. Additionally, the authors took a conservative approach in classifying PFF events, ensuring that their findings were not confounded by noise or ambiguous activity.

Weaknesses

Despite the impressive dataset, there are several methodological and interpretational concerns that limit the impact of the findings. Firstly, the virtual environment appears to be poorly enriched, relying mainly on wall patterns for visual cues, which raises questions about the generalizability of the results to more enriched environments. Prior studies have shown that environmental enrichment can significantly influence spatial coding, and it would be important to determine how a more immersive VR environment might alter the observed PFF dynamics. Secondly, the study relies on deconvolution methods in some cases to infer spiking activity from calcium signals without in vivo ground truth validation. This introduces potential inaccuracies, as deconvolution is an estimate rather than a direct measure of spiking, and any conclusions drawn from these inferred signals should be interpreted with caution. Thirdly, the figures would benefit from clearer statistical annotations and visual enhancements. For example, several plots lack indicators of statistical significance, making it difficult for readers to assess the robustness of the findings. Furthermore, the use of bar plots without displaying underlying data distributions obscures variability, which could be better visualized with violin plots or individual data points. The manuscript would also benefit from a more explicit breakdown of the proportion of place fields categorized as BTSP-like versus non-BTSP-like, along with clearer references to figures throughout the results section. Lastly, the authors' interpretation of their data, particularly regarding the sufficiency of large calcium transients for PFF induction, needs to be more cautious. Without direct confirmation that these transients correspond to actual BTSP events (including associated complex spikes and calcium plateau potentials), concluding that BTSP is not necessary or sufficient for PFF formation is speculative.

Reviewer #2 (Public review):

Summary:

The authors of this manuscript aim to investigate the formation of place fields (PFs) in hippocampal CA1 pyramidal cells. They focus on the role of behavioral time scale synaptic plasticity (BTSP), a mechanism proposed to be crucial for the formation of new PFs. Using in vivo two-photon calcium imaging in head-restrained mice navigating virtual environments, employing a classification method based on calcium activity to categorize the formation of place cells' place fields into BTSP, non-BTSP-like, and investigated their properties.

Strengths:

A new method to use calcium imaging to separate BTSP and non-BTSP place field formation. This work offers new methods and factual evidence for other researchers in the field.

The method enabled the authors to reveal that while many PFs are formed by BTSP-like events, a significant number of PFs emerge with calcium dynamics that do not match BTSP characteristics, suggesting a diversity of mechanisms underlying PF formation. The characteristics of place fields under the first two categories are comprehensively described, including aspects such as formation timing, quantity, and width.

Weaknesses:

There are some issues about data and statistics that need to be addressed before these research findings can be considered as rigorous conclusions.

While the authors mentioned 3 features of PF generated by BTSP during calcium imaging in the Introduction, the classification method used features 1 and 2. The confirmation by feature 3 in its current form is important but not strong enough.

Some key data is missing such as the excluded PFs, the BTSP/non-BTSP of each animal, etc

Impact:

This work is likely to provide a new method to classify BTSP and non-BTSP place field formation using calsium image to the field.

Reviewer #3 (Public review):

Summary:

In this manuscript, Sumegi et al. use calcium imaging in head-fixed mice to test whether new place fields tend to emerge due to events that resemble behavioral time scale plasticity (BTSP) or other mechanisms. An impressive dataset was amassed (163 sessions from 45 mice with 500-1000 neurons per sample) to study the spontaneous emergence of new place fields in area CA1 that had the signature of BTSP. The authors observed that place fields could emerge due to BTSP and non-BTSP-like mechanisms. Interestingly, when non-BTSP mechanisms seemed to generate a place field, this tended to occur on a trial with a spontaneous reset in neural coding (a remapping event). Novelty seemed to upregulate non-BTSP events relative to BTSP events. Finally, large calcium transients (presumed plateau potentials) were not sufficient to generate a place field.

Strengths:

I found this manuscript to be exceptionally well-written, well-powered, and timely given the outstanding debate and confusion surrounding whether all place fields must arise from BTSP event. Working at the same institute, Albert Lee (e.g. Epszstein et al., 2011 - which should be cited) and Jeff Magee (e.g. Bittner et al., 2017) showed contradictory results for how place fields arise. These accounts have not fully been put toe-to-toe and reconciled in the literature. This manuscript addresses this gap and shows that both accounts are correct - place fields can emerge due to a pre-existing map and due to BTSP.

We thank the Reviewer for his/her appreciation of the importance of our study. We have included the additional reference.

Weaknesses:

I find only three significant areas for improvement in the present study:

First, can it be concluded that non-BTSP events occur exclusively due to a global remapping event, as stated in the manuscript "these PFF surges included a high fraction of both non-BTSP- and BTSP-like PFF events, and were associated with global remapping of the CA1 representation"? Global remapping has a precise definition that involves quantifying the stability of all place fields recorded. Without a color scale bar in Figure 3D (which should be added), we cannot know whether the overall representations were independent before and after the spontaneous reset. It would be good to know if some neurons are able to maintain place coding (more often than expected by chance), suggestive of a partial-remapping phenomenon.

We have performed the analysis suggested by the Reviewer and determined what fraction of CA1PCs retained its original tuning property after the representation switch. We found that the remapping was essentially global, as only a small fraction (5.4%) of CA1PCs retained their pre-switch tuning curve after the switch. This is now described in the Results.

We now state in the figure legend for the former Figure 3D (now Figure 3F) that the color scale applies to all subpanels.

We would like to note that we do not conclude that non-BTSP events occur exclusively during global remapping – we have found a sizable fraction of PFF by non-BTSP mechanism also in the familiar environment with no signs of change in the population representation. We agree nonetheless that PFF is dominated by BTSP under these conditions, whereas the contribution of non-BTSP is larger during global remapping events.

Second, BTSP has a flip side that involves the weakening of existing place fields when a novel field emerges. Was this observed in the present study? Presumably place fields can disappear due to this bidirectional BTSP or due to global remapping. For a full comparison of the two phenomena, the disappearance of place fields must also be assessed.

In this study we focused on the birth of new PFs – yet, PFs not only form but also disappear constantly. The factors driving PF weakening are even less explored and understood than those driving PF birth. In fact, we observed (as illustrated by several examples in our MS) that many PFs weaken, or disappear completely during the course of an imaging session. These effects are sometimes accompanied by a new PFF event elsewhere (e.g. Figure 2 – figure supplement 2E bottom), whereas in other cases they are not (e.g. Figure 5A, middle). Similarly, some BTSP events seem to coincide with disappearance of another PF, but others are not (e.g. Figure 2A bottom, first PF along the track; Figure 3 – figure supplement 1A left, first PF). The picture is further complicated in the case of global remapping events (i.e. representation switches, Figure 3 – figure supplement 2B) that, by definition, include both new PFF and PF disappearance. We feel that exploration of the complex mechanisms at play in PF disappearance is outside the scope of the current study, but could be the subject of an interesting future investigation.

Finally, it would be good to know if place fields differ according to how they are born. For example, are there differences in reliability, width, peak rate, out-of-field firing, etc for those that arise due to BTSP vs non-BTSP.

We have analyzed several properties of the PFs and found no significant difference in either their width (BTSP: 46.4 ± 24.4 cm; non-BTSP: 50.4 ± 32.5 cm, p = 0.28) or peak rates (BTSP: 19.0 ± 14.7 a.u./s; non-BTSP: 21.4 ± 16.8 a.u./s, p = 0.27) or the out-of-field firing rates (BTSP: 0.64 ± 0.68 a.u./s; non-BTSP: 0.83 ± 1.25 a.u./s, p = 0.09, all unpaired t-test). We have included these data into the Results section.

Reviewer #1 (Recommendations for the authors):

Consider adding additional visual cues or environmental elements to the virtual reality (VR) setup to create a more enriched and immersive environment. Collect data from a couple of mice in the enriched environment and compare the PFF dynamics to the original environment. This would help determine whether the findings on PFF dynamics hold in a setting where spatial coding may be more robust. Including floor cues, distal visual markers, or varying textures might provide a more comprehensive understanding of the factors influencing BTSP-like and non-BTSP-like events.

We thank the Reviewer for her/his suggestion of analyzing data obtained from a more enriched VR environment compared to the one we used in our study. We have now included data obtained in a profoundly different VR environment, which did not have sparse dominant visual landmarks, but the entire wall was covered with a rich pattern with different shapes of different colors. Our data from 11 imaging sessions from 4 mice revealed BTSP- and non-BTSP-like PFF events with approximately the same ratio to that found in our regular maze. These results are described in the Results section and are presented in a new supplementary figure (Figure 2 – figure supplement 2). 

Wherever deconvolved spikes were used for analysis, provide a comparison of results obtained directly from the GCaMP ΔF/F signals versus those derived from the deconvolved spiking data. This could illustrate any differences and help readers understand the limitations and reliability of the inference method.

We have adopted a currently widely accepted method in the field to infer spikes from fluorescent traces using the Suite2p software package. All of our analyses were then performed on the inferred spikes. To address the concerns of the Reviewer, we analyzed the relationship between the peak [Ca²⁺] transients and inferred spike activity (new Figure 3 – figure supplement 1C-E). Our results clearly demonstrate a robust, highly significant correlation between these measures at the level of individual cells (new Figure 3 – figure supplement 1D) and the Spearman correlation coefficients show a distribution that is very different from random distributions (new Figure 3 – figure supplement 1E). From these, we conclude that using directly the fluorescent data would have resulted in largely similar PF detection and identification.

Improve the visual clarity of figures by enlarging key elements such as arrows that indicate BTSP-like events. Consider using colors that stand out more clearly to guide readers' attention. Include annotations of statistical significance directly on the figures (e.g., adding NS or * indicators) to make it clear which comparisons are statistically significant. This will help readers quickly interpret the data without needing to refer back to the text.

Based on the suggestion of the Reviewer, we have enlarged the arrows. We have also indicated statistical results on the figures. Because some of the results of factorial ANOVA tests are difficult to be comprehensively indicated on our plots, we kept the description of the statistical results in the legends as well. We hope that these alterations will make data interpretation easier.

Replace or supplement bar plots with violin plots or scatter plots that show the distribution of individual data points. This change would offer a clearer picture of data variability and underlying trends, aiding readers in assessing the robustness of the results.

We have changed the plots and now present all data points.

Add more detailed quantification in the results section, specifying the total number of newly formed place fields, the proportion that are categorized as BTSP-like versus non-BTSP-like, and how many events did not fit these categories. Explicitly state what fraction of the total recorded place field formations are represented by the 59 non-BTSP-like events mentioned, as this is currently difficult to discern.

The number of BTSP- and non-BTSP-like PFF events are given in the MS. As described in the Methods, after identifying BTSP- and non-BTSP-like PFF events using the shift and gain criteria, we have manually checked each of these ROIs and the spatial footprint of every new PFF events for these cells and excluded ROIs with non-soma-like shapes and activities with spurious footprints suggesting contamination, creating a ‘cleaned’ dataset. We did not perform such visual inspection and manual curation of every ROI’s spatial footprints that belong to the two additional categories (no gain with shift, gain without shift, 872 events). Since these classes are also overestimated without curation, we cannot provide a precise fraction of the BTSP- and non-BTSP-like PFF events from the total recorded PFF population. However, - assuming that factors leading to exclusion affect all groups equally - we can provide their fractions by comparing the numbers of newly born PFs in all categories before the visual inspections. In the normal maze, we found 806 candidate BTSP-like (52%),164 non-BTSP-like (10%) PFFs and an additional 593 PFs (38%) could not be included in these two groups [40 PFs (3%) with formation lap gain and backward shift but significant backward drift; 238 PFs (15%) with formation lap gain but without backward shift; 315 PFs (20%) with no formation lap gain but with backward shift]. These data have been included in the Methods.

Ensure that all statements describing specific findings are consistently linked to the appropriate figures and panels. There are instances in the text where results are discussed without clear references, which can make it challenging for readers to verify the data. For example, the section on population remapping in a novel environment should point directly to the relevant figure panels to guide readers.

We regret that our text was not linked properly to the appropriate figures. We corrected this during the revision.

Given that BTSP-like events are inferred rather than directly confirmed, it would be prudent to frame conclusions about their sufficiency in more tentative terms, acknowledging the limitations of the current data. Consider adding a discussion of potential future experiments that could confirm whether these large transients truly represent BTSP events, including evidence for complex spikes or calcium plateau potentials.

The Reviewer is correct that we do not have direct evidence that all large somatic Ca²⁺ events represent dendritic plateau potentials. Now we discuss this and other limitations in the MS (Discussion section).

Reviewer #2 (Recommendations for the authors):

Although the author has outlined three characteristics of place fields (PFs) generated by behavioral time scale synaptic plasticity (BTSP) during calcium imaging in the Introduction section, as follows: ' First, the prolonged CSB results in large [Ca²⁺] transient during the initial PFF event, typically followed by weaker Ca2+ signals on consecutive traversals through the PF. Second, due to the long and asymmetric temporal kernel of the plasticity (favoring potentiation of inputs active 1-2 seconds before the CSB) a substantial backward shift in the spatial position of the PF center can be observed on linear tracks after the formation lap. Third, the width of the new PF is generally proportional to the running speed of the animal during the PFF event.' Figure 3B, which displays the third feature of classified BTSP and non-BTSP data, serves as an important confirmation of the classification results using the first two features. Even though the Spearman correlation indicated a significant difference, the raw data distributions of BTSP and non-BTSP appear similar, suggesting that a distribution of bootstrap and more stringent confirmation should be conducted to be convincing.

As described in the MS, because of the difference in the number of events in the two groups, we randomly subsampled the BTSP-like events to the sample size of the non-BTSP-like PFF events 10000 times and performed regression analysis. This bootstrapping revealed that both the r and p values of the fit to the non-BTSP data fell outside the 95% confidence interval of the bootstrapped BTSP values, indicating that the difference between the groups was robust.

