Reviewer #3 (Public review):

Summary:

In the present work, the authors offer evidence for the therapeutic potential of trained immunity in the context of inflammatory bowel disease (IBD). Prior research has demonstrated that innate cells pre-treated (trained) with β-glucan show an enhanced pro-inflammatory response upon a second challenge with the same or different stimulus. While an increased immune response can be beneficial and protect against bacterial infections, there is also the risk that it will worsen symptoms in various inflammatory disorders.

Remarkably, the authors show that β-glucan training of bone marrow hematopoietic progenitors and peripheral monocytes mitigates the pro-inflammatory effects of colitis, with protection extending to naïve recipients of the trained cells. Additionally, the authors demonstrate that mice preconditioned with β-glucan have enhanced resistance to Staphylococcus aureus and Salmonella typhimurium infections, indicating heightened immune responses.<br /> Using a dextran sulfate sodium (DSS)-induced model of colitis, β-glucan pre-treatment significantly dampens disease severity. Importantly, the use of Rag1^-/- mice, which lack adaptive immune cells, confirms that the protective effects of β-glucan are mediated by innate immune mechanisms. Further, experiments using Ccr2^-/- mice underline the necessity of monocyte recruitment in mediating this protection, highlighting CCR2 as a key factor in the mobilization of β-glucan trained monocytes to inflamed tissues. In addition, β-glucan training highlights a distinct monocyte subpopulation with enhanced activation and phagocytic capacity. These monocytes, marked by increased expression of Cx3cr1, are suggestive of an increased ability to infiltrate inflamed colonic tissue and differentiate into macrophages.

Transcriptomic profiling reveals that β-glucan training upregulates genes associated with pattern recognition, antimicrobial defense, immunomodulation, and interferon signaling pathways, suggesting broad functional reprogramming of the innate immune compartment. Moreover, among the trained monocyte and macrophage subsets, gene expression signatures are associated with tissue and mucosal repair, suggesting a role in promoting resolution and regeneration following inflammatory insult. Furthermore, this was coupled with analysis of chromatin accessibility in publicly available data.

Strengths:

By employing a range of well-characterized murine models, the authors investigate specific mechanisms involved in the effects of β-glucan training. Furthermore, the study provides functional evidence that the protection conferred by the trained cells persists within the hematopoietic progenitors and can be transferred to naïve recipients. The integration of transcriptomic profiling allows the identification of changes in key genes and molecular pathways underlying the trained immune phenotype.

Weaknesses:

Further studies would benefit from investigating the cytokine responses of intestinal macrophages, particularly CX3CR1⁺ macrophages, following ex vivo stimulation of previously BCG-trained cells. Moreover, assessing the metabolic state of these macrophages would provide valuable insight into the mechanisms underlying trained immunity in this context.

Impact:

Overall, the authors present a mechanistically insightful investigation that advances our understanding of trained immunity in IBD. This is an important study that demonstrates that β-glucan-trained innate cells can confer protection against colitis and promote mucosal repair through trained-immunity related mechanisms. These findings underscore the potential of harnessing innate immune memory as a therapeutic approach for chronic inflammatory diseases.

Reviewer #3 (Public review):

Summary:

Several recent findings indicate that forces perpendicular to the microtubule accelerate kinesin unbinding, where perpendicular and axial forces were analyzed using the geometry in a single-bead optical trapping assay (Khataee and Howard, 2019), comparison between single-bead and dumbbell assay measurements (Pyrpassopoulos et al., 2020), and comparison of single-bead optical trap measurements with and without a DNA tether (Hensley and Yildiz, 2025).

Here, the authors devise an assay to exert forces along the microtubule axis by tethering kinesin to the microtubule via a dsDNA tether. They compared the behavior of kinesin-1, -2, and -3 when pulling against the DNA tether. In line with previous optical trapping measurements, kinesin unbinding is less sensitive forces when the forces are aligned with the microtubule axis. Surprisingly, the authors find that both kinesin-1 and -2 detach from the microtubule more slowly when stalled against the DNA tether than in unloaded conditions, indicating that these motors act as catch bonds in response to axial loads. Axial loads accelerate kinesin-3 detachment. However, kinesin-3 reattaches quickly to maintain forces. For all three kinesins, the authors observe weakly-attached states where the motor briefly slips along the microtubule before continuing a processive run.

Strengths:

These observations suggest that the conventional view that kinesins act as slip bonds under load, as concluded from single-bead optical trapping measurements where perpendicular loads are present due to the force being exerted on the centroid of a large (relative to the kinesin) bead, need to be reconsidered. Understanding the effect of force on the association kinetics of kinesin has important implications for intracellular transport, where the force-dependent detachment governs how kinesins interact with other kinesins and opposing dynein motors (Muller et al., 2008; Kunwar et al., 2011; Ohashi et al., 2018; Gicking et al., 2022) on vesicular cargoes.

Weaknesses:

The authors attribute the differences in the behaviour of kinesins when pulling against a DNA tether compared to an optical trap to the differences in the perpendicular forces. However, the compliance is also much different in these two experiments. The optical trap acts like a ~ linear spring with stiffness ~ 0.05 pN/nm. The dsDNA tether is an entropic spring, with negligible stiffness at low extensions and very high compliance once the tether is extended to its contour length (Fig. 1B). The effect of the compliance on the results is not fully considered in the manuscript.

Compared to an optical trapping assay, the motors are also tethered closer to the microtubule in this geometry. In an optical trap assay, the bead could rotate when the kinesin is not bound. The authors should discuss how this tethering is expected to affect the kinesin reattachment and slipping. While likely outside the scope of this study, it would be interesting to compare the static tether used here with a dynamic tether like MAP7 or the CAP-GLY domain of p150glued.

In the single-molecule extension traces (Fig. 1F-H; S3), the kinesin-2 traces often show jumps in position at the beginning of runs (e.g. the four runs from ~4-13 s in Fig. 1G). These jumps are not apparent in the kinesin-1 and -3 traces. What is the explanation? Is kinesin-2 binding accelerated by resisting loads more strongly than kinesin-1 and -3? In their response, the authors provide an explanation of the appearance of jumps due to limited imaging speeds. The authors state that the qualitative difference in the kinesin-2 traces compared to the kinesin-1 an -3 traces may be due to the specific rebinding kinetics of kinesin-2.

When comparing the durations of unloaded and stall events (Fig. 2), there is a potential for bias in the measurement, where very long unloaded runs cannot be observed due to the limited length of the microtubule (Thompson, Hoeprich, and Berger, 2013), while the duration of tethered runs is only limited by photobleaching. Was the possible censoring of the results addressed in the analysis? The authors addressed this concern by applying a Markov model to estimate the duration parameter.

The mathematical model is helpful in interpreting the data. To assess how the "slip" state contributes to the association kinetics, it would be helpful to compare the proposed model with a similar model with no slip state. Could the slips be explained by fast reattachments from the detached state? In their response, the authors addressed this question by explaining that a three-state model is required to model the recovery time distributions.

Reviewer #3 (Public review):

Summary:

This study reports KRH, a SaCas9 variant computationally engineered via UniDesign to recognize an expanded NNNRRT PAM with substantially enhanced editing efficiency at non-canonical sites. KRH achieves genome- and base-editing efficiencies comparable to or exceeding the evolution-derived KKH variant across multiple human cell types, demonstrating that computational design can effectively remodel PAM specificity while preserving nuclease activity.

Strengths:

The research follows a clear line of reasoning, and the results appear sound. The computational design strategy presented offers a valuable alternative to directed evolution, with potential applicability beyond Cas9 engineering.

Weaknesses:

The benchmarking of the UniDesign method is insufficient. How its performance compares to other protein design algorithms, whether the energy function parameters were systematically optimized, and if the design strategy can be generalized to other Cas9 orthologs or genome engineering tasks.

Reviewer #3 (Public review):

Summary:

The authors aimed to investigate whether disruption of intracrine steroid hormone metabolism contributes to meibomian gland dysfunction and proposed a "vicious cycle" of gland dysfunction and inflammation, using a global Had3b6 knockout mouse model. The work addresses an important aspect of MGD, but its impact may be limited unless the intracrine mechanism can be more clearly distinguished from systemic hormonal effects.

Strengths:

This study addressed an important question. The hormonal regulation of the meibomian gland has long been recognized. If clarified, the concept of local steroid metabolism influencing gland homeostasis could have implications for understanding disease mechanisms and identifying therapeutic targets.

Weaknesses:

The use of a global knockout makes it difficult to separate local intracrine effects from systemic hormonal changes, and key controls and hormone measurements are lacking.<br /> LPS-induced inflammation may not reflect the chronic nature of MGD.