In further analysis during the revision, we found that the PF width variance explained by distance from landmarks is substantially larger than the variance explained by the running speed during the formation lap. We performed a cross-validated analysis by these two factors (Figure 3D), which highlights that speed explains some of the PF width variance of BTSP-like PFFs, but none of the non-BTSP PFFs.

The proportions of the three types should be provided. page 6: ' Using a conservative approach, we categorized a new PF to be formed by a BTSP-like mechanism if it had both positive gain and negative shift values (Figure 2A; n = 310 new PFs), whereas new PFs exhibiting neither positive gain nor negative shift were considered as non-BTSP-like events (Figure 2B; n = 59). All other newly formed PFs (no-gain with backward shift and gain without backward shift) were excluded from further analysis.' The number of excluded newly formed PFs should be disclosed, as well as the distribution ratio of these three types in each animal.

The number of BTSP- and non-BTSP-like PFF events are given in the MS. As described in the Methods, after identifying BTSP- and non-BTSP-like PFF events using the shift and gain criteria, we have manually checked each of these ROIs and the spatial footprint of every new PFF events for these cells and excluded ROIs with non-soma-like shapes or spurious activities, creating a ‘cleaned’ dataset. We did not perform such visual inspection and manual curation of every ROI’s spatial footprints that belonged to the two additional categories (no gain with shift, gain without shift, 872 events). Since these classes are also overestimated without curation, we cannot provide a precise fraction of the BTSP- and non-BTSP-like PFF events from the total recorded PFF population. However, - assuming that factors leading to exclusion affect all groups equally - we can provide their fractions by comparing the numbers of newly born PFs in all categories before the visual inspections. In the normal maze, we found 806 candidate BTSP-like (52%),164 non-BTSP-like (10%) PFFs and an additional 593 PFs (38%) could not be included in these two groups [40 PFs (3%) with formation lap gain and backward shift but significant backward drift; 238 PFs (15%) with formation lap gain but without backward shift; 315 PFs (20%) with no formation lap gain but with backward shift]. These data have been included in the Methods.

Figure 2C, while showing an overall decrease in amplitude from the formation lap to the next lap, could benefit from a pairwise analysis of the corresponding formation lap and the following lap of each session to provide more convincing and detailed results.

We now present all data with connected lines across consecutive laps to illustrate the changes in each ROI. Our statistical analysis included the pairwise comparison of amplitudes.

The experiment's time range is broad (11-99 days); it is worth investigating whether different training intervals might influence the results.

Based on the suggestion of the Reviewer, we have analyzed the elapsed time and the number of sessions from the first training to the recording, and we demonstrate that there is no correlation of these parameters with the number of new PFFs. These data are now presented in Figure 2 – figure supplement 1C.

It is unclear whether the formation of place fields also generates characteristic features of dendritic properties.

It is not clear to us which ‘characteristic dendritic features of dendritic properties’ generated by PFF the Reviewer refers to. Since we did not image dendrites of individual CA1PCs, we have no information about dendritic properties of the neurons.

It may be necessary to add a clearer figure to illustrate the correlation between width and speed following the downsampling of non-BTSP-like events (refer to Figure 3B).

We have performed extensive additional analysis on the relationship of PF width with various behavioral factors, including the speed of the animal in the formation lap. Inspection of the PF width distributions along the track revealed a close association of PF width with the distance of the animal from the nearest visual landmark in the corridor, so that PFs close to landmarks were narrower than PFs between landmarks. We found that the PF width variance explained by distance from landmarks is substantially larger than the variance explained by the running speed during the formation lap. Nevertheless, there is a clear difference between BTSP-like and non-BTSP-like PFFs: running speed explains some variance in the case of BTSP-like PFFs, but none for non-BTSP-like PFFs.

We have included these findings into the Results section and created two new panels in Figure 3 (C, D) and Figure 3 – figure supplement 1 (A, B).

It is recommended that statistical results be labeled in the figures with n.s. or stars for better readability.

Based on the suggestion of the Reviewer, we have indicated statistical results on the figures. Because some of the results of factorial ANOVA tests are difficult to be comprehensively indicated on our plots, we kept the description of the statistical results in the legends as well. We hope that these alterations will make data interpretation easier. We hope that these alterations will make data interpretation easier.

eLife Assessment

This manuscript describes a useful study describing an interesting infection phenotype that differs between adult male and female zebrafish. The authors argue that male-biased expression of Cyp17a2 is implicated in mediating infection levels through STING and USP8 activity regulation. Thus, this study highlights an unexpected factor involved in antiviral immunity that could open new avenues of investigation for infection, metabolism, and other contexts. Although the manuscript presents some evidence supporting its main claims, the evidence for the main argument made in the study on sex dimorphism remains incomplete at this stage.

Reviewer #1 (Public review):

Summary:

In this manuscript, Lu & Cui et al. observe that adult male zebrafish are more resistant to infection and disease following exposure to Spring Viremia of Carp Virus (SVCV) than female fish. The authors then attempt to identify some of the molecular underpinnings of this apparent sexual dimorphism and focus their investigations on a gene called cytochrome P450, family 17, subfamily A, polypeptide 2 (cyp17a2) because it was among the genes that they found to be more highly expressed in kidney tissue from males than in females. Their investigations lead them to propose a direct connection between cyp17a2 and modulation of interferon signaling as the key underlying driver of the difference between male and female susceptibility to SVCV.

Strengths:

Strengths of this study include the interesting observation of a substantial difference between adult male and female zebrafish in their susceptibility to SVCV, and also the breadth of experiments that were performed linking cyp17a2 to infection phenotypes and molecularly to the stability of host and virus proteins in cell lines. The authors place the infection phenotype in an interesting and complex context of many other sexual dimorphisms in infection phenotypes in vertebrates. This study succeeds in highlighting an unexpected factor involved in antiviral immunity that will be an important subject for future investigations of infection, metabolism, and other contexts.

Weaknesses:

Weaknesses of this study include an indirect connection between the majority of experiments and the proposed mechanism underlying the sexual dimorphism phenotype, widespread reliance on over-expression when investigating protein-protein interaction and localization, and an insufficient amount of description of the data presented in the figures. Specific examples of areas for clarification or improvement include:

(1) Figure 10 outlines a mechanistic link between cyp17a2 and the sexual dimorphism the authors report for SVCV infection outcomes. The data presented on increased susceptibility of cyp17a2-/- mutant male zebrafish support this diagram, but this conclusion is fairly weak without additional experimentation in both males and females. The authors justify their decision to focus on males by stating that they wanted to avoid potential androgen-mediated phenotypes in the cpy17a2 mutant background (lines 152-156), but this appears to be speculation. It also doesn't preclude the possibility of testing the effects of increased cyp17a2 expression on viral infection in both males and females. This is of critical importance if the authors intend to focus the study on sexual dimorphism, which is how the introduction and discussion are currently structured.

(2) The authors present data indicating an unexpected link between cyp17a2 and ubiquitination pathways. It is unclear how a CYP450 family member would carry out such activities, and this warrants much more attention. One brief paragraph in the discussion (starting at line 448) mentions previous implications of CYP450 proteins in antiviral immunity, but given that most of the data presented in the paper attempt to characterize cyp17a2 as a direct interactor of ubiquitination factors, more discussion in the text should be devoted to this topic. For example, are there any known domains in this protein that make sense in this context? Discussion of this interface is more relevant to the study than the general overview of sexual dimorphism that is currently highlighted in the discussion and throughout the text.

(3) Figures 2-9 contain information that could be streamlined to highlight the main points the authors hope to make through a combination of editing, removal, and movement to supplemental materials. There is a consistent lack of clarity in these figures that could be improved by supplementing them with more text to accompany the supplemental figures. Using Figure 2 and an example, panel (A) could be removed as unnecessary, panel (B) could be exchanged for a volcano plot with examples highlighting why cyp17a2 was selected for further study and also the full dataset could be shared in a supplemental table, panel (C) could be modified to indicate why that particular subset was chosen for plotting along with an explanation of the scaling, panel (D) could be moved to supplemental because the point is redundant with panels (A) and (C), panel (E) could be presented as a heatmap, in panels (G) and (H) data from EPC cells could be moved to supplemental because it is not central to the phenotype under investigation, panels (J) to (L) and (N) to (P) could be moved to supplemental because they are redundant with the main points made in panels (M) and (Q). Similar considerations could be made with Figures 3-9

(4) The data in Figure 3 (A)-(C) do not seem to match the description in the text. That is, the authors state that cyp17a2 overexpression increases interferon signaling activity in cells, but the figure shows higher increases in vector controls. Additionally, the data in panel (H) are not described. What genes were selected and why, and where are the data on the rest of the genes from this analysis? This should be shared in a supplemental table.

(5) Some of the reagents described in the methods do not have cited support for the applications used in the study. For example, the antibody for TRIM11 (line 624, data in Figures 6 & 7) was generated for targeting the human protein. Validation for use of this reagent in zebrafish should be presented or cited. Furthermore, the accepted zebrafish nomenclature for this gene would be preferred throughout the text, which is bloodthirsty-related gene family, member 32.

Reviewer #2 (Public review):

The manuscript identified Cyp17a2 as a master regulator of male-biased antiviral immunity in a sex chromosome-free model (zebrafish) challenging established immunological paradigms.

Strengths:

(1) The bifunctional role of Cyp17a2 (host-directed STING stabilization and virus-directed P degradation) represents a significant conceptual advance.

(2) First demonstration of K33 chains as a critical regulatory switch for both host defense proteins and viral substrates.

(3) Comprehensive validation across biological scales: organismal (survival, histopathology), cellular (transcriptomics, Co-IPs), and molecular (ubiquitination assays, site-directed mutagenesis).

(4) Functional conservation in cyprinids (zebrafish and gibel carp) strengthens biological significance.

Weaknesses:

(1) Colocalization analyses (Figures 4G, 6I, 9D) require quantitative metrics (e.g., Pearson's coefficients) rather than representative images alone.

(2) Figure 1 survival curves need annotated statistical tests (e.g., "Log-rank test, p=X.XX")

(3) Figure 2P GSEA should report exact FDR-adjusted *p*-values (not just "*p*<0.05").

(4) Section 2 overextends on teleost sex-determination diversity, condensing to emphasize relevance to immune dimorphism would strengthen narrative cohesion.

(5) Limited discussion on whether this mechanism extends beyond Cyprinidae and its implications for teleost adaptation.

eLife Assessment

The study by Reed et al. provides fundamental findings and convincing evidence defining the topological changes that occur during tumorigenesis. The findings enhance the understanding of stable long-range connections among genes that reprogram cancer-related functions. Nevertheless, performing additional experiments is recommended.

Reviewer #1 (Public review):

Summary:

In their manuscript, Metz Reed and colleagues present an exceptionally thorough analysis of three-dimensional genome reorganization during breast cancer progression using the well-characterized MCF10 model system. The integration of high-resolution Micro-C contact maps with multi-omics profiling provides compelling insights into stage-specific dynamics of chromatin compartments, TAD boundaries, and looping events. The discovery that stable chromatin loops enable epigenetic reprogramming of cancer genes, while structural changes selectively drive metastasis-associated pathways, represents a significant conceptual advance. This work substantially deepens our understanding of genome topology in malignancy. To further enhance this impactful study, we offer the following constructive suggestions.

Strengths:

This work sets a benchmark for integrative 3D genomics in oncology. Its methodological sophistication and conceptual advances establish a new paradigm for studying nuclear architecture in disease.

Weaknesses:

Major Issues

(1) Functional tests would strengthen the observed links between structure and gene changes. For example, the COL12A1 gene loop formation correlates with its increased expression. Disrupting this loop using CRISPR-dCas9 at chr6 position 75280 kb could prove whether the loop causes COL12A1 activation. Such experiments would turn strong correlations into clear mechanisms.

(2) The H3K27ac looping idea needs deeper validation. Data suggests H3K27ac loss weakens loops without affecting CTCF. Testing how cohesin proteins interact with H3K27ac-modified sites would clarify this process. Degron systems could rapidly remove H3K27ac to observe real-time effects. Also, the AP-1 motifs found at dynamic loop sites deserve functional tests. Knocking down AP-1 factors might show if they control loop formation.

(3) Connecting findings to patient data would boost clinical relevance. The MCF10 model is excellent for controlled studies. Checking if TAD boundary weakening occurs in actual patient metastases would show real-world importance. Comparing primary and metastatic tumor samples from the same patients could reveal new structural biomarkers. If tissue is scarce, testing cancer cells with added stroma cells might mimic tumor environment effects.

Minor Issues

Adding a clear definition for static loops would help readers. For example, state that static loops show less than 10 percent contact change across replicates. In the ABC model analysis, removing promoter regions from the enhancer list would focus results on true long-range interactions. Briefly noting why this study sees TAD weakening while other cancer types show different patterns would provide useful context.

Reviewer #2 (Public review):

Employing the MCF10 breast-cancer progression series, the authors integrate high-resolution Micro-C chromatin-conformation capture with RNA-seq and ChIP-seq to delineate the sequential reorganization of compartments, topologically associated domains (TADs), and long-range loops across benign, pre-neoplastic, and metastatic states, and couple these 3D alterations to gene expression and enhancer activity. Four principal findings emerge: (i) largely static chromatin frameworks still gate differential gene output, with up-regulated loci most affected; (ii) enhancer-promoter contact strength covaries with transcriptional amplitude; (iii) 127 genes gain expression concomitant with increased chromatin contacts; and (iv) progression-associated genes acquire altered histone marks at distal enhancers that remain tethered by stable loops. While the conclusions are broadly supported, methodological and analytical refinements are required.

(1) Model representativeness.<br /> The long-term culture-adapted MCF10 genome harbours extensive aneuploidies and translocations. Validation of key COL12A1/WNT5A loop dynamics in an independent breast-cancer line (e.g., MDA-MB-231, T47D) or in patient-derived organoids/PDX models would strengthen generalizability.