Reviewer #3 (Public review):

Summary:

The manuscript by Kim et al. describes a MuSC subpopulation that loses VCam expression in geriatric muscle and shows reduced ability to contribute to muscle regeneration. They propose that this population underlies the reported decline of MuSCs in aged mice, suggesting that these cells remain present in geriatric muscle but are overlooked due to low or absent VCam expression. The identification of a subpopulation that changes with aging would be compelling and of interest to the field.

Strengths:

The authors employ a wide range of assays, from in vitro to in vivo systems, to characterize Vcam-low/negative cells from geriatric muscle. The loss of Vcam appears strong in geriatric mice. They further identify CD63 and CD200 as potential surface markers that remain stable with age, thereby enabling the isolation of MuSCs across different age groups.

Weaknesses:

Some issues remain before establishing whether this population represents a true functional subset or explains the reported decline in MuSC numbers in aged mice. Stronger fate assessment of Vcam-low/negative cells is needed to assess their propensity for cell death and whether this contributes to the conclusions. Comparisons include young, middle-aged, and geriatric mice, but not aged (~24 months) mice, which would help comparisons to previous reports of age-related MuSC decline. The suggestion that the Vcam-low/negative population reflects a senescence-like state remains unclear, as these cells display limited canonical senescence markers, exhibit reversible cell-cycle exit, and yet are reported to be sensitive to senolytic treatment. Validation of CD63 and CD200 as reliable age-independent MuSC markers requires further testing, specifically using the Pax7-YFP tracing model and co-labeling in geriatric mice. Finally, the grouping patterns in some analyses suggest that the Vcam-low/negative fraction may be present in only a subset of geriatric mice, raising the possibility that it reflects health status or pathology rather than a consistent aging-associated phenotype.

Reviewer #3 (Public review):

Summary:

This study aimed to investigate pseudouridylation across various RNA species in multiple bacterial strains using an optimized BID-seq approach. It examined both conserved and divergent modification patterns, the potential functional roles of pseudouridylation, and its dynamic regulation across different growth conditions.

Strengths:

The authors optimized the BID-seq method and applied this important technique to bacterial systems, identifying multiple pseudouridylation sites across different species. They investigated the distribution of these modifications, associated sequence motifs, their dynamics across growth phases, and potential functional roles. These data are of great interest to researchers focused on understanding the significance of RNA modifications, particularly mRNA modifications, in bacteria.

Reviewer #3 (Public review):

Zhang et al. investigated how germline tumors influence the development of neighboring wild-type (WT) germline stem cells (GSC) in the Drosophila ovary. They report that germline tumors generated by differentiation-arrested mutations (bam and bgcn) inhibit the differentiation of neighboring WT GSCs by arresting them in an undifferentiated state, resulting from reduced expression of the differentiation-promoting factor Bam. They find that these tumor cells produce low levels of the niche-associated signaling molecules Dpp and Gbb, which suppress bam expression and consequently inhibit the differentiation of neighboring WT GSCs non-cell-autonomously. Based on these findings, the authors propose that germline tumors mimic the niche to suppress the differentiation of the neighboring wild-type germline stem cells.

Strengths:

The study uses a well-established in vivo model to address an important biological question concerning the interaction between germline tumor cells and wild-type (WT) germline stem cells in the Drosophila ovary. If the findings are substantiated, this study could provide valuable insights that are applicable to other stem cell systems.

Weaknesses:

The authors have addressed some of my concerns in the revised submission. However, the data presented do not allow the authors to distinguish whether the failed differentiation of WT stem cells/germline cells results from "arrested differentiation due to the loss of the differentiation niche" or from "direct inhibition by tumor-derived expression of niche-associated molecules Dpp and Gbb". The critical supporting data, HCR in situ results, are not sufficiently convincing.

Reviewer #3 (Public review):

Summary:

This work uses a convolutional neural network to optimize a voltage clamp protocol to identify features and parameters from human pluripotent stem cell-derived cardiomyocytes.

Yang et al. introduce an innovative experimental framework that integrates computational modeling and deep learning to generate a digital twin of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs).

Strengths:

The major strength is the methodology used to bridge in silico prediction of cell behavior and mechanistic insights from the experimental dataset.

The approach used in this study represents a significant step toward precision medicine by enabling in silico prediction of cellular behavior and mechanistic insight from experimental datasets. The study addresses an important and timely challenge in stem cell-based and personalized medicine, and the authors compellingly leverage state-of-the-art methods alongside strong expertise in computational modeling and cardiac electrophysiology

Weaknesses:

While the overall approach is highly compelling and the potential impact is substantial, there are two areas where clarification and refinement, particularly in the phrasing and framing used throughout the manuscript, would further strengthen the work.

(1) While the overall goal of the study is compelling, the manuscript would benefit from clearer articulation of how the proposed framework is intended to be used in practice. In particular, it is not entirely clear whether the authors envision this approach as:

a) a method to extract population-level trends that, when paired with biological data, enhance statistical power and interpretability, or

b) a strategy capable of constructing a population-based model from limited single-cell recordings. If the latter is intended, additional guidance on the number of action potentials required per cell and the assumptions underlying this extrapolation would greatly clarify the scope and applicability of the method.

(2) The manuscript would also benefit from a clearer explanation of how electrophysiological heterogeneity observed in hPSC-CMs is linked to inter-patient variability. Although the authors state that this framework can be generalized to compare patient-specific hiPSC-CM lines, it remains unclear how this generalization is achieved, given the substantial sources of variability intrinsic to hiPSC-CMs (e.g., batch effects, reprogramming strategy, differentiation protocol, and maturation state). As acknowledged by the authors, addressing this level of variability likely requires large datasets; further clarification of how the proposed approach mitigates or accommodates these challenges would strengthen the translational claims.

Below are my suggestions that could help strengthen the claims in the manuscript:

(1) Adding a dedicated section describing the electrophysiological phenotype of the hPSC-CMs used in this study would help justify the choice of the underlying ionic model and the selection of the six ion currents analyzed. These currents are not only developmentally regulated but may also vary substantially across different hPSC-CM lines, which has implications for generalizability.

(2) If feasible, inclusion of patch-clamp data from an additional hPSC-CM line would significantly strengthen the claim that this framework can harmonize and generalize across datasets and cell sources.

(3) The authors note that the experimental cells exhibited high variability in action potential morphology. This is an important observation that directly supports the motivation for the study and should be explicitly presented, even if only in the supplementary materials.

(4) In the hERG-blocker experiments, further clarification is needed regarding the biological relevance of the reported 3% incidence of early afterdepolarizations (EADs). Additionally, an interrupted sentence in this section makes it unclear whether the goal is to demonstrate that the digital twin can capture rare arrhythmic risk events or whether the digital twin is necessary to determine whether this level of risk is clinically meaningful.

(5) The manuscript states that some action potentials were excluded from the experimental dataset. A brief explanation of the exclusion criteria, along with guidance on how to distinguish high-quality from low-quality recordings, would improve transparency and reproducibility.

Reviewer #3 (Public review):

Summary:

The paper describes the structure of gp5.4, the spike tip of phage T4. This structure was released in the PBD in 2013. The paper further investigates the role of this protein in virion assembly, stability, and infection by comparing the behaviour of the WT phage and a phage without the protein, resulting from an amber mutation in the phage genome. A competition assay between the WT and mutant phage shows a clear increase in the fitness of the WT. A further screening of a transposon bank allowed for the identification of a host strain that is resistant to the mutant phage while still sensitive to the WT phage.

Strengths:

(1) Beautiful structure, at very high resolution (1.15 Å).

(2) Very sophisticated microbiology experiments to allow mutant phage characterisation and dissect the role of the spike tip in phage fitness.

Weaknesses:

(1) The paper is very descriptive, and the lack of a general conclusion, not to say discussion, is frustrating. What do the findings of the paper bring to the knowledge of infection? What would be the fate of the spike and tip? A discussion in the context of the data available in the literature would greatly increase the interest of the paper.

(2) Why didn't the authors include the description of the structure of the homologous Pvc10 and PhiKV gp5.4 in complex with gp5ß, which they also solved a while ago?

(3) Because microbiology is sophisticated, special care should be taken to introduce the strains used (both E. coli and T4). E.g. it is still not clear to me what the difference is between the supF and the supD coli strains in terms of mutant phage produced (both should produce T4(5.4am)-gp5.4?).

(4) For the same reason, strains should always be called by the same name.

(5) In some sections, the conclusion seems lost in the description of controls (e.g. in the "The spike is translocated into the periplasmic space during infection" paragraph).

Appraisal:

The authors show that the sharp tip of the membrane-perforating tube of T4 contractile tail contributes to perforating the outer membrane. In particular, this protein is necessary in a host bearing mutated LPS.

PlayStation 3 received an update in March 2026 to allow people to continue using it as a Blu-Ray player. It was first released in November 2006.

Reviewer #3 (Public review):

Summary:

Gosselin et al. use the sybody technology to study effects of in vivo inhibition of the Bacillus subtilis SMC complex. Smc proteins are central DNA binding elements of several complexes that are vital for chromosome dynamics in almost all organisms. Sybodies are selected from three different libraries of the single domain antibodies, using the "transition state" mutant Smc. They identify 14 such mutant sybodies that are lethal when expressed in vivo, because they prevent proper function of Smc. The authors present evidence suggesting that all obtained sybodies bind to a coiled-coil region close to the Smc "neck", and thereby interfere with the Smc activity cycle, as evidenced by defective ATPase activity when Smc is bound to DNA.<br /> The study is well done and presented and shows that the strategy is very potent in finding a means to quickly turn off a protein's function in vivo, much quicker than depleting the protein.

The authors also draw conclusions on the molecular mode of action of the SMC complex. The provide a number of suggestive experiments, but in my view mostly indirect evidence for such mechanism.

My main criticism is that the authors have used a single - and catalytically trapped form of SMC. They speculate why they only obtain sybodies from one library, and then only identify sybodies that bind to a rather small part of the large Smc protein. While the approach is definitely valuable, it is biassed towards sybodies that bind to Smc in a quite special way, it seems. Using wild type Smc would be interesting, to make more robust statements about the action of sybodies potentially binding to different parts of Smc.

Line 105: Alternatively, the other libraries did not produce good binders or these sybodies were 106 not stably expressed in B. subtilis. This could be tested using Western blotting - I am assuming sybody antibodies are commercially available. However, this test is not important for the overall study, it would just clarify a minor point.

Fig. 2B: is odd to count Spo0J foci per cells, as it is clear from the images that several origins must be present within the fluorescent foci. I am fine with the "counting" method, as the images show there is a clear segregation defect when sybodies are expressed, I believe the authors should state, though, that this is not a replication block, but failure to segregate origins.

Testing binding sites of sybodies to the SMC complex is done in an indirect manner, by using chimeric Smc constructs. I am surprised why the authors have not used in vitro crosslinking: the authors can purify Smc, and mass spectrometry analyses would identify sites where sybodies are crosslinked to Smc. Again, I am fine with the indirect method, but the authors make quite concrete statements on binding based on non-inhibition of chimeric Smc; I can see alternative explanations why a chimera may not be targeted.

Smc-disrupting sybodies affect the ATPase activity in one of two ways. Again, rather indirect experiments. This leads to the point Revealing Smc arm dynamics through synthetic binders in the discussion. The authors are quite careful in stating that their experiments are suggestive for a certain mode of action of Smc, which is warranted.

In line 245, they state More broadly, the study demonstrates how synthetic binders can trap, stabilize, or block transient conformations of active chromatin-associated machines, providing a powerful means to probe their mechanisms in living cells. This is off course a possible scenario for the use of sybodies, but the study does not really trap Smc in a transient conformation, at least this is not clearly shown.

Overall, it is an interesting study, with a well-presented novel technology, and a limited gain of knowledge on SMC proteins.

Significance:

The work describes the gaining and use of single-binder antibodies (sybodies) to interfere with the function of proteins in bacteria. Using this technology for the SMC complex, the authors demonstrate that they can obtain a significant of binders that target a defined region is SMC and thereby interfere with the ATPase cycle.

The study does not present a strong gain of knowledge of the mode of action of the SMC complex.

Reviewer #3 (Public review):

Summary:

Tkacik et al. systematically characterized all three RAF kinase isoforms in vitro with all three types of RAF inhibitors (Type I, I1/2, and II) to investigate the mechanism underlying paradoxical activation.

In this study, the authors reconstituted heterodimers of A-, B-, and C-RAF kinase domains bound to non-phosphorylable MEK1 (SASA), mimicking the monomeric auto-inhibited state of RAF. These "RAF monomers" were tested for MEK phosphorylation with an increasing concentration of all three types of RAF inhibitors (Type I, I1/2, and II). This study is reminiscent of a previous study of the same team measuring RAF kinase activity in the presence of all three types of inhibitors in the context of dimeric RAF isoforms stabilized by 14-3-3 proteins (Tkacik et al 2025 JBC). RAF monomers had little to no activity at low concentrations of inhibitors (consistent with their "monomeric state"). Addition of type I1/2 inhibitor did not induce paradoxical activation as, in this context, they do not induce RAF dimerization required for activation, as observed by MP. Addition of type I and type II inhibitors led to paradoxical activation consistent with the RAF dimerization induced by these inhibitors, as observed by MP. Interestingly, type II inhibitors induced activation only for B- and C-RAF and not A-RAF.

At high concentrations of type II inhibitors, kinase activity is inhibited with a strong or weak positive cooperativity for BRAF and CRAF, respectively. This observation is very similar to what the authors previously observed with their dimeric RAF system. Interestingly, when the NtA motif is modified by phosphomimetic mutations in A- and C-Raf, basal kinase activity is stronger, but most importantly, inhibitor-induced paradoxical activation is much stronger with both type I and II inhibitors. This demonstrates that mutation of the NtA motif of ARAF and CRAF sensitized them to paradoxical activation by type II inhibitors.

The authors also tested the effect of ATP in the paradoxical activation observed in their RAF "monomer" system. As previously published in their assay with 14-3-3 stabilized dimeric RAF, the authors observed an expected shift of the IC50 with Type I inhibitors, while Type II inhibitors seem to behave as a non-competitive inhibitor. The authors next reconstituted the MAP kinase pathway (with RAF monomers at the top of the phosphorylation cascade) to test paradoxical activation amplification. Again, Type I1/2 inhibitors did not induce paradoxical activation, while Type I and II inhibitors did. The authors tested the inhibitors with FL auto-inhibited RAF/MEK/14-3-3 complexes, where, contrary to the "RAF monomers" experiments, FL B- and C-RAF were not paradoxically activated but were inhibited by all three types of inhibitors.

Overall, Tkacik et al. tackle an important question in the field for which definitive experiments and thorough biochemical investigation to understand the molecular mechanisms for the inhibitor-induced paradoxical activation are still missing, and of high importance for future drug development.

Strengths:

The biochemical experiments here are rigorously executed, and the results obtained are highly informative in the field to decipher the intricate mechanisms of RAF activation and inhibitor-induced paradoxical activation.

Weaknesses:

The interpretation of the results in the context of the current state of the art is ambiguous and raises questions about the relevance of introducing a new model for inhibitor-induced paradoxical activation, particularly since the findings presented here do not clearly contradict established paradigms. I believe some clarification and precision are required.

Main comments:

(1) Figure 2:

The authors comment on the expected greater increase (for a cascade assay) in the magnitude of ERK phosphorylation compared to what was observed for MEK phosphorylation. However, this observation might be reflective of the stoichiometries used in the assay, with 40 times more MEK compared to RAF concentration (250nm vs 6nM), which might favour pERK vs pMEK.

- The authors should clarify their rationale for the protein concentration used in this assay and explain how protein stoichiometry was taken into account for the interpretation of their results.

- In addition, the authors should justify comparing pMEK and pERK TR-FRET values when different anti-phospho antibodies were used. Antibodies may have distinct binding affinities for their epitopes. Could this not lead to differences in FRET signal amplitudes that complicate direct comparison?

(2) Supplementary Figure 2:

The author mentioned that the inhibitors did not activate the FL auto-inhibited RAF complexes; however, they did inhibit the TR-FRET signal.

- Can the authors comment on the origin of the observed basal activity? Would the authors expect self-release of the RAF kinase protein from the auto-inhibited state in the absence of RAS, leading to dimerization and activation? Alternatively, do the inhibitors at low-concentration relieve the auto-inhibited state, thereby driving dimerization and activation?

- Did the author test the addition of RAS protein in their in vitro system to determine whether "soluble" RAS is sufficient to release the protective interactions with RBD/CRD/14-3-3 and lead to inhibitor-induced paradoxical activation of FL RAF?

(3) Figure 5B:

The authors said that the Kd values obtained from their MP assay are consistent with prior studies of RAF homodimerization and RAF:MEK heterodimerization. While this is true from the previous studies of RAF:MEK interaction by BLI (performed from the same team), the Kd of isolated RAF kinase homodimerization has been measured around ~30µM by AUC in the cited ref (24,27 & 37).

- The authors should discuss the discrepancy between their Kd of homodimerization and the reported Kd values in the literature. At the concentration used for MP, it is surprising to observe RAF dimerization while the Kd of homodimerization has been measured at ~30µM (in the absence of MEK).

- Would the authors expect the presence of MEK to influence the homodimerization affinity for the isolated KD?

(4) Conclusions:

Several times in the introduction and the conclusion, the authors suggest that the negative allostery model (where "inhibitor binding to one protomer of the dimer promotes an active but inhibitor-resistant conformation in the other") is a model that applies to all types of RAF inhibitors (I, I1/2, and II).