(2) The study remains purely correlative; no perturbation experiments are conducted to demonstrate causal roles of chromatin loops on gene expression. CRISPR interference (CRISPR-Cas9-KRAB/HDAC) or enhancer deletion/inversion should be applied to 3-5 pivotal loops (e.g., COL12A1, WNT5A) to test their impact on target-gene expression and cellular phenotypes (e.g., proliferation, migration).

(3) The manuscript lacks integration with clinical datasets. Integrate TCGA-BRCA data to assess whether elevated COL12A1/WNT5A expression associates with overall survival (OS) or distant metastasis-free survival (DMFS).

Reviewer #3 (Public review):

Summary:

The authors tackle an important problem: defining the topological changes that occur during tumorigenesis. To study this, they use an established stepwise cell model of breast cancer. A strength of their study is a careful, robust differential analysis of topological features across each cell state, which is presented clearly and rigorously. They define changes in compartmentalization, TAD structure, and chromatin looping. Intriguingly, when the authors integrate differential gene expression with chromatin looping, they see that most differentially regulated genes are not involved in loop changes, suggesting that changes in promoter or enhancer chromatin marks may play a bigger role in regulating transcription than differential loops. The differential topology analysis and its integration with transcription is very well done- one of the best versions of this I have read in the 3D genome field! However, the paper is framed largely as a cancer biology study, and it teaches us much less about this. I am worried that some of the trends for each topologic feature are not going to be consistent across the pre-malignant-malignant-metastatic spectrum and would like the authors to soften some of their claims a bit regarding how this clarifies our understanding of cancer evolution.

Weaknesses:

Major Concerns:

(1) The integration of gene expression and chromatin loops is intriguing. The authors' differential analysis, however, omits consideration of genes that are on and simply further upregulated versus genes that transition on/off or off/on. It would be nice to see the authors break out looping patterns for these two different patterns of regulation, as it may be instructive regarding the rules for how EP loops govern transcription.

(2) Given the paucity of differential loops at the majority of genes whose expression changes, the authors should examine chromatin subcompartments, as these may associate more with differential transcription.

(3) The authors could push their TAD analysis further by integrating it with transcription. Can they look at genes and their enhancers that span these altered boundaries to see if these shifts impact transcription?

(4) The progression of cancer critically goes from a benign -> pre-malignant -> malignant -> metastatic series of steps. The AT1 line is described as 'premalignant' and thus the authors' series omits a malignant line. While I think adding such a sample is an unreasonable request at this point (as it would have had to have been studied in 'batch' with these other samples), the authors should acknowledge that they omit this step and spend some time discussing the genetic, morphologic, and phenotypic features for their 3 conditions. The images in Figure 1S aren't particularly useful- they don't tell the reader that these cells are malignant/benign. The karyotypic data are intriguing but not fully analyzed, so it is hard to know what true phenotype these cells represent. For example, malignant means DCIS/invasive carcinoma - so then what does this pre-malignant cell model represent? The described alteration in the AT1 line is a Ras oncogene, so in some sense, the transition to this line really is just +/- Ras. The authors could spend some time thinking about the effects of Ras specifically on the 3D genome.

Kathryn Zeiler is co-Editor-in-Chief of MetaROR working with Ludo Waltman, a co-author of the article and co-Editor-in-Chief of MetaROR

The authors present a descriptive analysis of preprint review services. The analysis focuses on the services’ relative characteristics and differences in preprint review management. The authors conclude that such services have the potential to improve the traditional peer review process. Two metaresearchers reviewed the article. They note that the background section and literature review are current and appropriate, the methods used to search for preprint servers are generally sound and sufficiently detailed to allow for reproduction, and the discussion related to anonymizing articles and reviews during the review process is useful. The reviewers also offered suggestions for improvement. They point to terminology that could be clarified. They suggest adding URLs for each of the 23 services included in the study. Other suggestions include explaining why overlay journals were excluded, clarifying the limitation related to including only English-language platforms, archiving rawer input data to improve reproducibility, adding details related to the qualitative text analysis, discussing any existing empirical evidence about misconduct as it relates to different models of peer review, and improving field inclusiveness by avoiding conflation of “research” and “scientific research.”

The reviewers and I agree that the article is a valuable contribution to the metaresearch literature related to peer review processes.

This manuscript examines preprint review services and their role in the scholarly communications ecosystem.  It seems quite thorough to me. In Table 1 they list many peer-review services that I was unaware of e.g. SciRate and Sinai Immunology Review Project.

To help elicit critical & confirmatory responses for this peer review report I am trialling Elsevier’s suggested “structured peer review” core questions, and treating this manuscript as a research article.

Introduction

1. Is the background and literature section up to date and appropriate for the topic?

   Yes.

2. Are the primary (and secondary) objectives clearly stated at the end of the introduction?

   No. Instead the authors have chosen to put the two research questions on page 6 in the methods section. I wonder if they ought to be moved into the introduction – the research questions are not methods in themselves. Might it be better to state the research questions first and then detail the methods one uses to address those questions afterwards? [as Elsevier’s structured template seems implicitly to prefer.

Methods

1. Are the study methods (including theory/applicability/modelling) reported in sufficient detail to allow for their replicability or reproducibility?

   I note with approval that the version number of the software they used (ATLAS.ti) was given.

   I note with approval that the underlying data is publicly archived under CC BY at figshare.

   The Atlas.ti report data spreadsheet could do with some small improvement – the column headers are little cryptic e.g. “Nº  ST “ and “ST” which I eventually deduced was Number of Schools of Thought and Schools of Thought (?)   

   Is there a rawer form of the data that could be deposited with which to evidence the work done? The Atlas.ti report spreadsheet seemed like it was downstream output data from Atlas.ti. What was the rawer input data entered into Atlas.ti? Can this be archived somewhere in case researchers want to reanalyse it using other tools and methods.

   I note with disapproval that Atlas.ti is proprietary software which may hinder the reproducibility of this work. Nonetheless I acknowledge that Atlas.ti usage is somewhat ‘accepted’ in social sciences despite this issue.

   I think the qualitative text analysis is a little vague and/or under-described: “Using ATLAS.ti Windows (version 23.0.8.0), we carried out a qualitative analysis of text from the relevant sites, assigning codes covering what they do and why they have chosen to do it that way.” That’s not enough detail. Perhaps an example or two could be given? Was inter-rater reliability performed when ‘assigning codes’ ? How do we know the ‘codes’ were assigned accurately?

2. Are statistical analyses, controls, sampling mechanism, and statistical reporting (e.g., P-values, CIs, effect sizes) appropriate and well described?

   This is a descriptive study (and that’s fine) so there aren’t really any statistics on show here other than simple ‘counts’ (of Schools of Thought) in this manuscript. There are probably some statistical processes going on within the proprietary qualitative analysis of text done in ATLAS.ti but it is under described and so hard for me to evaluate. 

Results

1. Is the results presentation, including the number of tables and figures, appropriate to best present the study findings?

   Yes. However, I think a canonical URL to each service should be given.  A URL is very useful for disambiguation, to confirm e.g. that the authors mean this Hypothesis (www.hypothes.is) and NOT this Hypothesis (www.hyp.io). I know exactly which Hypothesis is the one the authors are referring to but we cannot assume all readers are experts 😊

   Optional suggestion: I wonder if the authors couldn’t present the table data in a slightly more visual and/or compact way? It’s not very visually appealing in its current state. Purely as an optional suggestion, to make the table more compact one could recode the answers given in one or more of the columns 2, 3 and 4 in the table e.g. "all disciplines = ⬤ , biomedical and life sciences = ▲, social sciences =  ‡  , engineering and technology = † ". I note this would give more space in the table to print the URLs for each service that both reviewers have requested.

   ———————————————————————————————

   | Service name | Developed by | Scientific disciplines | Types of outputs |

   | Episciences | Other | ⬤ | blah blah blah. |

   | Faculty Opinions | Individual researcher | ▲ | blah blah blah. |

   | Red Team Market | Individual researcher | ‡ | blah blah blah. |

   ———————————————————————————————

   The "Types of outputs" column might even lend themselves to mini-colour-pictograms (?) which could be more concise and more visually appealing? A table just of text, might be scientifically 'correct' but it is incredibly dull for readers, in my opinion.

2. Are additional sub-analyses or statistical measures needed (e.g., reporting of CIs, effect sizes, sensitivity analyses)?

   No / Not applicable. 

Discussion

1. Is the interpretation of results and study conclusions supported by the data and the study design?

   Yes.

2. Have the authors clearly emphasized the limitations of their study/theory/methods/argument?

   No. Perhaps a discussion of the linguistic/comprehension bias of the authors might be appropriate for this manuscript. What if there are ‘local’ or regional Chinese, Japanese, Indonesian or Arabic language preprint review services out there? Would this authorship team really be able to find them?

Additional points:

• Perhaps the points made in this manuscript about financial sustainability (p24) are a little too pessimistic. I get it, there is merit to this argument, but there is also some significant investment going on there if you know where to look. Perhaps it might be worth citing some recent investments e.g. Gates -> PREreview (2024) https://content.prereview.org/prereview-welcomes-funding/  and Arcadia’s $4 million USD to COAR for the Notify Project which supports a range of preprint review communities including Peer Community In, Episciences, PREreview and Harvard Library.  (source: https://coar-repositories.org/news-updates/coar-welcomes-significant-funding-for-the-notify-project/ ) 

• Although I note they are mentioned, I think more needs to be written about the similarity and overlap between ‘overlay journals’ and preprint review services. Are these arguably not just two different terms for kinda the same thing? If you have Peer Community In which has it’s overlay component in the form of the Peer Community Journal, why not mention other overlay journals like Discrete Analysis and The Open Journal of Astrophysics.   I think Peer Community In (& it’s PCJ) is the go-to example of the thin-ness of the line the separates (or doesn’t!) overlay journals and preprint review services. Some more exposition on this would be useful.

Thank you very much for the opportunity to review the preprint titled “Preprint review services: Disrupting the scholarly communication landscape?” (https://doi.org/10.31235/osf.io/8c6xm) The authors review services that facilitate peer review of preprints, primarily in the STEM (science, technology, engineering, and math) disciplines. They examine how these services operate and their role within the scholarly publishing ecosystem. Additionally, the authors discuss the potential benefits of these preprint peer review services, placing them in the context of tensions in the broader peer review reform movement. The discussions are organized according to four “schools of thought” in peer review reform, as outlined by Waltman et al. (2023), which provides a useful framework for analyzing the services. In terms of methodology, I believe the authors were thorough in their search for preprint review services, especially given that a systematic search might be impractical.

As I see it, the adoption of preprints and reforming peer review are key components of the move towards improving scholarly communication and open research. This article is a useful step along that journey, taking stock of current progress, with a discussion that illuminates possible paths forward. It is also well-structured and easy for me to follow. I believe it is a valuable contribution to the metaresearch literature.

On a high level, I believe the authors have made a reasonable case that preprint review services might make peer review more transparent and rewarding for all involved. Looking forward, I would like to see metaresearch which gathers further evidence that these benefits are truly being realised.

In this review, I will present some general points which merit further discussion or clarification to aid an uninitiated reader. Additionally, I raise one issue regarding how the authors framed the article and categorised preprint review services and the disciplines they serve. In my view, this problem does not fundamentally undermine the robust search, analyses, and discussion in this paper, but it risks putting off some researchers and constrains how broadly one should derive conclusions.

General comments

Some metaresearchers may be aware of preprints, but not all readers will be familiar with them. I suggest briefly defining what they are, how they work, and which types of research have benefited from preprints, similar to how “preprint review service” is clearly defined in the introduction.

Regarding Waltman et al.’s (2023) “Equity & Inclusion” school of thought, does it specifically aim for “balanced” representation by different groups as stated in this article? There is an important difference between “balanced” versus “equitable” representation, and I would like to see it addressed in this text.

Another analysis I would like to see is whether any of the 23 services reviewed present any evidence that their approach has improved research quality. For instance, the discussion on peer review efficiency and incentives states that there is currently “no hard evidence” that journals want to utilise reviews by Rapid Reviews: COVID-19, and that “not all journals are receptive” to partnerships. Are journals skeptical of whether preprint review services could improve research quality? Or might another dynamic be at work?

The authors cite Nguyen et al. (2015) and Okuzaki et al. (2019), stating that peer review is often “overloaded”. I would like to see a clearer explanation by what “overloaded” means in this context so that a reader does not have to read the two cited papers.

To the best of my understanding, one of the major sticking points in peer review reform is whether to anonymise reviewers and/or authors. Consequently, I appreciate the comprehensive discussion about this issue by the authors.

However, I am only partially convinced by the statement that double anonymity is “essentially incompatible” with preprint review. For example, there may be, as yet not fully explored, ways to publish anonymous preprints with (a) a notice that it has been submitted to, or is undergoing, peer review; and (b) that the authors will be revealed once peer review has been performed (e.g. at least one review has been published). This would avoid the issue of publishing only after review is concluded as is the case for Hypothesis and Peer Community In.

Additionally, the authors describe 13 services which aim to “balance transparency and protect reviewers’ interests”. This is a laudable goal, but I am concerned that framing this as a “balance” implies a binary choice, and that to have more of one, we must lose an equal amount of the other. Thinking only in terms of “balance” prevents creative, win-win solutions. Could a case be made for non-anonymity to be complemented by a reputation system for authors and reviewers? For example, major misconduct (e.g. retribution against a critical review) would be recorded in that system and dissuade bad actors. Something similar can already be seen in the reviewer evaluation system of CrowdPeer, which could plausibly be extended or modified to highlight misconduct.

I also note that misconduct and abusive behaviour already occur even in fully or partially anonymised peer review, and they are not limited to the review or preprints. While I am not aware of existing literature on this topic, academics’ fears seem reasonable. For example, there is at least anecdotal testimonies that a reviewer would deliberately reject a paper to retard the progress of a rival research group, while taking the ideas of that paper and beating their competitors to winning a grant. Or, a junior researcher might refrain from giving a negative review out of fear that the senior researcher whose work they are reviewing might retaliate. These fears, real or not, seem to play a part in the debates about if and how peer review should (or should not) be anonymised. I would like to see an exploration of whether de-anonimisation will improve or worsen this behaviour and in what contexts. And if such studies exist, it would be good to discuss them in this paper.