However, from my understanding and all the references cited by the authors, this model only applies to type I1/2 inhibitors, where indeed the aC IN conformation in the second (inhibitor-free) protomer of the RAF dimer might be incompatible with the type I1/2 inhibitors inducing aC OUT conformation. The type I and type II inhibitors are aC IN inhibitors and are expected to bind both protomers from RAF dimers with similar affinities. Therefore, the negative allostery model does not apply to the type I and type II inhibitors. The difference in the mechanism of action of inhibitors is even used to explain the difference in the concentration range in which inhibitor-induced activation is observed in cells. The description of the state of the art in this study is confusing and does not help to properly understand their argumentation to revise the established model for paradoxical RAF activation.

- Can the authors clarify their analysis of the state of the art on the different mechanisms of action for the paradoxical activation of RAF by the different types of RAF inhibitors?

5) Conclusions:

"Our results suggest that negative allostery (or negative cooperativity) is not a requisite feature of paradoxical activation. The type I and type II inhibitors studied here induce RAF dimers and exhibit paradoxical activation but do so without evidence of negative cooperativity, nor do they appear to inhibit intentionally engineered RAF dimers with negative cooperativity (25). Indeed, type II inhibitors exhibit apparent positive cooperativity while type I inhibitors are non-cooperative inhibitors of RAF dimers (25)."

- Can the authors explain how results on the paradoxical activation induced by type I and type II inhibitors inform or challenge a model that specifically applies to type I1/2 inhibitors?

The authors often refer to their previous study (reference 25), where they tested the inhibition of all three types of inhibitors with engineered RAF dimers. While I agree with the authors that in reference 25 the Type I and type II inhibitors inhibit RAF dimers without exhibiting negative cooperativity (as expected from the literature and the current model), the authors did observe some negative cooperativity for Type I1/2 inhibitors in their study most particularly for the type I1/2 PB (with hill slope ranging from -0.4 to -0.9, indicative of negative cooperativity).<br /> While the observations that type II inhibitors display positive cooperativity is both novel and very interesting, from what I understand the results from thakick et al 2025 and the current study appear more in line with the current paradigm in the field (which describe paradoxical activation with negative cooperativity for type I1/2 inhibitors and no negative cooperativity for the Type I and II inhibitors) rather than disapproving of the current model and supporting for a new model.

- In this context, can the authors clarify how their results challenge the current model for paradoxical activation?

(6) Conclusions:

The authors describe the JAB34 experiment from Poulikakos et al. 2010 to conclude that "While this experiment cleanly demonstrates inhibitor-induced transactivation of RAF dimers, it is important to recognize that the differential inhibitor sensitivity of the two subunits in this experiment is artificial - it is engineered rather than induced by inhibitor binding as the negative allostery model proposes."

Indeed, the JAB34 experiment demonstrated the inhibitor-induced transactivation, but the Poulikakos et al. 2010 study does not discuss differential inhibitor sensitivity. The negative allostery model was proposed later by poulikakos team in other papers (Yao et al 2015 and Karoulia et al, 2016), in which JAB34 was not used.

- Can the authors clarify how the JAB34 experiments question differential inhibitor sensitivity?

(7) Conclusions:

"Considering that the conformation required for binding of type I.5 inhibitors destabilizes RAF dimers, it is unclear how an inhibitor binding to one protomer would be able to transmit an allosteric change to the opposite protomer, if that inhibitor's binding causes the existing dimer to dissociate."

- The authors should comment on whether 14-3-3 proteins might overcome negative regulation by type I1/2 inhibitors, similar to what has been shown for ATP, which acts as a dimer breaker like type I1/2 inhibitors.

(8) Conclusions:

"Furthermore, the complex effects of type I.5 inhibitors on dimer stability and the clear resistance of active RAF dimers to these inhibitors complicates interpretation of inhibition data - weak or incomplete inhibition of an enzyme can be difficult to discern from true negative cooperativity (43). As we discuss below, the clear resistance of RAF dimers to type I.5 inhibitors is alone sufficient to explain their ineffective inhibition during paradoxical activation, without invoking negative allostery."

- The authors should explain how they reconcile this statement and their proposal of a new model that does not rely on negative allostery with their previous findings showing negative cooperativity for RAF dimer inhibition with type I1/2 inhibitors.

(9) Conclusions:

Here, the authors propose a new universal model to explain paradoxical activation of RAF by all types of RAF inhibitors:<br /> " Our findings here, in light of structural studies of RAF complexes and prior cellular investigations of paradoxical activation, lead us to a model for paradoxical activation that does not rely on negative allostery and is consistent with activation by diverse inhibitor classes. In this model, the open monomer complex is the target of inhibitor-induced paradoxical activation (Figure 6). Binding of ATP to the RAF active site stabilizes the inactive conformation of the open monomer, which disfavors dimerization. Displacement of ATP by an ATP-competitive inhibitor, irrespective of class, alters the relative N- and C-lobe orientations of the kinase to promote dimerization (30, 35). Once dimerized, inhibitor dissociation from one or both sides of the dimer would allow phosphorylation and activation of MEK."

From my understanding, the novelty of this new model is twofold: a) the open monomer is the target of the inhibitor-induced paradoxical activation and b) once dimerized, inhibitor dissociation from one or both sides of the dimer would allow phosphorylation and activation of MEK.

Novelty a) implies, as the authors stated, that "Inhibitor-induced activation and inhibition act on distinct species - activation on the open monomer and inhibition on the 14-3-3-stabilized dimer". The authors should explain what they mean by "activation of the open monomer", while only RAF dimers are catalytically active (except for BRAF V600E mutant)?

For novelty b), the authors should explain more clearly what experimental results support this new model.

Reviewer #4 (Public review):

This is a very interesting study, where the authors discovered two neuroendocrine signaling circuits with opposite effects on organismal longevity elicited by motor neurons at different ages.

Interestingly, both systems employ the same neurotransmitter (that is, acetylcholine) and signal the intestine. However, one has effects on early life to shorten lifespan whereas the other system is activated in mid-life to extend lifespan. At the mechanistic level, this bidirectional regulation is possible through the recruitment of two different ACh receptors in the gut: ACR-6 and GAR-3. The authors found that ACR-6 expression in the intestine is restricted to early life, whereas GAR-3 expression in the gut is confined to mid-late life. Interestingly, ACR-6 modulates the transcription factor DAF-16, but GAR-3 regulates HSF-1.

The study combines different approaches, including inducible systems (AID) which are critical for the conclusions of the paper. The conclusions are well supported by the experiments and results. The data provide a potential mechanism for the temporal control of lifespan and shed light on the complex role of the nervous system in organismal aging. These results can have important implications to understand how organismal aging is regulated in a temporal manner by cell non-autonomous mechanisms.

The paper has significantly improved after addressing all the Reviewers' comments and I did not observe significant weaknesses in the study.

developing effective criteria can be challenging. It requires deeper critical thinking.

comparison and contrast can challenge people’s common ideas.

This means that comparison and contrast is not only about similarities and differences, but also about how we classify things.

Reviewer #3 (Public review):

Significance of the Findings:

The study by Liu et al. presents a novel method, DNA-O-MAP, which combines locus-specific hybridisation with proximity biotinylation to isolate specific genomic regions and their associated proteins. The potential significance of this approach lies in its purported ability to target genomic loci with heightened specificity by enabling extensive washing prior to the biotinylation reaction, theoretically improving the signal-to-noise ratio when compared with other methods such as dCas9-based techniques. Should the method prove successful, it could represent a notable advancement in the field of chromatin biology, particularly in establishing the proteomes of individual chromatin regions-an extremely challenging objective that has not yet been comprehensively addressed by existing methodologies.

Strength of the Evidence:

The evidence presented by the authors is somewhat mixed, and the robustness of the findings appears to be preliminary at this stage. While certain data indicate that DNA-O-MAP may function effectively for repetitive DNA regions, a number of the claims made in the manuscript are either unsupported or require further substantiation. There are significant concerns about the resolution of the method, with substantial biotinylation signals extending well beyond the intended target regions (megabases around the target), suggesting a lack of specificity and poor resolution, particularly for smaller loci. Furthermore, comparisons with previous techniques are unfounded since the authors have not provided direct comparisons with the same mass spectrometry (MS) equipment and protocols. Additionally, although the authors assert an advantage in multiplexing, this claim appears overstated, as previous methods could achieve similar outcomes through TMT multiplexing. Therefore, while the method has potential, the evidence requires more rigorous support, comprehensive benchmarking, and further experimental validation to demonstrate the claimed improvements in specificity and practical applicability.