I found it interesting that almost all preprint review services claim to be complementary to, and not compete with, traditional journal-based peer review. The methodology described in this article cannot definitely explain what is going on, but I suspect there may be a connection between this aversion to compete with traditional journals, and (a) the skepticism of journals towards partnering with preprint review services and (b) the dearth of publisher-run options. I hypothesise that there is a power dynamic at play, where traditional publishers have a vested interest in maintaining the power they hold over scholarly communication, and that preprint review services stress their complementarity (instead of competitiveness) as a survival mechanism. This may be an avenue for further metaresearch.

To understand preprints from which fields of research are actually present on the services categorised under “all disciplines,” I used the Random Integer Set Generator by the Random.org true random number service (https://www.random.org/integer-sets/) to select five services for closer examination: Hypothesis, Peeriodicals, PubPeer, Qeios, and Researchers One. Of those, I observed that Hypothesis is an open source web annotation service that allows commenting on and discussion of any web page on the Internet regardless of whether it is research or preprints. Hypothesis has a sub-project named TRiP (Transparent Review in Preprints), which is their preprint review service in collaboration with Cold Spring Harbor Laboratory. It is unclear to me why the authors listed Hypothesis as the service name in Table 1 (and elsewhere) instead of TRiP (or other similar sub-projects). In addition, Hypothesis seems to be framed as a generic web annotation service that is used by some as a preprint review tool. This seems fundamentally different from others who are explicitly set up as preprint review services. This difference seems noteworthy to me.

To aid readers, I also suggest including hyperlinks to the 23 services reviewed in this paper. My comments on disciplinary representation in these services are elaborated further below.

One minor point of curiosity is that several services use an “automated tool” to select reviewers. It would be helpful to describe in this paper exactly what those tools are and how they work, or report situations where services do not explain it.

Lastly, what did the authors mean by “software heritage” in section 6? Are they referring to the organisation named Software Heritage (https://www.softwareheritage.org/) or something else? It is not clear to me how preprint reviews would be deposited in this context.

Respecting disciplinary and epistemic diversity

In the abstract and elsewhere in the article, the authors acknowledge that preprints are gaining momentum “in some fields” as a way to share “scientific” findings. After reading this article, I agree that preprint review services may disrupt publishing for research communities where preprints are in the process of being adopted or already normalised. However, I am less convinced that such disruption is occurring, or could occur, for scholarly publishing more generally.

I am particularly concerned about the casual conflation of “research” and “scientific research” in this article. Right from the start, it mentions how preprints allow sharing “new scientific findings” in the abstract, stating they “make scientific work available rapidly.” It also notes that preprints enable “scientific work to be accessed in a timely way not only by scientists, but also…” This framing implies that all “scholarly communication,” as mentioned in the title, is synonymous with “scientific communication.” Such language excludes researchers who do not typically identify their work as “scientific” research. Another example of this conflation appears in the caption for Figure 1, which outlines potential benefits of preprint review services. Here, “users” are defined as “scientists, policymakers, journalists, and citizens in general.” But what about researchers and scholars who do not see themselves as “scientists”?

Similarly, the authors describe the 23 preprint review services using six categories, one of which is “scientific discipline”. One of those disciplines is called “humanities” in the text, and Table 1 lists it as a discipline for Science Open Reviewed. Do the authors consider “humanities” to be a “scientific” discipline? If so, I think that needs to be justified with very strong evidence.

Additionally, Waltman et al.’s four schools of thought for peer review reform works well with the 23 services analysed. However, at least three out of the four are explicitly described as improving “scientific” research.

Related to the above are how the five “scientific disciplines” are described as the “usual organisation” of the scholarly communication landscape. On what basis should they be considered “usual”? In this formulation, research in literature, history, music, philosophy, and many other subjects would all be lumped together into the “humanities”, which sit at the same hierarchical level as “biomedical and life sciences”, arguably a much more specific discipline. My point is not to argue for a specific organisation of research disciplines, but to highlight a key epistemic assumption underlying the whole paper that comes across as very STEM-centric (science, technology, engineering, and math).

How might this part of the methodology affect the categories presented in Table 1? “Biomedical and life sciences” appear to be overrepresented compared to other “disciplines”. I’d like to see a discussion that examines this pattern, and considers why preprint review services (or maybe even preprints more generally) appear to cover mostly the biomedical or physical sciences.

In addition, there are 12 services described as serving “all disciplines”. I believe this paper can be improved by at least a qualitative assessment of the diversity of disciplines actually represented on those services. Because it is reported that many of these service stress improving the “reproducibility” of research, I suspect most of them serve disciplines which rely on experimental science.

I randomly selected five services for closer examination, as mentioned above. Of those, only Qeios has demonstrated an attempt to at least split “arts and humanities” into subfields. The others either don’t have such categories altogether, or have a clear focus on a few disciplines (e.g. life sciences for Hypothesis/TRiP). In all cases I studied, there is a heavy focus on STEM subjects, especially biology or medical research. However, they are all categorised by the authors as serving “all disciplines”.

If preprint review services originate from, or mostly serve, a narrow range of STEM disciplines (especially experiment-based ones), it would be worth examining why that is the case, and whether preprints and reviews of them could (or could not) serve other disciplines and epistemologies.

It is postulated that preprint review services might “disrupt the scholarly communication landscape in a more radical way”. Considering the problematic language I observed, what about fields of research where peer-reviewed journal publications are not the primary form of communication? Would preprint review services disrupt their scholarly communications?

To be clear, my concern is not just the conflation of language in a linguistic sense but rather inequitable epistemic power. I worry that this conflation would (a) exclude, minoritise, and alienate researchers of diverse disciplines from engaging with metaresearch; and (b) blind us from a clear pattern in these 23 services, that is their strong focus on the life sciences and medical research and a discussion of why that might be the case. Critically, what message are we sending to, for example, a researcher of 18th century French poetry with the language and framing of this paper? I believe the way “disciplines” are currently presented here poses a real risk of devaluing and minoritising certain subject areas and ways of knowing. In its current form, I believe that while this paper is a very valuable contribution, one should not derive from it any conclusions which apply to scholarly publishing as a whole.

The authors have demonstrated inclusive language elsewhere. For example, they have consciously avoided “peer” when discussing preprint review services, clearly contrasting them to “journal-based peer review”. Therefore, I respectfully suggest that similar sensitivity be adopted to avoid treating “scientific research” and “research” as the same thing. A discussion, or reference to existing works, on the disciplinary skew of preprints (and reviews of them) would also add to the intellectual rigour of this already excellent piece.

Overall, I believe this paper is a valuable reflection on the state of preprints and services which review them. Addressing the points I raised, especially the use of more inclusive language with regards to disciplinary diversity, would further elevate its usefulness in the metaresearch discourse. Thank you again for the chance to review.

Signed:

Dr Pen-Yuan Hsing (ORCID ID: 0000-0002-5394-879X)

University of Bristol, United Kingdom

Data availability

I have checked the associated dataset, but still suggest including hyperlinks to the 23 services analysed in the main text of this paper.

Henriques, S. O., Rzayeva, N., Pinfield, S., & Waltman, L. (2023, October 13). Preprint review services: Disrupting the scholarly communication landscape?. https://doi.org/10.31235/osf.io/8c6xm

Authors:

• Susana Henriques (Research on Research Institute (RoRI) Centre for Science and Technology Studies (CWTS), Leiden University, Leiden, the Netherlands Scientific Research Department, Azerbaijan University of Architecture and Construction, Baku, Azerbaijan) s.oliveira@cwts.leidenuniv.nl
• Narmin Rzayeva (Research on Research Institute (RoRI) Information School, University of Sheffield, Sheffield, UK) n.rzayeva@cwts.leidenuniv.nl
• Stephen Pinfield (Research on Research Institute (RoRI) Centre for Science and Technology Studies (CWTS), Leiden University, Leiden, the Netherlands) s.pinfield@sheffield.ac.uk
• Ludo Waltman waltmanlr@cwts.leidenuniv.nl

Preprint review services: Disrupting the scholarly communication landscape

Ludo Waltman is Editor-in-Chief of MetaROR working with Adrian Barnett, a co-author of the article and a member of the editorial team of MetaROR.

In this article the authors use a discrete choice experiment to study how health and medical researchers decide where to publish their research, showing the importance of impact factors in these decisions. The article has been reviewed by two reviewers. The reviewers consider the work to be robust, interesting, and clearly written. The reviewers have some suggestions for improvements. One suggestion is to emphasize more strongly that the study focuses on the health and medical sciences and to reflect on the extent to which the results may generalize to other fields. Another suggestion is to strengthen the embedding of the article in the literature. Reviewer 2 also suggests to extend the discussion of the sample selection and to address in more detail the question of why impact factors still persist.

In "Researchers Are Willing to Trade Their Results for Journal Prestige: Results from a Discrete Choice Experiment", the authors investigate researchers’ publication preferences using a discrete choice experiment in a cross-sectional survey of international health and medical researchers. The study investigates publishing decisions in relation to negotiation of trade-offs amongst various factors like journal impact factor, review helpfulness, formatting requirements, and usefulness for promotion in their decisions on where to publish. The research is timely; as the authors point out, reform of research assessment is currently a very active topic. The design and methods of the study are suitable and robust. The use of focus groups and interviews in developing the attributes for study shows care in the design. The survey instrument itself is generally very well-designed, with important tests of survey fatigue, understanding (dominant choice task) and respondent choice consistency (repeat choice task) included. Respondent performance was good or excellent across all these checks. Analysis methods (pMMNL and latent class analysis) are well-suited to the task. Pre-registration and sharing of data and code show commitment to transparency. Limitations are generally well-described.

In the below, I give suggestions for clarification/improvement. Except for some clarifications on limitations and one narrower point (reporting of qualitative data analysis methods), my suggestions are only that – the preprint could otherwise stand, as is, as a very robust and interesting piece of scientific work.

1. Respondents come from a broad range of countries (63), with 47 of those countries represented by fewer than 10 respondents. Institutional cultures of evaluation can differ greatly across nations. And we can expect variability in exposure to the messages of DORA (seen, for example, in level of permeation of DORA as measured by signatories in each country, https://sfdora.org/signers/)..%3B!!NVzLfOphnbDXSw!HdeyeHHei6yWQHFjhN3deSSfp82ur9i9JNOLEVOYZN0BvyslUO2S8DlvjBbautmafJEvlUsxQZbT0JLQX7lO8EcOYtZsJkA%24&data=05%7C02%7Ca.l.brasil.varandas.pinto%40cwts.leidenuniv.nl%7C9f47a111adec49d04bb608dd0614ae94%7Cca2a7f76dbd74ec091086b3d524fb7c8%7C0%7C0%7C638673408085242099%7CUnknown%7CTWFpbGZsb3d8eyJFbXB0eU1hcGkiOnRydWUsIlYiOiIwLjAuMDAwMCIsIlAiOiJXaW4zMiIsIkFOIjoiTWFpbCIsIldUIjoyfQ%3D%3D%7C0%7C%7C%7C&sdata=by5mhPfSM0MFFG9LE2iiYjdtSs5IhvpuukqVv%2FLak2s%3D&reserved=0 "https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.com%2Fv3%2F__https%3A%2F%2Fsfdora.org%2Fsigners%2F).%3B!!NVzLfOphnbDXSw!HdeyeHHei6yWQHFjhN3deSSfp82ur9i9JNOLEVOYZN0BvyslUO2S8DlvjBbautmafJEvlUsxQZbT0JLQX7lO8EcOYtZsJkA%24&data=05%7C02%7Ca.l.brasil.varandas.pinto%40cwts.leidenuniv.nl%7C9f47a111adec49d04bb608dd0614ae94%7Cca2a7f76dbd74ec091086b3d524fb7c8%7C0%7C0%7C638673408085242099%7CUnknown%7CTWFpbGZsb3d8eyJFbXB0eU1hcGkiOnRydWUsIlYiOiIwLjAuMDAwMCIsIlAiOiJXaW4zMiIsIkFOIjoiTWFpbCIsIldUIjoyfQ%3D%3D%7C0%7C%7C%7C&sdata=by5mhPfSM0MFFG9LE2iiYjdtSs5IhvpuukqVv%2FLak2s%3D&reserved=0") In addition, some contexts may mandate or incentivise publication in some venues using measures including IF, but also requiring journals to be in certain databases like WoS or Scopus, or having preferred journal lists). I would suggest the authors should include in the Sampling section a rationale for taking this international approach, including any potentially confounding factors it may introduce, and then adding the latter also in the limitations.

2. Reporting of qualitative results: In the introduction and methods, the role of the focus groups and interviews seems to have been just to inform the design of the experiment. But then, results from that qualitative work then appear as direct quotes within the discussion to contextualise or explain results. In this sense though, the qualitative results are being used as new data. Given this, I feel that the methods section should include description of the methods and tools used for qualitative data analysis (currently it does not). But in addition, to my understanding (and this may be a question of disciplinary norms – I’m not a health/medicine researcher), generally new data should not be introduced in the discussion section of a research paper. Rather the discussion is meant to interpret, analyse, and provide context for the results that have already been presented. I personally hence feel that the paper would benefit from the qualitative results being reported separately within the results section.