Reviewer #3 (Public review):

Summary:

In their manuscript, Koch et al. describe a novel strategy to synchronize cells of the budding yeast Saccharomyces cerevisiae in metaphase I and metaphase II, thereby facilitating comparative analyses between these meiotic stages. This approach, termed SynSAC, adapts a method previously developed in fission yeast and human cells that enables the ectopic induction of a synthetic spindle assembly checkpoint (SAC) arrest by conditionally forcing the heterodimerization of two SAC components upon addition of the plant hormone abscisic acid (ABA). This is a valuable tool, which has the advantage that induces SAC-dependent inhibition of the anaphase promoting complex without perturbing kinetochores. Furthermore, since the same strategy and yeast strain can be also used to induce a metaphase arrest during mitosis, the methodology developed by Koch et al. enables comparative analyses between mitotic and meiotic cell divisions. To validate their strategy, the authors purified kinetochores from meiotic metaphase I and metaphase II, as well as from mitotic metaphase, and compared their protein composition and phosphorylation profiles. The results are presented clearly and in an organized manner.

Significance:

Koch et al. describe a novel methodology, SynSAC, to synchronize budding yeast cells in metaphase I or metaphase II during meiosis, as well and in mitotic metaphase, thereby enabling differential analyses among these cell division stages. Their approach builds on prior strategies originally developed in fission yeast and human cells models to induce a synthetic spindle assembly checkpoint (SAC) arrest by conditionally forcing the heterodimerization of two SAC proteins upon addition of abscisic acid (ABA). The results from this manuscript are of special relevance for researchers studying meiosis and using Saccharomyces cerevisiae as a model. Moreover, the differential analysis of the composition and phosphorylation of kinetochores from meiotic metaphase I and metaphase II adds interest for the broader meiosis research community. Finally, regarding my expertise, I am a researcher specialized in the regulation of cell division.

Reviewer #3 (Public review):

Summary

This study provides a detailed investigation of neural auditory responses and spontaneous movements in infants listening to music. Analyses of EEG data (event-related potentials and steady-state responses) first highlighted that infants at 3, 6 and 12 months of age and adults showed enhanced auditory responses to music than shuffled music. 6-month-olds also exhibited enhanced P1 response to high-pitch vs low-pitch stimuli, but not the other groups. Besides, whole body spontaneous movements of infants were decomposed into 10 principal components. Kinematic analyses revealed that the quantity of movement was higher in response to music than shuffled music only at 12 months of age. Although Granger causality analysis suggested that infants' movement was related to the music intensity changes, particularly in the high-pitch condition, infants did not exhibit phase-locked movement responses to musical events, and the low movement periodicity was not coordinated with music.

Strengths

This study investigates an important topic on the development of music perception and translation to action and danse. It targets a crucial developmental period that is difficult to explore. It evaluates two modalities by measuring neural auditory responses and kinematics, while cross-modal development is rarely evaluated. Overall, the study fills a clear gap in the literature.

Besides, the study uses state-of-the-art analyses. Detailed investigations were performed, as well as exploratory analyses in supplementary information. The discussion is rich in neurodevelopmental interpretations and comparisons with the literature. All steps are clearly detailed. The manuscript is very clear, well-written and pleasant to read. Figures are well-designed and informative. The authors' responses to previous reviews are also detailed and informative.

Comments on revisions:

The authors answered all my questions.

Reviewer #3 (Public review):

Summary:

In this manuscript, Guin et al. use a CRISPR KO screen of ~1000 candidates in two human cell lines along with high-throughput image analysis to demonstrate that orderly progression through mitosis shapes centromere organization. They identify ~50 genes that perturb centromere clustering when depleted in both RPE1 and HCT116 cells and validate many of these hits using RNAi. They then use auxin-mediated acute depletion of four factors (NCAPH2, KI67, SPC24 and NUF2) to demonstrate that their effects on centromere clustering require passage through mitosis. They further suggest that lack of these factors during mitosis leads to disorganization of centromeres on the mitotic spindle and these effects persist in the subsequent interphase. Overall, the manuscript is clear, well-written, the experiments performed are appropriate and the data is interpreted accurately. In my opinion, the main strength of this manuscript is the discovery of several hits associated with altered centromere clustering. These hits will serve as a solid foundation for future work investigating the functional significance of centromere clustering in human cells. On the other hand, how the changes in centromere clustering relate to other aspects of interphase genome architecture (A/B compartments, chromosome territories etc) remains unclear and represents the main limitation of this manuscript.

Reviewer #3 (Public review):

Summary:

The authors aim to investigate the mechanisms of anxiety. The paper focuses on supramammillary nucleus (SuM) based on a fos screen and recordings showing that footshock and social defeat stress increases activity in this region. Using activity-dependent tagging, they show that reactivation of stress-activated neurons in SuM has an anxiety-like effect, reducing open-arm exploration in the elevated zero task. They then investigate the ventral subiculum as a potential source of anxiety-related information for SuM. They show that ventral subiculum (vSub) inputs to SuM are more strongly activated than dSub when mice explore open arms of the elevated zero. Finally, they show that DREADD-mediated inhibition of vSub-SuM projections alleviates stress-enhanced anxiety. Overall the results provide good evidence that SuM contains a stress-activated neuronal population whose later activity increases anxiety-like behavior. It further provides evidence that vSub projects to SuM are activated by stress and their inhibition alleviates some effects of stress.

Strengths:

Strengths of this paper include the use of convergent methods (e.g., fos plus electrode recordings, footshock and social defeat) to demonstrate that the SuM is activated by different forms of stress. The activity-dependent tagging experiment shows that footshock-activated SuM neurons are reactivated by social defeat but not sucrose is also compelling because it provides evidence that SuM neurons are driven by some integrative aspect of stress rather than by a simple sensory stimulus.

Weaknesses:

The strength of some evidence is judged to be incomplete. The paper provides good evidence that SuM contains stress-responsive neurons, and the activity of these neurons increases some measure of anxiety-like behavior. However, the evidence that the vSub-SuM projection "encodes anxiety" and that the SuM is a key regulator of anxiety is judged to be incomplete. I am not convinced that the identified SuM cells have a specific anxiety function. As the authors mention in the introduction, SuM regulates exploration and theta activity. Since theta potently regulates hippocampal function, there is the concern that SuM manipulations could have broad effects beyond anxiety-like behavior.

Reviewer #3 (Public review):

Summary:

Protein complexes, like the GID/CTLH-type E3 ligase, adopt a complex three-dimensional structure, which is of functional importance. Several domains are known to be involved in shaping the complexes. Structural information based on cryo-EM is available, but its resolution does not always provide detailed information on protein-protein interactions. The work by van gen Hassend and Schindelin provides additional structural data based on crystal structures.

Strengths:

The work is solid and very carefully performed. It provides high-resolution insights into the domain architecture, which helps to understand the protein-protein interactions on a detailed molecular level. They also include mutant data and can thereby draw conclusions on the specificity of the domain interactions. These data are probably very helpful for others who work on a functional level with protein complexes containing these domains.

Weaknesses:

The manuscript contains a lot of useful, very detailed information. This information is likely very helpful to investigate functional and regulatory aspects of the protein complexes, whose assembly relies on the LisH-CTLH-CRA modules. However, this goes beyond the scope of this manuscript.

Comments on revisions:

I am fine with the revised version of the manuscript.

Reviewer #3 (Public review):

Summary:

Sleep is typified by a behavioural attenuation of responsiveness to external stimuli (higher arousal thresholds). There are various mechanisms through which sensory perception could be dampened, and while thalamic and cortical gate points have been well studied, the focus here is on peripheral ones - at the level of the olfactory bulb (OB). While something conceptually similar has been shown in insects, this paper represents an important contribution to understanding attenuation of sensory perception during rodent sleep and anaesthesia.

This paper shows that respiration-locked potentials and gamma activity in the olfactory bulb, which are important for olfactory coding, are diminished during sleep and when under anaesthesia compared to wake. Further, this state-dependent activity in OB is likely to be locally generated. Using a tracheotomy procedure aimed to dissociate nasal airflow from natural inhalations, authors demonstrate that local field potentials (LFPs) in the OB phase lock with artificially generated air pulses (delivered into the nasal cavity) during the active phase of anaesthesia but not during a more passive state. LFPs did not synchronise with respiratory signals during either anaesthesia state. Lastly, the authors showed that as delta power increased (typical of slow-wave-sleep), the coherence between nasal inhalation rhythms and OB LFP coherence decreased, indicating that as rats experienced something akin to slow-wave-sleep (during anaesthesia), disconnection from the external environment could be augmented. Taken together, the authors argue that the change in activity observed in the olfactory bulb during sleep and anaesthesia provides a non-permissive state for sensory processing and manifests as sensory dissociation

Strengths:

The manuscript is well-written, and the experiments are thorough. Experiments examining coupling of nasal respiration with OB potentials and delta activity are particularly interesting as they point to augmented sensory disconnection during a sleep phase typically associated with higher arousal thresholds.

Weaknesses:

(1) An experiment addressing the following points, is missing:

Does odour stimulation that wakes up a subject restore gamma activity and respiration-locked potentials?

Is OB/respiration desynchrony maintained when presented with a non-rousing stimulus?