3. Impact factors – Discussion section: While there is interesting new information on the relative trade-offs amongst other factors, the most emphasised finding, that impact factors still play a prominent role in publication venue decisions, is hardly surprising. More could perhaps be done to compare how the levels of importance reported here differ with previous results from other disciplines or over time (I know a like-for-like comparison is difficult but other studies have investigated these themes, e.g., https://doi.org/10.1177/01655515209585). In addition, beyond the question of whether impact factors are important, a more interesting question in my view is why they still persist. What are they used for and why are they still such important “driver[s] of researchers’ behaviour”? This was not the authors’ question, and they do provide some contextualisation by quoting their participants, but still I think they could do more to contextualise what is known from the literature on that to draw out the implications here. The attribute label in the methods for IF is “ranking”, but ranking according of what and for what? Not just average per-article citations in a journal over a given time frame. Rather, impact factors are used as a proxy indicators of less-tangible desirable qualities – certainly prestige (as the title of this article suggests), but also quality, trust (as reported by one quoted focus group member “I would never select a journal without an impact factor as I always publish in journals that I know and can trust that are not predatory”, p.6), journal visibility, importance to the field, or improved chances of downstream citations or uptake in news media/policy/industry etc. Picking apart the interactions of these various factors in researchers’ choices to make use of IFs (which is not in all cases bogus or unjustified) could add valuable context. I’d especially recommend engaging at least briefly with more work from Science and Technology Studies - especially Müller and de Rijcke’s excellent Thinking with Indicators study (doi: 10.1093/reseval/rvx023), but also those authors other work, as well as work from Ulrike Felt, Alex Rushforth (esp https://doi.org/10.1007/s11024-015-9274-5), Björn Hammerfelt and others.

4. Disciplinary coverage: (1) A lot of the STS work I talk about above emphasises epistemic diversity and the ways cultures of indicator use differ across disciplinary traditions. For this reason, I think it should be pointed out in the limitations that this is research in Health/Med only, with questions on generalisability to other fields. (2) Also, although the abstract and body of the article do make clear the disciplinary focus, the title does not. Hence, I believe the title should be slightly amended (e.g., “Health and Medical Researchers Are Willing to Trade …”)

This manuscript reports the results of an interesting discrete choice experiment designed to probe the values and interests that inform researchers’ decisions on where to publish their work.

Although I am not an expert in the design of discrete choice experiments, the methodology is well explained and the design of the study comes across as well considered, having been developed in a staged way to identify the most appropriate pairings of journal attributes to include.

The principal findings to my mind, well described in the abstract, include the observations that (1) researchers’ strongest preference was for journal impact factor and (2) that they were prepared to remove results from their papers if that would allow publication in a higher impact factor journal. The first of these is hardly surprising – and is consistent with a wide array of literature (and ongoing activism, e.g. through DORA, CoARA). The second is much more striking – and concerning for the research community (and its funders). This is the first time I have seen evidence for such a trade-off. 

Overall, the manuscript is very clearly written. I have no major issues with the methods or results. However, I think but some minor revisions would enhance the clarity and utility of the paper.

First, although it is made clear in Table 1 that the researchers included in the study are all from the medical and clinical sciences, this is not apparent from the title or the abstract. I think both should be modified to reflect the nature of the sample. In my experience researchers in these fields are among those who feel most intensely the pressure to publish in high IF journals. The authors may want also to reflect in a revised manuscript how well their findings may transfer to other disciplines.

Second, in several places I felt the discussion of the results could be enriched by reference to papers in the recent literature that are missing from the bibliography. These include (1) Muller and De Rijcke’s 2017 paper on Thinking with Indicators, which discusses how the pressure of metrics impacts the conduct of research (https://doi.org/10.1093/reseval/rvx023); (2) Bjorn Brembs’ analysis of the reliability of research published in prestige science journals (https://www.frontiersin.org/journals/human-neuroscience/articles/10.3389/fnhum.2018.00376/full; and (3) McKiernan’s et al.’s examination of the use of the Journal Impact Factor in academic review, promotion, and tenure evaluations (https://pubmed.ncbi.nlm.nih.gov/31364991/). 

Third, although the text and figures are nicely laid out, I would recommend using a smaller or different font for the figure legends to more easily distinguish them from body text.

As a statistician, I am in strong agreement on the widespread inappropriate use of statistical inference (page 2) and the importance of software. I also strongly agree that “independent critical inspection [is] particularly challenging” (page 3). I also strongly agree that “The main difficulty in achieving such audits is that none of today’s scientific institutions consider them part of their mission”, as this is everyone’s problem and nobody’s problem.

I also agree that automation has encouraged standardisation and I have personally supported standardisation because some practices are so bad that many authors need to be “standardised”. However, I’ve also felt frustration at the sometimes fussy requirements when uploading R packages to CRAN (https://cran.r-project.org/). Similarly, some blanket changes from CRAN seem pedantic. There’s likely a balance between reducing poor practice and becoming too prescriptive.

In terms of transparency (section 2.4) I did think about the “Verbose=TRUE” option that I sometimes see in R. I tend to turn this on, as it’s good to see more of the workings, but perhaps the default is off? I did look at some packages using the google search: “verbose site:cran.r-project.org/web/packages”. I was also reminded of the difference between Bayesian and frequentist statistical modelling. Frequentist modelling often uses maximum likelihood to create parameter estimates, which usually runs quickly to create the estimates. In contrast, Bayesian methods often use MCMC, which is often slow and creates long chains of estimates; however, the chains will show if the likelihood does not have a clear maximum, which is usually from a badly specified model, whereas the maximum likelihood simply finds any peak. Frustratingly, I often get more push back from reviewers when using Bayesian methods, whereas in my opinion it should be the other way around as the Bayesian estimates have shown far more of the inner workings.

Some reflection on the growing use of AI to write software may be worthwhile. Presumably this could be more standardised, but there are other concerns. Using automation to check code could also be worthwhile.

For section 3, I thought that more sharing of code would mean “more eyeballs”, but the sharing needs to be done in FAIR way.

I wondered if highly-used software should get more scrutiny. Peer review is a scarce resource, so is likely better directed towards high use software. Andrew Gelman recently put forward a similar argument for checking published papers when they reach 250 citations: https://statmodeling.stat.columbia.edu/2025/02/26/pp/.

I agreed with the need for effort (page 19) and wondered if this paper could call for more effort.

Minor comments:

• typo “asses” on page 7.

• “supercomputers are rare”, should this be “relatively rare” or am I speaking from a privileged university where I’ve always had access to supercomputers.

• I did think about “testthat” at multiple points whilst reading the paper (https://testthat.r-lib.org/)

• Can badges on github about downloads and maturity help (page 7)? Although, far from all software is on github.

This summary article does not present new data or experiments but instead takes a broad look at automated reasoning and software. Reviewer #1 thought the article needed much more detail, including citations, examples, screenshots and figures. They were concerned about strong generalisations that were lacking evidence and have provided places where they wanted these details. Reviewer #2 considers the differences between reviewability and the practicalities of reviewing everything, and how being easily able to build-on other software acts as a kind of reproducibility. In my own editorial review, I generally enjoyed reading the paper and it prompted some interesting thoughts on trade-offs with standardisation and the level of detail shown to users for statistical code.

Thank you for submitting this paper. I think the paper requires substantial, major revisions to be published. Throughout the paper I noted many instances where references or examples would help make the intent clear. I also think the message of the paper would benefit from several figures to demonstrate workflows or ideas. The figures presented are essentially tables, and I think the message could be made clearer for the reader if they were presented as flow charts or at least with clear numbering to hook the ideas to the reader - e.g., Figures 1 & 2 would benefit from having numbers on the key ideas.

The paper is lacking many instances of citation, and at times reads as though it is an essay delivering an opinion. I'm not sure if this is the type of article that the journal would like, but two examples of sentences missing citations are:

1. "Over the last two decades, an unexpectedly large number of peer-reviewed findings across many scientific disciplines have been found to be irreproducible upon closer inspection." (Introduction, page 2)

2. "A large number of examples cited in this context involves faulty software or inappropriate use of software" (Introduction, page 3)

Two examples of sentences missing examples are:

1. Experimental software evolves at a much faster pace than mature software, and documentation is rarely up to date or complete (in Mature vs. experimental software, page 7). Could the author provide more examples of what "experimental software" is? There is also consistent use of universal terms like "...is rarely up to date or complete", which would be better phrased as "is often not up to date or complete"

2. There are various techniques for ensuring or verifying that a piece of software conforms to a formal specification.

Overall the paper introduces many new concepts, and I think it would greatly benefit from being made shorter and more concise, with adding some key figures for the reader to refer back to to understand these new ideas. The paper is well written, and it is clear the author is a great writer, and has put a lot of thought into the ideas. However it is my opinion that because these ideas are so big and require so much unpacking, they are also harder to understand. The reader would benefit from having more guidance to come back to understand these ideas.

I hope this review is helpful to the author.

In his article Establishing trust in automated reasoning (Hinsen, 2023) Hinsen argues that much of current scientific software lacks reviewability. Because scientific software has become such a central part of many scientific endeavors he worries that unreviewed software might contain mistakes which will never be spotted and consequently taint the scientific record. To illustrate this worry he cites issues with reproductions in different fields of science, which are often subsumed under the umbrella term of reproducibility crises. These crises, though not uncontested, have varied sources. In the field of social psychology reproducibility issues can for example often be traced to errors in statistical analyses, while shifting baselines and data leakage lead to problems in ML. Hinsen is only concerned with errors in scientific software. He suggests that potential errors could be spotted more easily if scientific software would be more reviewable. Thus he proposes five criteria against which reviewability could be judged. I will not discuss them in detail in this commentary and refer the interested reader to Hinsen (2023, section 2) for an extensive discussion. I note though, that the five criteria are meant to ensure an ideal type of reproducibility which Hinsen defines as follows: “Ideally, each piece of software should perform a well-defined computation that is documented in sufficient detail for its users and verifiable by independent reviewers.” (Hinsen, 2023, p.2). I take the upshot of these criteria to be that one could assert the reviewability of a piece of software before actually doing the review. They could thus function, perhaps contrary to Hinsen’s open science convictions, as a gatekeeping device in a peer review process for software. An editor could ”desk reject” software for not fulfilling the criteria before even sending it out to potential reviewers. If I am correct in this interpretation then we should entertain the same caution with them as we do with preregistration.

To be fair, Hinsen envisions a software review process which differs from current peer review with its acknowledged defects in several ways. He says, ”Developing suitable intermediate processes and institutions for reviewing such software is perhaps possible, but I consider it scientifically more appropriate to restructure such software into a convivial collection of more situated modules, possibly supported by a shared wide-spectrum layer.” (Hinsen, 2023, p.16).

Convivial software in turn is supposed to augment ”its users’ agency over their computation.” (Hinsen, 2023, p.16). This gives us a hint about the kind of user Hinsen has in mind – it is the software developer as a user. His concept of reviewability aims to make software transparent only to this kind of user (see Hinsen, 2023, p.20). In one of his many comparisons of scientific software to science, he notes that ”[. . . ] the main intellectual artifacts of science, i.e. theories and models, have always been convivial.” (Hinsen, 2023, p.9) and we can guess that he wants this to be the case for software too. But, if at all, scientific theories and models only have ever been convivial for scientists. The comparison also works the other way around, science as much as software is heavily fragmented into modules (disciplines). Scientists have always relied on the results of other scientists – they often have done and still do so without reviewing them. Has this hindered progress? I think one would be hard pressed to answer such a question in general for science, and perhaps it is the same for scientific software.

As Hinsen admits formal peer review is a quite novel addition to scientific methodology, being enforced on a larger scale only since the past fifty years or so. Science has progressed many years without, so we could ask why scientific software should not do likewise. Hinsen’s answer of course has to do with how he grades such software with respect to his reviewability criteria – obviously, most of it scores badly. Most scientific software is neither reviewed nor reviewable, Hinsen claims. This he considers a defect, because only reviewable software has to potential of being reviewed. Many practical considerations he discusses actually speak against the hope that most reviewable software will actually be reviewed. Still, without reviewability, it is hard, if not impossible, to spot mistakes. A case that was recently brought to my attention emphasizes this point. In Beheim et al. (2021) it is pointed out that a statistical analysis imputed missing values in an archaeo-historical database with the number 0. But for the statistical model (and software!) in use 0 had a different meaning than not available. This casts doubt on the conclusion that was drawn from the model. Beheim et al. were only able to spot this assumption because the code and data were available for review1. Cases like this abound and are examples for invisible programming values that philosopher James Moor discussed in the context of computer ethics (see Moor, 1985, The invisibility factor). Hinsen calls such values “tacit assumptions made by software developers” (Hinsen, 2023, p.3). We might speculate though, what would have happened if this questionable result had been incorporated into the scientific canon. Would later scientists really have continued building on it without ever realizing their shaky foundations? Or would the whole edifice have had to face the tribunal of experience at some point and crumbled? Perhaps the originating problem would never have been found and a whole research program would have been abandoned, perhaps a completely different part would have been blamed and excised – hard to say!

But maybe reviewability can also serve a different aim than establishing trust in the results of certain pieces of scientific software. Perhaps, it facilitates building on and incorporating pieces of such software in other projects. Its purpose could be more instrumental than epistemic. Although Hinsen seems to worry more about the epistemic problems coming with lack of reviewability, many points he makes implicitly deal with practical problems of software engineering. Whoever has fought against jupyter notebooks with legacy python requirements can immediately relate to his wish for keeping the execution environment as small as possible. For Hinsen software is actually defined by its execution environment (Hinsen, 2023, p.11), thus the complete environment must be available for its reviewability2. Software cannot be really seen as a separate entity and a review always reviews the whole environment. Analogously to Quine-Duhem we could call this situation review holism. But review holism might be less problematic than its scientific cousin suggests. We might not actually need to explicitly review the whole system. Perhaps it is sufficient if we achieve frictionless reproducibility (see Donoho, 2024), that is, other people can more or less easily incorporate and built on the software in question. Firstly if other software which incorporates the software in question works, it already is a type of successful reproduction. Secondly, the process of how software evolves might weed out any major errors, whatever errors remain are perhaps just irrelevant. In all fairness it has to be said that Hinsen does not think this is the case with current software. He argues that ”Software with a small defect, on the other hand, can introduce unpredictable errors in both kind and magnitude, which neither a domain expert nor a professional programmer or computer scientist can diagnose easily.” (Hinsen, 2023, p.13). But if that is the case then Hinsen’s later recourse to reliabilist-style justifications for software correctness is blocked too. We are in a situation for which the late Humphreys coined the term strange error (Rathkopf & Heinrichs, 2023, p.5). Strange errors are a challenge for any reliabilist account of justification because their magnitude can easily overwhelm arduously collected reliability assurances. If computational reliabilism was just reliabilism, and Hinsen seems to take it as such3, it would suffer from this problem too. But computational reliabilism has an additional internalist component, which explicitly allows for the whole toolbox of ”rationalist” software verification methods. If possible we should learn something about our tools other than their mere reliability. As Hacking said, ”[To understand] whether one sees through a microscope, one needs to know quite a lot about the tools.” (Hacking, 1981, p.135).

I would go so far and say that, if available, internalist justificiations are preferable to reliabilistic guarantees. It is only the case that often they are not and then we might content ourselves with the guarantees reliabilism provides. I said might content here, because such guarantees are unlikely to satisfy the skeptic. Obviously strange errors are always a possibility and no finite observation of correct software behaviour can completely rule them out. But in practice such concerns tend to fade over time, although they provide opportunity for unchecked philosophically skepticism. Many discussions about software opacity feed from such skepticism and this is what I tried to balance with computational reliabilism. In this spirit computational reliabilism was an attempt to temper theoretical skeptics in philosophy, not to give normative guidance to software engineering practice. My view was always that practice has the last say over philosophical concerns. If the emerging view in software engineering practice now is that more skepticism is appropriate, I will happily concur. But I should like to remind the practitioner that evidence for such skepticism has to be given in practice too, mere theoretical possibilities are not sufficient to establish it.

Reviewability does not mean reviewed. And only reviews can give us trust - or so we might think. As Hinsen acknowledges we should not expect that a majority of scientific software will ever be reviewed. Does this mean we cannot trust the results from such software? Above I tried to sketch a way out of this conundrum: We can view reviewability as advocated by Hinsen as a way to enable frictionless reproducibility, which in turn lets us built upon software, incorporate it in our own projects and use its results. As long as it works in a practically fulfilling way, this might be all the reviewing we need.

Notes

1A statistician once told me, that one glance at the raw data of this example immediately made clear to him that whatever problem there was with imputation, the data would never have supported the desired conclusions in any way. One man’s glance is another’s review.

2Hinsen’s definition of software closely parallels that of Moor, who argued that computer programs are a relation between a computer, a set of instructions and an activity (Moor, 1978, p.214).

3Hinsen characterizes computational reliabilism as follows, ”As an alternative source of trust, they propose computational reliabilism, which is trust derived from the experience that a computational procedure has produced mostly good results in a large number of applications.” (Hinsen, 2023, p.10)

References

Beheim, B., Atkinson, Q. D., Bulbulia, J., Gervais, W., Gray, R. D., Henrich, J., Lang, M., Monroe, M. W., Muthukrishna, M., Norenzayan, A., Purzy- cki, B. G., Shariff, A., Slingerland, E., Spicer, R., & Willard, A. K. (2021). Treatment of missing data determined conclusions regarding moralizing gods. Nature, 595 (7866), E29–E34. https://doi.org/10.1038/s41586-021-03655-4

Donoho, D. (2024). Data Science at the Singularity. Harvard Data Science Re- view, 6 (1). https://doi.org/10.1162/99608f92.b91339ef

Hacking, I. (1981). Do We See Through a Microscope? Pacific Philosophical Quarterly, 62 (4), 305–322. https://doi.org/10.1111/j.1468-0114.1981.tb00070.x

Hinsen, K. (2023, July). Establishing trust in automated reasoning. https:// doi.org/10.31222/osf.io/nt96q

Moor, J. H. (1978). Three Myths of Computer Science. The British Journal for the Philosophy of Science, 29 (3), 213–222. https://doi.org/10.1093/bjps/29.3.213

Moor, J. H. (1985). What is computer ethics? Metaphilosophy, 16 (4), 266–275. https://doi.org/10.1111/j.1467-9973.1985.tb00173.x

Rathkopf, C., & Heinrichs, B. (2023). Learning to Live with Strange Error: Be- yond Trustworthiness in Artificial Intelligence Ethics. Cambridge Quarterly of Healthcare Ethics, 1–13. https://doi.org/10.1017/S0963180122000688

different societal risks = different things to vote on

I was surprised by how peaceful and normal the setting of the village feels at the start of the story, which makes the story's violent ending even more shocking. This contrast builds suspense and emphasizes the horror of blind tradition.

This seems important because in the story it shows how authority figures can enforce traditions and maintain control even when the practice is harmful.

This had confused me initially because the villagers follow a ritual they barely remember. It highlights blind conformity and how people can follow traditions without questioning their purpose.

This is a very crucial moment that highlights the human cost of blindly following tradition. Tessie's protest underscores the story's central theme: unquestioned social practices can lead to injustice and harm.

Please check that all icons show as squares, for instance the arrows before "see figure" and the bullet points in the challenges and opportunities

Please amend the endnote for figure B-1, endnote should be 4 and not "xx"

DOI: 10.3390/epigenomes9030034

Resource: The Cancer Genome Atlas (RRID:SCR_003193)

Curator: @scibot

SciCrunch record: RRID:SCR_003193

What is this?

DOI: 10.1038/s41591-025-03928-9

Resource: The European Genome-phenome Archive (EGA) (RRID:SCR_004944)

Curator: @scibot

SciCrunch record: RRID:SCR_004944

What is this?

Truly a Catch-22.

Built on consent, rather than scraped without their knowledge.

The "new vision" for A.I. as a tool for minorities to reclaim digital spaces.

Both A.I. and colonization cause language to trend towards globalization--that is, the spread of dominant languages and decline of minority languages.

Maori people were able to surveil and protect their data from big US tech companies by having the power to decide who does/ does not receive access

US tech companies have little interest in letting Maori people reconnect with their native customs , but instead, offer this "privilege" at a steep cost

I found this paragraph extremely important due to the fact that isolating these technologies is reshaping universal relationships.

In order to preserve their language they had to hide it from big tech companies

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public review):

Summary:

The question of how central nervous system (CNS) lamination defects affect functional integrity is an interesting topic, though it remains a subject of debate. The authors focused on the retina, which is a relatively simple yet well-laminated tissue, to investigate the impact of afadin - a key component of adherens junctions on retinal structure and function. Their findings show that the loss of afadin leads to significant disruptions in outer retinal lamination, affecting the morphology and localization of photoreceptors and their synapses, as illustrated by high-quality images. Despite these severe changes, the study found that some functions of the retinal circuits, such as the ability to process light stimuli, could still be partially preserved. This research offers new insights into the relationship between retinal lamination and neural circuit function, suggesting that altered retinal morphology does not completely eliminate the capacity for visual information processing.

Strengths:

The retina serves as an excellent model for investigating lamination defects and functional integrity due to its relatively simple yet well-organized structure, along with the ease of analyzing visual function. The images depicting outer retinal lamination, as well as the morphology and localization of photoreceptors and their synapses, are clear and well-described. The paper is logically organized, progressing from structural defects to functional analysis. Additionally, the manuscript includes a comprehensive discussion of the findings and their implications.

Weaknesses:

While this work presents a wealth of descriptive data, it lacks quantification, which would help readers fully understand the findings and compare results with those from other studies. Furthermore, the molecular mechanisms underlying the defects caused by afadin deletion were not explored, leaving the role of afadin and its intracellular signaling pathways in retinal cells unclear. Finally, the study relied solely on electrophysiological recordings to demonstrate RGC function, which may not be robust enough to support the conclusions. Incorporating additional experiments, such as visual behavior tests, would strengthen the overall conclusions. 

We would like to thank the reviewer for the thoughtful and valuable comments that helped us to further improve the manuscript. We have revised the manuscript to address the following three points in response to the reviewer's comments.

While this work presents a wealth of descriptive data, it lacks quantification, which would help readers fully understand the findings and compare results with those from other studies.

In response, we quantified the position of each retinal cell type and measured retinal thickness in the cHet and cKO mice at 1M, as presented in Figures 2F–M. To reflect these additions, we have included explanatory text in the revised manuscript (see lines 507–533).

Furthermore, the molecular mechanisms underlying the defects caused by afadin deletion were not explored, leaving the role of afadin and its intracellular signaling pathways in retinal cells unclear.

As AJ components, such as catenin and cadherin, are known to be associated with several signaling pathways, including Notch and Wnt signals (PMID: 37255594), we speculated that these pathways might be disrupted in the afadin cKO retina. Since these pathways are involved in cell proliferation, we examined the number of progenitor cells in the afadin cKO retina at developmental stages P1, P3, and P5 (new Figure S6C, see lines 868-870). No significant differences were observed at any of these stages. We also quantified the number of each retinal cell type at P14 when differentiation is complete. In the cKO retina, the number of BCs significantly increased, whereas the number of photoreceptors significantly reduced (new Figure S4C, see lines 620-622). To our knowledge, activation or inactivation of any AJ-associated signaling pathway does not reproduce the cell fate alterations observed in the afadin cKO retina. These findings suggest that the above pathways related to AJ may be unchanged in the cKO retina. However, we cannot exclude the possibility that multiple signaling pathways may be affected simultaneously or other pathways affected in the cKO retina.

Finally, the study relied solely on electrophysiological recordings to demonstrate RGC function, which may not be robust enough to support the conclusions. Incorporating additional experiments, such as visual behavior tests, would strengthen the overall conclusions.

We appreciate the reviewer’s insightful suggestion. To more robustly evaluate visual function in the cKO mice, we performed optomotor response (OMR) and visual cliff tests using cHet, cKO, and optic nerve crush (ONC) mice with Aki Hashio, Yuki Emori, and Mao Hiratsuka. We added their name as co-authors to the new manuscript. In the OMR test, cKO mice exhibited fewer responses to visual stimuli than cHet mice but significantly more than ONC mice. Furthermore, although no significant difference was detected between cKO and ONC mice in the visual cliff test, some cKO mice displayed cautious behavior suggestive of depth perception. These results indicate that cKO mice retain partial visual function, which is consistent with the MEA analysis. We have included these data as the new Figure 8 and incorporated the findings into the revised manuscript in the Introduction (lines 130-131 and 133-134), Methods (lines 378-406), Results (lines 775-816), and Discussion sections (lines 1026-1035).

Reviewer #2 (Public review):

Summary:

Ueno et al. described substantial changes in the afadin knockout retina. These changes include decreased numbers of rods and cones, an increased number of bipolar cells, and disrupted somatic and synaptic organization of the outer limiting membrane, outer nuclear layer, and outer plexiform layer. In contrast, the number and organization of amacrine cells and retinal ganglion cells remain relatively intact. They also observed changes in ERG responses and RGC receptive fields and functions using MEA recordings.<br /> Strengths:

The morphological characterization of retinal cell types and laminations is detailed and relatively comprehensive.

Weaknesses:

(1) The major weakness of this study, perhaps, is that its findings are predominantly descriptive and lack any mechanistic explanation. As afadin is key component of adherent junctions, its role in mediating retinal lamination has been reported previously (see PMCID: PMC6284407). Thus, a more detailed dissection of afadin's role in processes, such as progenitor generation, cell migration, or the formation of retinal lamination would provide greater insight into the defects caused by knocking out afadin.

Thank you for valuable comments. We agree with the reviewer's point that findings are predominantly descriptive and lack any mechanistic explanation. However, we would like to clarify that the study cited in the comment (PMCID: PMC6284407) analyzed the role of afadin in dendritic stratification of direction-selective RGCs within the IPL, where “lamination” refers to the layering of RGC dendrites in the IPL. Here, we analyzed the function of afadin in the laminar construction of the overall retina.

In response to the reviewer’s comment, we have added new analyses addressing retinal lamination, as well as the number and spatial distribution of progenitor cells, during development in the cKO retina. These new results are shown in Figures 4E, 9C–F, S5A–C, and S6C of the revised manuscript, and corresponding explanations added in the revised text (lines 643–662 and 855–870).

(2) The authors observed striking changes in the numbers of rods, cones, and BCs, but not in ACs or RGCs. The causes of these distinct changes in specific cell classes remain unclear. Detailed characterizations, such as the expression of afadin in early developing retina, tracing cell numbers across various early developmental time points, and staining of apoptotic markers in developing retinal cells, could help to distinguish between defects in cell generation and survival, providing a better understand of the underlying causes of these phenotypes.

Thank you for the insightful comment. Following the reviewer’s suggestion, we quantified the number of retinal cell types at P14 when cell differentiation is complete (new Figure S4C). At P14, the numbers of photoreceptors and BCs were significantly reduced in the cKO retina, while Müller glia, which was significantly reduced at 1M, showed no difference. We further examined the number of rods and BCs at P1, P3, and P5 (new Figures S4E, F). No significant differences were detected at P1 or P3, however, at P5, rod marker expression was significantly decreased, while the number of BCs was significantly increased. These results suggest that the defects in cell fate determination of BCs and rods begin to emerge between P3 and P5, a period for which rods and BCs actively differentiate. We speculate that cells originally destined to become rods may instead differentiate into BCs in the cKO retina. In addition, we found a significant increase in apoptotic cells at P1, P3, P5, and P14 (new Figure S6B). Furthermore, Müller glia and rod photoreceptors showed significantly greater reduction at 1M compared to P14, suggesting that the reduction in Müller glia observed at 1M may be due to post-differentiation cell death. These are presented in Figures S4C, S4E–F, and S6B, and described in the revised manuscript (lines 620-635 and 827-838).

(3) Although the total number of ACs or RGCs remains unchanged, their localizations are somewhat altered (Figures 2E and 4E). Again, the cause of the altered somatic localization in ACs and RGCs is unclear.

Thank you for the valuable question. In response to the reviewer’s comment, we analyzed the position of RGCs and ACs in the developing cKO retina. In the cKO retina at P1, retinal cells were organized into distinct multicellular compartments with clear boundaries, and acellular regions extending to the outer retinal surface were observed at these boundaries. These acellular regions contained dendritic processes of RGCs and ACs, which are components of the IPL, indicating that elements of the IPL extended vertically across the retina. As development progressed, the compartment boundaries gradually shifted toward the inner retina. At P14, the IPL was mainly located on the inner retina, as in the normal retina. However, some IPL structures remained in the outer retina and may correspond to the acellular patches. We have included the above data in the revised manuscript as Figures S5A and S5B and revised the manuscript to include this point (lines 643-660).