Is waking upon stimulus delivery less likely as delta activity increases and coherence between OB/respiratory rhythms weakens?

(2) Many of the experiments are performed under anaesthesia, which I understand is for practical reasons. While authors are forthcoming about limitations of using anaesthesia in lieu of natural sleep states, I would have preferred to see more experiments performed on sleeping animals.

Reviewer #3 (Public Review):

Summary:

72 subjects, and 144 hemispheres, from the Human Connectome Project had their parietal sulci manually traced. This identified the presence of previous undescribed shallow sulci. One of these sulci, the ventral supralateral occipital sulcus (slocs-v), was then demonstrated to have functional specificity in spatial orientation. The discussion furthermore provides an eloquent overview of our understanding of the anatomy of the parietal cortex, situating their new work into the broader field. Finally, this paper stimulates further debate about the relative value of detailed manual anatomy, inherently limited in participant numbers and areas of the brain covered, against fully automated processing that can cover thousands of participants but easily misses the kinds of anatomical details described here.

Strengths:

- This is the first paper describing the tertiary sulci of the parietal cortex with this level of detail, identifying novel shallow sulci and mapping them to behaviour and function.

- It is a very elegantly written paper, situating the current work into the broader field.

- The combination of detailed anatomy and function and behaviour is superb.

Reviewer #3 (Public review):

Summary:

Fan et al utilize large omics data sets to give an overview of proteomic and gene expression changes after 4 moths of intermittent fasting (IF) in liver, muscle and brain tissue. They describe common and district pathways altered under IF across tissues using different analysis approaches. Main conclusions presented are the variability in responses across tissues with IF. Some common pathways were observed, but there were notable distinctions between tissues.

Strengths:

(1) The IF study was well conducted and ran out to 4 months which was a nice long-term design.

(2) The multi omics approach was solid and additional integrative analysis was complementary to the illustrate the differential pathways and interactions across tissues.

(3) The authors did not over-step their conclusions and imply an overreached mechanism.

Weaknesses:

The weaknesses, which are minor, include use of only male mice and the early start (6 weeks) of the IF treatment. However, the authors have provided justification on why they chose male mice and the time points used in the study.

Reviewer #3 (Public review):

Summary:

The manuscript by Nishimura et al. examines the behavioural and neural mechanisms of stress-enhanced fear responding (SEFR) and stress-enhanced fear learning (SEFL). Groups of stressed (4 x shock exposure in a context) vs non-stressed (context exposure only) animals are compared for their fear of an unconditioned tone, and context, as well as their learning of new context fear associations. Shock of higher intensity led to higher levels of unlearned stress-enhanced fear expression. Immediate early gene analysis uncovered the PVT as a critical neural locus, and this was confirmed using fiber photometry, with stressed animals showing an elevated neural signal to an unconditioned tone. Using a gain and loss of function DREADDs methodology, the authors provide convincing evidence for a causal role of the PVT in SEFR.

Strengths:

(1) The manuscript uses critical behavioural controls (no stress vs stress) and behavioural parameters (0.25mA, 0.5mA, 1mA shock). Findings are replicated across experiments.

(2) Dissociating the SEFR and SEFL is a critical distinction that has not been made previously. Moreover, this dissociation is essential in understanding the behavioural (and neural) processes that can go awry in fear.

(3) Neural methods use a multifaceted approach to convincingly link the PVT to SEFR: from Fos, fiber photometry, gain and loss of function using DREADDs.

Weaknesses:

No weaknesses were identified by this reviewer; however, I have the following comments:

A closer examination of the Test data across time would help determine if differences may be present early or later in the session that could otherwise be washed out when the data are averaged across time. If none are seen, then it may be worth noting this in the manuscript.

Given the sex/gender differences in PTSD in the human population, having the male and female data points distinguished in the figures would be helpful. I assume sex was run as a variable in the statistics, and nothing came as significant. Noting this would also be of value to other readers who may wonder about the presence of sex differences in the data.

Comments on revisions:

Following revision, this reviewer felt all of the above comments were addressed.

Reviewer #3 (Public review):

In the manuscript, the authors generated several mutant plants defective in the eIF4E family proteins and detected cassava brown streak viruses (CBSVs) infection in these mutant plants. They found that CBSVs induced significantly lower disease scores and virus accumulation in the double mutant plants. Furthermore, they identified important conserved amino acid for the interaction between eIF4E protein and the VPg of CBSVs by yeast two hybrid screening. The experiments are well designed, however, some points need to be clarified:

(1) The authors reported that the ncbp1 ncbp2 double mutant plants were less sensitive to CBSVs infection in their previous study, and all the eIF4E family proteins interact with VPg. In order to identify the redundancy function of eIF4E family proteins, they generated mutants for all eIF4E family genes, however, these mutants are defective in different eIF4E genes, they did not generate multiple mutants (such as triple, quadruple mutants or else) except several double mutant plants, it is hard to identify the redundant function eIF4E family genes.

(2) The authors identified some key amino acids for the interaction between eIF4E and VPg such as the L51, it is interesting to complement ncbp1 ncbp2 double mutant plants with L51F form of eIF4E and double check the infection by CBSVs.

Comments on revisions:

The reviewer understand Cassava is not a model plant, it is hard for the authors to generate multiple genetic mutant plants for experiments, so nothing was done to respond to the comments raised by the reviewer.

Reviewer #3 (Public review):

Summary:

This study by Mercer et al. investigates how inhibitory modulation of basolateral amygdala neurons expressing Vglut1 and projecting to the nucleus accumbens (Vglut1BLA→NAc) influences motivated behavior in both appetitive and aversive tasks. Using a combination of whole-cell electrophysiology, chemogenetic inhibition and behavioral tests, the authors demonstrate that (1) reward conditioning increases inhibitory synaptic input and reduces intrinsic excitability of Vglut1BLA→NAc neurons, (2) chemogenetic inhibition of these neurons enhances the number of conditioned approaches in a Pavlovian task and the number of nosepoke responses in an instrumental task, elevates reward valuation, and increases fear discrimination and (3) these effects are linked to salience assignment and associative strength, rather than altered learning or reversal flexibility. The work challenges the classical excitatory function usually reported about the BLA projection to the NAc and highlights an interesting and thought-provoking result. Nevertheless, the study does not address the potential effect of their manipulation on motoric impulsivity, nor did they provide a theoretical framework explaining this unorthodox yet interesting effect.

Strengths:

The study establishes the initial finding with a correlational approach that informs a causal study. They find convincingly that Pavlovian conditioning induces an increase in inhibitory inputs onto Vglut1BLA→NAc neurons that leads to reduced excitability. Causality is studied using a powerful dual recombinase chemogenetic strategy to selectively inhibit this population of Vglut1BLA→NAc neurons and determine the effect on different behavioral tasks. The use of different tasks provides convergence on their effect. This surprising finding provokes interest and will stimulate further investigation into the mechanisms underlying these effects.

Weaknesses:

Several important aspects of the evidence remain incomplete.

(1) First, an important aspect of the underlying processes at play remains to be investigated. In all behavioral tasks, the authors find that their manipulation increases responding that they interpret as a facilitation of learning. However, none of the appetitive tasks include a control stimulus that could address the specificity of their effect. Given that on the Pavlovian task, responding to the CS is almost 100%, I suspect that their manipulation may induce motoric impulsivity. This aspect would clearly benefit from additional controls.

(2) Second, I have several interrogations about the time-resolved probability of port entries (PSTHs).

a) There is a mismatch between the results presented in Figure 1. Panel D shows a peak of responses on the PSTH at ~2s on day 5 (my remark applies to all days), suggesting that the average should lie around this value. However, panel C reports a latency to respond at ~4sec. Could the authors double-check their PSTHs?

b) More generally, the fact that in the Pavlovian task all PSTHs show a peak at almost exactly 2 sec is quite surprising and raises questions about how they are constructed. Sure, the most salient event is the water drop occurring 2s after cue onset. Yet, if mice responded only to these drops, the peak response should occur at 2s+reaction time, which is not the case. Figure 2 shows that on the first acquisition day, responding is already centered around 2s and does not decrease with learning, except for treated animals.

(3) Several methodological flaws are present.

a) The authors need to report clearly the statistics. In most cases, the statistical test used is mentioned in the figure caption with a single P-value. Thus, on two-way ANOVAs, I do not know whether the P-value relates to the interaction, the main effects, or the post-hoc tests.

b) Another important issue is related to the average time-resolved z-score probability of port entries. The bin size used, the smoothing (that is much too strong), and the baseline period used to calculate the z-score are absent from the methods.

(4) This study reports that manipulating 70% of the glutamatergic projection to the NAc induces an effect opposed to what has been previously reported in many different studies. Such a surprising finding deserves a more elaborate discussion about the mechanism that could be at play.