(4) One conclusion that the authors emphasise is that the function of RGCs remains detectable despite a major disrupted outer plexiform layer. However, the organization of the inner plexiform layer remains largely intact, and the axonal innervation of BCs remains unchanged. This could explain the function integrity of RGCs. In addition, the resolution of detecting RGCs by MEA is low, as they only detected 5 clusters in heterozygous animals. This represents an incomplete clustering of RGC functional types and does not provide a full picture of how functional RGC types are altered in the afadin knockout.

We appreciate the reviewer’s insightful comments. Although our clustering of RGC subtypes in afadin cHet retinas resulted in only five clusters, the key finding of our study is the preservation of RGC receptive fields in afadin cKO retinas, despite severe photoreceptor loss (reduced to about one-third of normal) and disruption of photoreceptor-bipolar cell synapses in the OPL. This suggests that even with crucial damage to the OPL, the primary photoreceptor-bipolar-RGC pathway can still function as long as the IPL remains intact. Moreover, the presence of rod-driven responses in RGCs indicates that the AII amacrine cell-mediated rod pathway may also continue to function. We agree that our functional clustering in afadin cHet retinas was incomplete. However, we guess that the absence of RGCs with fast temporal responses in afadin cKO retinas may not simply be due to the loss of specific RGC subtypes but due to disrupted synaptic connections between photoreceptors and fast-responding BCs. Furthermore, the structural abnormalities in retinal lamination in afadin cKO retinas may alter RGC response properties, making strict functional classification less meaningful. We would like to emphasize the finding that disruption of the retinal lamination in afadin cKO retinas leads to the absence of RGCs with fast temporal response properties, rather than focusing solely on the classification of RGC subtypes.

Minor Comments:

(1) Line 56-67: "Overall, these findings provide the first evidence that retinal circuit function can be partially preserved even when there are significant disruptions in retinal lamination and photoreceptor synapses" There is existing evidence showing substantial adaption in retinal function when retinal lamination or photoreceptor synapses are disrupted, such as PMCID: PMC10133175.

Thank you for your comment. We agree that the original sentence was ambiguous in its wording, and we have revised it to clarify our intended meaning (lines 48-50):

"Overall, these findings provide the first evidence that retinal circuit function can be partially preserved even when there are significant disruptions in both retinal lamination and photoreceptor synapses."

In response, we have cited this study and added the following sentence to the Discussion section of the revised manuscript. The paper you mentioned is crucial for discussing and considering the results of our study. We have cited this study and added the following sentence to the Discussion section of the revised manuscript (lines 910-915):

“Furthermore, RFs of RGCs are also detected in several mouse models of retinitis pigmentosa, in which rod photoreceptors are degenerated and surviving cone photoreceptors lose their OS discs and pedicles, instead forming abnormal processes resembling synaptic dendrites (Barhoum et al., 2008; Ellis et al., 2023; Scalabrino et al., 2022).”

(2) Line 114-115: "we focused on afadin, which is a scaffolding protein for nectin and has no ortholog in mice." The term "Ortholog" is misused here, as the mouse has an afadin gene. Should the intended meaning be that afadin has no other isoforms in mouse?

Thank you for pointing it out. As we misused "Ortholog" as "Paralog", we revised the sentence (line 108).

Recommendations for the authors:

(1) The introduction to afadin is insufficient. Please provide more background information about this protein.

Following the reviewer’s recommendations, we expanded the Introduction in the revised manuscript to provide a more detailed background on afadin, as follows (lines 108-119):

“Afadin regulates the localization of nectin, which initiates cell–cell adhesion and promotes AJ formation by recruiting the cadherin–catenin complex. (Ohama et al., 2018; Takai and Nakanishi, 2003). In addition, afadin interacts with various cell adhesion and signaling molecules, as well as the actin cytoskeleton, and contributes to the accumulation of β-catenin, αE-catenin, and E-cadherin at AJs (Sakakibara et al., 2018; Sato et al., 2006). Afadin KO mice exhibit severe disruption of AJs in the ectoderm, along with other developmental defects, leading to embryonic lethality (Ikeda et al., 1999; Zhadanov et al., 1999). Conditional deletion of afadin in RGCs leads to disruption of dendrites in ON-OFF direction-selective RGCs (Duan et al., 2018). However, the effect of afadin loss on retinal lamination, circuit formation, and function is poorly understood.”

(2) In Figure 1A (Bottom), regarding the peptide+ image, what does the green signal represent?

The green signal observed in the peptide+ image represents the background and non-specific staining. We have added the sentence to the legend of Figure 1A in the revised manuscript (lines 1067-1068).

(3) In the RESULTS section on page 17, the statement "Nectin-1, unlike nectin-2 and nectin-3, was partially co-localized with afadin at the OPL and IPL, in addition to the OLM" suggests that nectin-2 is also expressed at the IPL, as shown in Figure S1A. Providing high-power images, similar to those in Figure S1B, could help readers clearly recognize the staining signals.

Following your suggestion, we added higher-magnification images of Nectin-2 signals in the IPL to Figure S1A and included the following clarification in the Figure legend (lines 1356-1358):

“Nectin-2 and nectin-3 were localized in the OLM. The Nectin-2 signal in the IPL was insufficient for reliable assessment of its localization and colocalization.”

(4) Figure S2A requires an uncropped scan of the membrane after Western blotting to demonstrate that there are no non-specific bands when using this afadin antibody, which was also utilized for IHC.

We revised the new Figure S2C to include the uncropped membrane scan. Faint non-specific bands were observed in the Western blot, consistent with detecting non-specific signals in immunostaining using the anti-afadin antibody pre-absorbed with its antigen peptide.

(5) IHC staining is necessary to demonstrate the knockout of afadin in retinal cells, as the paper does not show Cre expression in the retinal cells of the Dkk3-Cre mouse line. This would also help verify the specificity of the afadin antibody.

In the cKO retina, the laminar structure was disrupted, and the background signal was generally high, making it difficult to reliably assess whether afadin expression was lost using immunostaining with the anti-afadin antibody. Therefore, in addition to the Western blot analysis already presented, we evaluated Cre activity in the Dkk3-Cre mouse line by crossing it with the R26-H2B-EGFP reporter line. Cre-mediated recombination was observed in all retinal cells at P0 and 1M. We have added these results to a revised Figure S2A and B and included explanatory text in the revised manuscript (lines 455–458).

(6) Why is the outer nuclear layer (ONL) severely impaired in the cKO mice when afadin is not expressed in this layer? Additionally, given that afadin is highly expressed in the inner plexiform layer (IPL), why does the cKO not affect its structure?

We speculate that the AJ defect in the outer retina during development may cause severe disruption of the ONL in afadin cKO mice. As shown in new Figure 9, ectopic AJs and aberrant position of mitotic cells were observed in the P0 cKO retina. These defects caused abnormal cell migration and position, resulting in the ONL disruption. On the other hand, in the IPL, afadin and other cell adhesion molecules may function redundantly, and thus, the IPL structure would be kept intact in the afadin cKO retina. We have added this interpretation to the Discussion section of the revised manuscript (lines 998–1005).

(7) In the RESULTS section on page 20, the authors state, "We further investigated adherens junctions (AJs) in the cKO retina by immunostaining with OLM adherens junction markers β-catenin, N-cadherin, and nectin-1. We found that these signals were dispersed in the cKO retina (Figure S2C)." It appears that β-catenin, N-cadherin, and nectin-1 can still be detected in the cKO retina.

We agree with the reviewer that β-catenin, N-cadherin, and nectin-1 can still be detected in the cKO retina. We used the term 'dispersed' to indicate that the signal was “scattered” rather than “disappeared”. To avoid confusion, we have revised the wording in the revised manuscript (line 499).\

(8) In Figure 3, please indicate where the zoomed-in images were captured from the low-power images. Additionally, point out the locations of zoomed-in images in other figures as well.

Following the reviewer’s suggestion, we updated Figures 2D, 3A-C, 4A, S2D, S3A, S3D, S3E, and S5D. The related Figure legends have also been revised.

(9) The authors should include individual data points in all statistical graphics to provide a clearer presentation of the data.

As suggested by the reviewers, we have revised all statistical graphs to display individual data points. Furthermore, the statistical analysis of synapse counts in Figures 3E, 3F, and S3C has been changed to linear mixed models (LMM) or generalized LMM to account for the variability in the number of synapses within individual mice.

(10) In the RESULTS section on page 23, the statement "These data indicate that the rosette-like structure in the cKO may be an ectopic IPL, termed 'acellular patches'". What is the mechanism that may cause the rosette-like structure to translocate from the IPL to the outer region of the retina?

Thank you for raising a valuable question. To clarify the mechanism of acellular patch formation in the cKO mice, we analyzed the position of RGCs and ACs in the developing cKO retina. In the cKO retina at P1, retinal cells were organized into distinct multicellular compartments with clear boundaries, and acellular regions extending to the outer retinal surface were observed at these boundaries. These acellular regions contained dendritic processes of RGCs and ACs, which are components of the IPL, indicating that elements of the IPL extended vertically across the retina. As development progressed, the compartment boundaries gradually shifted toward the inner retina. At P14, the IPL was mainly located on the inner retina, as in the normal retina. However, some IPL structures remained in the outer retina and may correspond to the acellular patches. We have included these findings in the revised manuscript as Figures S5A and S5B and added the corresponding description to the text (lines 643–665).

(11) Is the blood vessel structure normal in the cKO retina? Could this impact the survival of retinal cells?

Thank you for your valuable comment. We performed immunostaining with an anti-CD31 antibody, a marker for blood vessels, as shown in the new Figure S2G. No apparent differences were observed in the cKO retina. We have added the following description to the revised manuscript (lines 539–543):

“It has been reported that defects in the distal processes of Müller glia are associated with abnormal retinal vasculature (Shen et al., 2012). Thus, we immunostained the cKO retina with anti-CD31, a blood vessel marker, but no apparent vascular abnormalities were detected (Figure S2G).”

(12) In the RESULTS section on pages 26-29, there is a lot of statistical information included in parentheses. It would be more concise to place this information in the figure legends, if possible.

Following the reviewer's suggestion, we have moved the statistical information from the main text (pages 26–29) to the corresponding Figure legends.

(13) In the RESULTS section on page 28, the authors state, "On the other hand, the inner retina was apparently normal, and both the inner nuclear layer (INL) and IPL could be recognized." However, in Fig 7A, it appears that the INL is mixed with the ONL and cannot be clearly identified.

We agree with the reviewer that the INL is mixed with the ONL and cannot be clearly identified. Accordingly, we have revised the description in the text (lines 740–742) as follows:

“On the other hand, the inner retina was apparently normal, and both the IPL and the proximal part of the INL could be recognized.”.

(14) It is mentioned in the manuscript that "The receptive field (RF) area in the cKO retinas was significantly smaller than that in the cHet retinas." Is there an impairment in the dendritic fields of RGCs in the cKO retina that could lead to a smaller RF?

Thank you for asking an interesting question. The dendritic field reflects the region where presynaptic cells can form synaptic contacts, whereas the receptive field is dynamically shaped by spatiotemporal excitatory and inhibitory inputs, gap junctions, and membrane properties of the dendrites. Consequently, the size of the dendritic field does not necessarily correspond to that of the receptive field. Moreover, the disruption of the retinal lamination in the afadin cKO retina may alter the morphology of RGC dendritic fields—even when RNA expression levels are identical—which makes it difficult to exactly compare the morphology of the same RGC subtype between afadin cHet and afadin cKO retinas. Additionally, due to the presence of over 40 RGC subtypes and the rosette-like structures in the afadin cKO retina, it is challenging to trace the complete dendritic arborization of individual RGCs. For these reasons, we rather hesitate to compare the dendritic field size and the receptive field size.

(15) Figure 7H was not cited in the corresponding section of the main text.

Thank you for pointing it out. We have added a citation of Figure 7H in the revised manuscript (line 759).

(16) In Figure 8C, is there a difference in the number of pHH3+ mitotic cells between the cHet and cKO mice?

We quantified the number of pHH3-positive cells in the cKO retina at P0, as shown in the new Figure 9B. The number of mitotic cells was significantly increased in the cKO retina (see lines 853-855). In contrast, the number of BrdU-labeled progenitor cells at P1, P3, and P5 was not significantly different between cHet and cKO retinas, as presented in the new Figure S6C. These results suggest that although the total number of progenitor cells remain unchanged in cKO retinas, the M phase may be prolonged.

(17) The results related to Figure 8 should be moved to a location before Figure 5, as Figure 8 is also related to the lamination defects.

In the original manuscript, Figures 2–7 presented the phenotypes observed in the cKO retina, while Figure 8 addressed the possible cause of the lamination defects. Since the revised Figure 8 presents behavioral tests evaluating visual function, the phenotypic analyses are presented in the revised Figures 2–8. In response to the reviewers’ comments, we further analyzed the distribution of mitotic and progenitor cells during development and included these results as revised Figure 9.

(18) In the DISCUSSION section on page 32, the authors state, "A few photoreceptor-bipolar cell-retinal ganglion cell (BC-RGC) pathways (vertical pathways of the retina) are inferred to be maintained in the cKO retina." The authors could verify this using retrograde transsynaptic tracing with a pseudorabies virus injected into the superior colliculus.

Thank you for your interesting suggestion. This is an important point, and the recommended experiment idea sounds excellent. We attempted this analysis; however, the virus injected into the superior colliculus successfully labeled RGCs but failed to reach BCs and photoreceptors in normal mice. We guess that light stimulation evoked RGC firings evidently show that the photoreceptor-bipolar cell-retinal ganglion cell (BC-RGC) pathways function.