Reviewer #3 (Public review):

Summary

In this study, the authors investigated how inference about the current task context is encoded in the cortex, using MEG measurements. Using the same behavioral task that was initially developed for an fMRI study to identify the loci of task context representation, the current results complement and extend the previous study by identifying the candidate regions that are important for the inference process, not just for encoding the end product. They reported widespread modulation of cortical activity by uncertainty in evidence and volatility of task context changes. In comparison, modulation correlated with the decision variable underlying the task context inference process was more restricted to the parietal and visual cortices, particularly in alpha-band activity.

Strengths:

(1) The normative model provides a solid computational foundation for disambiguating quantities related to decision variables from those related to task factors (e.g., uncertainty and volatility).

(2) The MEG technique allows examination of cortical activity that is modulated by the temporally evolving decision variable.

(3) Rigorous modeling efforts, including comparisons of well-reasoned alternative/reduced models and examinations of diagnostic features using participant-matched simulations.

Weaknesses:

(1) There are two major surprises in the results that raise concerns about how to interpret these data. The first is the absence of modulation of prefrontal cortical activities by prior or posterior. As the authors acknowledged, there are extensive single-neuron recording data (e.g., from the Miller group) demonstrating the presence of task rule modulation in the monkey PFC and prior representation in the PFC in the mouse study that they cited. The second surprise is that the strongest modulation of prior/posterior/evidence was almost always observed in the visual cortex, in contrast to the common embodied cognition assumption. A more elaborated discussion about these discrepancies would help contextualize the current results.

(2) It is not clear why the effects in Figures 2D and E dipped before responses, which is not expected from any of the models. This could potentially affect the interpretation of the MEG signals in late-post-cue or pre-response periods.

(3) The definitions of the different periods (e.g., early/late post-cue) are vague, making it hard to assess the functional relevance of the signals. For example, is the difference between the early pre-response map in Figure 5B and the late evidence map in Figure 4B due to completely non-overlapping time periods? A diagram of the timing definitions for different task periods would be helpful.

(4) Perhaps related to #2, it is puzzling that evidence encoding is absent in the visual cortex during the early post-cue period.

(5) The presentation and discussion of results related to correlated variability assume that the readers have already read their previous paper. A little more elaboration of the significance of this measurement would be helpful.

Reviewer #3 (Public review):

Summary:

This paper develops a model to account for flexible and context-dependent behaviors, such as where the same input must generate different responses or representations depending on context. The approach is anchored in the hippocampal place cell literature. The model consists of a module X, which represents context, and a module H (hippocampus), which generates "sequences". X is a binary attractor RNN, and H appears to be a discrete binary network, which is called recurrent but seems to operate primarily in a feedforward mode. H has two types of units (those that are directly activated by context, and transition/sequence units). An input from X drives a winner-take-all activation of a single unit H_context unit, which can trigger a sequence in the H_transition units. When a new/unpredicted context arises, a new stable context in X is generated, which in turn can trigger a new sequence in H. The authors use this model to account for some experimental findings, and on a more speculative note, propose to capture key aspects of contextual processing associated with schizophrenia and autism.

Strengths:

Context-dependency is an important problem. And for this reason, there are many papers that address context-dependency - some of this work is cited. To the best of my knowledge, the approach of using an attractor network to represent and detect changes in context is novel and potentially valuable.

Weaknesses:

The paper would be stronger, however, if it were implemented in a more biologically plausible manner - e.g., in continuous rather than discrete time. Additionally, not enough information is provided to properly evaluate the paper, and most of the time, the network is treated as a black box, and we are not shown how the computations are actually being performed.

Reviewer #3 (Public review):

Summary:

In the paper "Deep mutational scanning reveals pharmacologically relevant insights into TYK2 signaling and disease", the authors perform a comprehensive deep mutational scan of the kinase TYK2, a protein of pharmacological interest due to its central role in multiple immune-related phenotypes. The study assesses two key functional phenotypes: protein abundance and IFN-α-dependent signaling. The signaling assays were conducted across a dose-response range under various inhibitor conditions, allowing for an in-depth characterization of TYK2 activity and regulation. Both the experimental design and data analysis were executed with rigor and transparency, yielding a dataset that appears highly reliable. The authors provide strong evidence and a scientifically grounded interpretation of their results.

The paper presents the results of a deep mutational scan based on two assays: an IFN-α-stimulated signaling assay and a protein abundance assay. These measurements are further supported by variant classifications from AlphaMissense and ClinVar, providing a framework for functional interpretation. Building on these data, the authors propose four potential pharmacological applications of their screening system at the end of the first results section.

First, they demonstrate that the combined analysis of abundance and IFN-α signaling identifies potential allosteric sites, focusing on variants with normal protein stability but reduced signaling activity. Through this approach, they detect two previously uncharacterized allosteric regions (Results Section 2).

Second, they explore how the screen can be used to predict variant-specific drug responses or resistance mechanisms (Results Section 3). This is achieved through assays involving two different inhibitors, which reveal both resistance- and potentiation-associated variants.

Third, they assess the relative functional consequences of ligand and inhibitor dosing by performing IFN-α and inhibitor dose-response experiments (1, 10, and 100 U/mL IFN-α; IC99 and IC75 inhibitor concentrations; Results Section 3).

Finally, the authors investigate how specific human variants, such as P1104A and I684S, may inform therapeutic modality selection (Results Section 4). Although these variants exhibit no detectable effect on IFN-α signaling within this experimental system, they substantially impact protein abundance. By integrating data from the UK Biobank, the authors further demonstrate that protective effects against autoimmune disease are associated with altered protein abundance rather than differences in IFN-α signaling, highlighting the distinct mechanistic basis of TYK2's clinical relevance.

Strengths:

Overall, we found this paper rigorous, well-written, and easy to follow. As such, we think this is an exceptional example of a deep mutational scanning manuscript, and this dataset will be invaluable to the field. We particularly appreciate that the authors could explore sensitivity to inhibitor concentration across multiple doses of the inhibitor.

Weaknesses:

Despite the authors' rigorous experimentation and thoughtful interpretation, the study leaves several important mechanistic questions unresolved, as is common in any study. While the data provide clear functional patterns, the underlying biophysical and biochemical explanations remain insufficiently explored. For instance, in point 1, the identification of two novel allosteric sites is intriguing, yet the paper does not elaborate on the structural basis or mechanistic rationale for their regulatory effects. In point 2, resistance and potentiation variants are described for two distinct inhibitors, but it remains unclear why certain variants respond specifically to one compound and not the other. In point 3, higher inhibitor concentrations appear to diminish allosteric interactions, though the reasons why some sites are affected while others are not are left unexplained. Finally, in point 4, the observation that protein abundance, but not IFN-α signaling, correlates with autoimmune protection is compelling but mechanistically ambiguous. These gaps do not detract from the technical excellence of the work; rather, they highlight opportunities for future studies to clarify the molecular and pharmacological mechanisms underlying TYK2 regulation and to deepen the translational insights drawn from this comprehensive mutational scan. We hope that the authors could provide more direction and mechanistic context in the discussion section to guide readers toward these next steps.

Reviewer #3 (Public review):

Summary:

Yu et al harness the capabilities of mesoscopic 2P imaging to record simultaneously from populations of neurons in several visual cortical areas and measure their correlated variability. They first divide neurons in 65 classes depending on their tuning to moving gratings. They found the pairs of neurons of the same tuning class show higher noise correlations (NCs) both within and across cortical areas. Based on these observations and a model they conclude that visual information is broadcast across areas through multiple, discrete channels with little mixing across them.<br /> NCs can reflect indirect or direct connectivity, or shared afferents between pairs of neurons, potentially providing insight on network organization. While NCs have been comprehensively studied in neurons pairs of the same area, the structure of these correlations across areas is much less known. Thus, the manuscripts present novel insights on the correlation structure of visual responses across multiple areas.

Strengths:

The measurements of shared variability across multiple areas are novel. The results are mostly well presented and many thorough controls for some metrics are included.

Weaknesses:

I have concerns that the observed large intra class/group NCs might not reflect connectivity but shared behaviorally driven multiplicative gain modulations of sensory evoked responses. In this case, the NC structure might not be due to the presence of discrete, multiple channels broadcasting visual information as concluded. I also find that the claim of multiple discrete broadcasting channels needs more support before discarding the alternative hypothesis that a continuum of tuning similarity explains the large NCs observed in groups of neurons.

Specifically:

Major concerns:

(1) Multiplicative gain modulation underlying correlated noise between similarly tuned neurons

(1a) The conclusion that visual information is broadcasted in discrete channels across visual areas relies on interpreting NC as reflecting, direct or indirect connectivity between pairs, or common inputs. However, a large fraction of the activity in the mouse visual system is known to reflect spontaneous and instructed movements, including locomotion and face movements, among others. Running activity and face movements are one of the largest contributors to visual cortex activity and exert a multiplicative gain on sensory evoked responses (Niell et al , Stringer et al, among others). Thus, trial-by-fluctuations of behavioral state would result in gain modulations that, due to their multiplicative nature, would result in more shared variability in cotuned neurons, as multiplication affects neurons that are responding to the stimulus over those that are not responding ( see Lin et al , Neuron 2015 for a similar point).