Reviewer #2 (Public review):

Summary:

Ueno et al. described substantial changes in the Afadin knockout retina. These changes include decreased numbers of rods and cones, an increased number of bipolar cells, and disrupted somatic and synaptic organization of the outer limiting membrane, outer nuclear layer, outer plexiform layer. In contrast, the number and organization of amacrine cells and retinal ganglion cells remain relatively intact. They also observed changes in ERG responses, RGC receptive fields and functions, and visual behaviors. The morphological and function characterization of retinal cell types and laminations is detailed and relatively comprehensive.

Reviewer #1 (Public review):

Summary:

The question of how central nervous system lamination defects affect functional integrity is an interesting yet debated topic. The authors investigated the role of afadin, a key adherens junction scaffolding protein, in retinal lamination and function using a retina-specific conditional knockout mouse model. Their findings show that the loss of Afadin caused severe outer retinal lamination defects, disrupting photoreceptor morphology, synapse numbers, and cell positioning, as demonstrated by histological analysis. Despite these structural impairments, retinal function was partially preserved: mERG detected small a- and b-waves, retinal ganglion cells responded to light, and behavioral tests confirmed residual visual function. This research offers new insights into the relationship between retinal lamination and neural circuit function, suggesting that altered retinal morphology does not completely eliminate the capacity for visual information processing.

Strengths:

The study effectively employs the well-organized laminar structure of the retina as an accessible model for investigating afadin's role in lamination within the central nervous system. High-quality histological, immunostaining, and electron microscopy images clearly reveal structural defects in the conditional knockout mice. The revised manuscript significantly enhances the findings by incorporating robust quantitative analyses of cell positioning, retinal thickness, and cell numbers, as well as new assessments of developmental defects. Additionally, new behavioral tests, including the optomotor response and visual cliff tests, have been introduced. Together with electrophysiological recordings, these additions compellingly demonstrate the partial preservation of visual function despite severe structural disruptions.

Weaknesses:

Overall, the study of the mechanisms remains weak. While the authors addressed concerns about molecular mechanisms by examining cell proliferation potentially related to Notch and Wnt signaling (Figure S6C, lines 868-870), the findings are largely negative (no significant changes in progenitor cell numbers), and the discussion of alternative pathways remains speculative.

eLife Assessment

This study demonstrates that conditional knockout of afadin disrupts retinal laminar organization and reduces the number of photoreceptors, while preserving certain aspects of retinal ganglion cell structure and light responsiveness. The work is valuable and well-supported by revised figures and comprehensive data on retinal cell types, lamination patterns, and visual functio. The findings are solid and intriguing, and the study provides insights into the relationship between retinal lamination and neural circuit function.

Oates consistently uses first person language and makes an effort to make this writing very personal. I think this is to make a more impactful argument because it shows more emotion, and therefore could mean more to the reader. But is this just a normal practice for this form of writing? Or is it specific to this, setting it aside from other writings?

Terra nullius = territory which legally belongs to no state, but as the article states, is a legal fiction that was used to justify colonization.

AI's promised innovation only benefits those that bias is in favor of

Does this sentence need a qualifier about the type of task?

key difference between deep learning and computational neuroscience.

There is a lot of mention of biological and neurological sciences in the history of deep learning. However, a group that may be should get some recognition in this field are the computational chemists. Computational chemists, since the 70's, have been using sophisticated techniques (LCAO, density functional theory, coupled cluster theory, etc.) to develop models for multi-particle systems. These have aspects of parameter fitting, updating weights through convolution layers, and feedback loops for updating models.

This is interesting. How important is the concept of 'depth' in deep learning?

A feedback loop that updates the prior based on new information to eventually reach a good posterior.

Are the simpler representations some sort of 'building blocks'?

The 'representations' displayed here are just transformations of the dataset. With multidimensional data, it is perhaps also important to recognize how the data was generated, and if there are causal hints as to which representation to utilize.

You may also view the table form as in the abstract definition of a function

$$ f = {(1, \sqrt{2}), (2, \pi), (3, -1)} \subseteq {1, 2, 3}\times \mathbb{R}. $$

deneme

Very much giving "we created a problem, but don't worry, we can create a solution to our problem!" But worse because the problem doesn't even really exist yet.

For those that are privileged, the biggest problem with A.I. is a doomsday hypothetical scenario that A.I. currently does not have any capacity whatsoever to act on, rather than the present.

This tracks with the popularity of right wing content I've been seeing for a while (for example, manosphere podcasts).

Google says a different story happened than what Gebru claimed but won't release investigation results? Hmm...

👏 Well put.

This!!! A.I. is essentially regurgitating what its been fed, and if it is fed on data by humans with certain biases it will return outputs with bias.

Reminds me of the most recent case I saw of the ramifications of this technology in criminal policing: Trevis Williams, who was wrongly arrested for a sex crime he didn't commit due to a false match with the NYPD's A.I. facial recognition technology.

The article also mentions Robert WIlliams, who I also remember pretty clearly because his case had a bit more virality since it is largely considered the first case of these false matches. The ramifications of that lack of diversity have already happened.

The comparison and accusation of systematic bias is 100% true

I find it interesting that french is spoken so much around the world. It's interesting due to the fact that from where I've always lived there are always many more hispanic languages. I view European languages as incredibly foreign and rare.

The french language is used to share all different types of media and culture.

They author is trying to convey that people all over the world speak french.

I believe this text is meant to inform people about the diversity among french people and people who speak french.

The evolution of how many people speak each language over time.

This conclusion reflects on how literacy shaped they writer's life and future. They wonder if their success in school and acceptance to UCf would have been possible without their literacy sponsors and multiliteracies. The final message is hopeful, no matter how confusing life gets, literacy has taught them to adapt, keep going, and "don't panic."

This section shows how being on the debate team helped a writer build new literacies. They learned not just to write and speak clearly, but also to use strategies, read signals, and control arguments, similar to Mirabelli's idea of power struggles in communication. Debate even shaped how the writer approaches writing today, always thinking about evidence and possible weaknesses in their arguments.

Pasqualin highlights the impact and importance of learning the many different types of literacies the world has to offer. The way he has shared his experience has such power and gives learning the many different types of literacies a new meaning.

The writer shows how different jobs helped them develop "multiliteracies." As a busboy, they learned technical language about food and drinks, but also how to read body language. Later, working at a pet store, they learned scientific terms about animals and how to communicate with customers. These examples connect to Mirabelli's ideas about how service jobs require complex literacy skills, even if people think of them as "low-skilled."

This shows how the writer learned to love reading. At school they felt left out and called "stupid," but home their mom pushed them to keep trying. With her help and support, they became confident in reading and saw their mom as their biggest influence.

This is a great motto to live by/remember. We live in a world where one small thing could lead us to spiral and question ourselves. Where if a drastic change happens, if we go through something traumatic, or if something just simply doesn’t go the way we plan it to, our own little world just stops. I find that most people try to constantly make sense of it when these things happen, but learning that these things don’t always need to make sense to us can drastically change the way we live our lives.

This makes me wonder, if his mom had taken a different approach to helping him learn, would his love for reading and writing be different or would it be the same? Would it spark his love for a different subject rather than reading and writing?

I like how he uses one of his favorite books as evidence of his reading habits, making the overall narrative more humorous and creative.

I really like how Pasqualin connects personal experience with a larger idea of literacy. It makes the essay feel relatable rather than academic.

The use of dialogue and body language humanizes the narrative and makes it more approachable. I like his writing style because it helps the reader be more engaged.

When I read any big assignments, I like doing Cornell notes so I can understand what I have read, and I put my questions down right away.

My attention span is all over the place but I noticed when I had a good book I enjoyed reading more. My writing was also effected!

Trying to read when you're tired makes it hard to stay focused or remember what you read. It is easier for me to stay focused in the day with a full stomach so there is no distractions.

I agree that learning to communicate in the genres of your workplace is important. Every field has its own way of writing and speaking.

Is there a word/encyclopedia entry for this? We need it.

I am blind and it took me too long to realize where this was located but Great Syllabus! love the attention to detail

This isn't only a teacher problem, its a student problem. If teachers are having a hard time, so are the students. These problems affect everybody, even those who we're trying to help and educate.

Many problems educators face day to day isn't within the classroom. The reason for many so called burnouts come from up higher.

This statistic comes as no surprise. I know many teachers have grown frustrated due to these factors. Many have grown frustrated, though of leaving the profession, and giving up.

Absolutely. We need to have a reliable team, parents, administrators, staff members, etc. if we want our students to succeed.

I believe that as a school district, we are lucky enough to get a para if someone is having extreme behaviors, but some behaviors go without being helped because they are "mild", but it can be a lot of work to continue to correct these behaviors and exhausting.

All of those things in the graph are so valid. There is so much that goes into teacher that people don't understand so it makes sense that it causes so much stress.

Exactly. If are experiencing burnout, students are the most affected by it.

I think that there is not enough time for a teacher to plan all the time for the class size that they have.

I don't think teachers should have to be the one to enforce this rule, this rule should already be enforced before it even gets to the teacher.

I think we need to find more efficient ways to help teachers with stress and burnout. I think teachers are relied on too much, they are asked to put too much on their shoulders.

Although I agree that teachers should be paid more, I don't think teacher retention should depend on pay. Teacher don't go into teaching for the pay anyways so I think this is a hard thing to say.

This is where the Special Education teachers are getting burnt out this most for what I have seen.

In this set of sentences, the author is trying to describe the almost unnatural beauty of this woman. To do so the author goes into extreme detail, describing the clothing that she wears as "spotless linen" and "dye from the bats of Alexandria" The author also describes her features "fresh and slender" and "rondure of her bosom showed whiter and more untouched" meaning the that her chest was round and clean. The words Fresh and White describe her cleanliness. Overall these sentences describe her beauty which ties later to the story that no one, not even the queen was more fair and beautiful than this woman.

it's not to say chatgpt is completely bad, it has resources that can help you expand and improve on your original work with it still being your original work and not falsified.

the brief contained false cases, why include fake cases knowing they might be looked into? all to proceed a lawsuit

it is indeed a "existential threat to humanity" the more we lean on chat the less we are capable of thinking for ourselves since we'll come to believe that chatgpt automatically "knows everything".

main topic of article

how is he not aware of a document under his own name?

I am definitely most intrigued by this section because he is teaching the students how to read rhetorically. This stood out to me the most because I agree that this is how kids should be learning to read as they will need to use this type of reading in the future.

The lack of context in the tests will affect the students because they are not having to critically think. All of the tests they are having to read are not real situations or have a real purpose, this means that they are not gonna learn how to read in a critical way.

To me this definitely sticks with me. throughout all of high school and middle school I have struggled with reading. Being able to read is way more important than getting a 36 on the ACT. You use reading in your everyday unlike the standardized test questions.

Its crucial for staff to find ways around standard based grading for success. If it's to lenient, students will fail to do work and more importantly to learn. If to strict, there's really no point at all to the change.

This is the great thing about standards based grading. Students wont always, even not often, fully grasp the concept the first time around. With the second time around, and with more practice and opportunities, it gives the students a chance to really master the skill and content. Rather then just getting bye.

This is what grade based grading is about. Students completing work and turning it in. It doesn't reflect how a student really understands content.

I agree 100%. Even growing up for me when social media was fairly new, the toll it took on many people's mental health because of Instagram especially was crazy and so sad to see.

Plus, they may hear it from someone else's phone, but think it's theirs. They are constantly flipping their phones to check.

Absolutely! That is huge. Once COVID hit, people became more attached to cell phones than ever before.

This sentence I really like. As a student I sometimes feel like my teachers are competing with social media and my phone so I have to imagine it's the same for them.

Colloquialism is a word or phrase that is not formal or literary, typically one used in ordinary or familiar conversation.

This is a very powerful statement. I feel like this would make some people uncomfortable due to how true it is.

Satirical means containing or using satire.

Ubiquitous means present, appearing, or found everywhere.

a-splenic (if the word must be split between 2 lines)

(Globin gene duplication, either alpha- or beta-globin genes, can also cause an imbalance in the abundance of alpha- and beta-globin proteins, also resulting in pathology.)

chains, also called the alpha-globin gene cluster,

chains, also called the beta-globin gene cluster,

I think that this can be used as a tool to study, I have see math teachers have students ask ChatGPT to give them 10 practice problems over the lesson/chapter for a great use of their time and also helps students study

They may not judge you, but it's also still a computer. It's not a real person who has feelings and may prevent you from doing something that you shouldn't do. For example, if you are fighting with a friend and you hit them for no reason, you can manipulate ChatGPT to be on your side.

One thing I wonder is if AI will eventually take away some jobs in education.

I am all for lightening the work load for teachers because we have so many things to do constantly. Making work a little easier by creating it to your style is very intriguing. As a teacher, you still would need to proof read it and fix it if needed.

Another helpful tool that personalizes students' learning with their scores from beginning of the year testing is IXL. Many of the teachers utilize that in my district for math and reading.

I have never heard of these tools and would be curious to know more about them.

That is a huge struggle. I've noticed that students are getting flagged for using "--" to separate sentences. What if they are flagged but it's actually their work?

AI can be so helpful with homework, IF we use it correctly. It could be so great for students if they are at home, really having a tough time with a math problem. AI could help the student solve the problem in a way that doesn't give them the answer, but instead they are only using it to copy the answers.

I agree with this but I think it depends on the student. some students are going to wait to do the hardest things until the last minute.

Time management needs to be started as early as possible, it is probably the longest to figure out.

Very interesting to read, I have not heard this before myself but am not shocked to hear it here

I found this the most interesting because policies for student safety requires careful, community-specific planning.

Although cyberbullying may stop, traditional in-person bullying might come back full force.

Pair any restriction with instruction on self-regulation and healthy digital habits.

This pushes educators away from quick fixes and toward goal-driven policy design.

Clearly state whether the goal is academic focus, safety, or social wellbeing. Keep in mind different goals need different approaches.

quality over quantity (while meeting word count requirements)

Tone is so important to the purpose of your writing and knowing when it's appropriate for casual language or not is just as important.