In the new version of the manuscript, behavioral modulations are explicitly considered in Figure S8. New analyses show that most of the variance of the neuronal responses is driven by the stimulus, rather than by behavioural variable. However, they new analyses still do not address if the shared noise correlation in cotuned neurons is also independent of behavioral modulations .

As behavioral modulations are not considered this confound affects the conclusions and the conclusion that activity in communicated unmixed across areas ( results in Figure 4), as it would result in larger NCs the more similar the tuning of the neurons is, independently of any connectivity feature. It seems that this alternative hypothesis can explain the results without the need of discrete broadcasting channels or any particular network architecture and should be addressed to support the main claims.

(2) Discrete vs continuous communication channels<br /> (2a) One of the author's main claims is that the mouse cortical network consists of discrete communication channels, as stated in teh title of the paper. This discreteness is based on an unbiased clustering approach on the tuning of neurons, followed by a manual grouping into six categories with relation to the stimulus space. I believe there are several problems with this claim. First, this clustering approach is inherently trying to group neurons and discretise neural populations. To make the claim that there are 'discrete communication channels' the null hypothesis should be a continuous model. An explicit test in favor of a discrete model is lacking, i.e. are the results better explained using discrete groups vs. when considering only tuning similarity? Second, the fact that 65 classes are recovered (out of 72 conditions) and that manual clustering is necessary to arrive at the six categories is far from convincing that we need to think about categorically different subsets of neurons. That we should think of discrete communication channels is especially surprising in this context as the relevant stimulus parameter axes seem inherently continuous: spatial and temporal frequency. It is hard to motivate the biological need for a discretely organized cortical network to process these continuous input spaces.

Finally, as stated in point 1, the larger NCs observed within groups than across groups might be due to the multiplicative gain of state modulations, due to the larger tuning similarity of the neurons within a class or group.

Reviewer #3 (Public review):

Summary:

This study uses computational modeling of a spiking network of E-I with homeostatic inhibitory plasticity and aims to show that self-organized criticality that arises from the homeostatic mechanism can result in representational drift as well as reliable stimulus representation, because the geometric representation of stimuli remains restricted.

Strengths:

This paper provides a framework to link critical spontaneous state, homeostatic inhibitory plasticity, representational drift, and stimulus population response reliability

Weaknesses:

The study does not show a causal (or necessary/ sufficient) relationship between criticality at the spontaneous state, representational drift, and reliable stimulus presentation. The study only reports an observation that these features could co-exist. However, it does not show how the criticality of the spontaneous state could restrict the manifold for stimulus response.

Reviewer #5 (Public review):

Summary:

This study investigates the molecular mechanisms underlying the maintenance of neuropathic pain, specifically focusing on the role of mRNA translation in the spinal cord. Using the Spared Nerve Injury (SNI) model, the authors demonstrate that while both transcription and translation are active in the early phase, the chronic phase (day 63) is uniquely characterized by a shift toward translational control. They identify spinal inhibitory neurons, particularly parvalbumin-positive interneurons, as key sites of this translational regulation.

Strengths:

Technical Rigor: The use of Ribo-seq and TRAP-seq allows for a high-resolution view of the "translatome," which more accurately reflects the functional protein output than standard mRNA-seq.Novelty: The study uncovers that reducing a single translation initiation factor (eIF4E) specifically in the CNS is sufficient to provide long-lasting relief from established chronic pain.Addressing Disinhibition: The electrophysiological evidence showing that increased translation in PV+ neurons reduces their excitability provides a clear mechanism for the "spinal disinhibition" typically seen in chronic pain.

Weaknesses:

Cell-Type Sufficiency: New experiments in the revision show that while inhibiting translation in PV+neurons restores their individual excitability, it is not sufficient on its own to reverse behavioral pain hypersensitivity. This suggests that the maintenance of chronic pain likely involves translational changes across a broader network of cell types, including other inhibitory neurons or non-neuronal cells like microglia. -This does not have to be resolved in the current study, but providing some framework to account for potential mechanisms might help the audience.

Reviewer #3 (Public review):

Summary:

The manuscript by Patel et al investigates the hypothesis that CDHR1a on photoreceptor outer segments is the binding partner for PCDH15 on the calyceal processes, and the absence of either adhesion molecule results in separation between the two structures, eventually leading to degeneration. PCDH15 mutations cause Usher syndrome, a disease of combined hearing and vision loss. In the ear, PCDH15 binds CDH23 to form tip links between stereocilia. The vision loss in less understood. Previous work suggested PCDH15 is localized to the calyceal processes, but the expression of CDH23 is inconsistent between species. Patel et al suggest that CDHR1a (formerly PCDH21) fulfills the role of CDH23 in the retina.

The experiments are mainly performed using the zebrafish model system. Expression of Pcdh15b and Cdhr1a protein is shown in the photoreceptor layer through standard confocal and structured illumination microscopy. The two proteins co-IP and can induce aggregation in vitro. Loss of either Cdhr1a or Pcdh15, or both, results in degeneration of photoreceptor outer segments over time, with cones affected primarily.

The idea of the study is logical given the photoreceptor diseases caused by mutations in either gene, the comparisons to stereocilia tip links, and the protein localization near the outer segments. The work here demonstrates that the two proteins interact in vitro and are both required for ongoing outer segment maintenance. The major novelty for this paper would be the demonstration that Pcdh15 localized to calyceal processes interacts with Cdhr1a on the outer segment, thereby connecting the two structures. Unfortunately, the data as presented are inadequate proof of this model.

Strengths:

The in vitro data to support the ability of of Pcdh15b and Cdhr1a to bind is well done. The use of pcdh15b and cdhr1a single and double mutants is also a strength of the study, especially being that this would be the first characterization of a zebrafish cdhr1a mutant.

This is a large body of data.

Weaknesses:

(1) I have serious concerns about the quality of the imaging here. The premise that cdhr1a/pcdh15 juxtaposition is evidence for the two proteins mediating the connection between outer segments and calyceal processes requires very careful microscopy. The SIM images have two major issues - one being that the red and green channels are misaligned and the other being evidence of bleed through between the channels. This is obvious in Fig 2A but likely true across all the panels in Fig 2, and possibly applies to confocal images in Fig 1 as well. The co-labelling with actin shows very uneven, punctate staining for actin bundles.

(2) The newly added TEM and transverse sections include colored regions that obscure the imaging.

(3) The quantification should be done with averages from individual fish. Counting individual measurements as single data points artificially inflates the significance. Also, the cone subtypes are still lumped together for analysis despite their variable sizes.

(4) I highlighted previously that the measurement of calyceal processes was incorrect. The redrawn labels in Fig 7 are now more accurate, although still difficult to interpret. However, the quantification in Fig 7O is exactly the same. How can that be if the measurement region is now different?

(5) Lower magnification views would provide context for the TEM data.

(6) The statement describing the separation between calyceal processes and the outer segment in the mutants is still not backed up by the data.

(7) The authors state "from the fact that rod CPs are inherently much smaller than cone CPs". This is now referenced, but incorrectly. Also, the issue of pigment interference was not addressed.

(8) The images in panels B-F of the Supplemental Figure are uncannily similar, possibly even of the same fish at different focal planes.

Reviewer #3 (Public review):

Summary:

This study's core conclusions are well-supported by data. It is shown that co-folding outperforms docking in known ligand pose/affinity prediction (validated by RMSD and IC₅₀ correlation), struggles with false-positive discrimination in virtual screens (lower AUC values), and is complementary to docking (non-correlated errors, distinct strengths in drug discovery stages).

Strengths:

(1) Unprecedented prospective design with 557 novel Mac1-ligand complexes ensures rigorous, independent evaluation of co-folding methods.

(2) Comprehensive comparison of 3 co-folding tools (AlphaFold3, Chai-1, Boltz-2) with DOCK3.7 across diverse targets and metrics enables nuanced performance assessment.

(3) The study clearly demonstrates complementary roles of co-folding (superior pose/affinity prediction for known ligands) and docking (better hit prioritization), and addresses deep learning memorization concerns via ligand similarity analysis.

Weaknesses:

(1) Limited generalization to diverse protein families (e.g., no ion channels/transporters).

(2) Ambiguity in the mechanism underlying co-folding's failure to predict rare conformational changes.

(3) Virtual screen comparison is unbalanced (docking-prioritized hit lists bias results).

Reviewer #3 (Public review):

The authors state that infection of cells by the alphaherpesviruses HSV-1 or PRV leads to a proteosome-dependent reduction in levels of HDAC1 and HDAC2 and that this leads to chromatin hyperacetylation, a DNA damage response, and greater replication of these viruses. Previously, other authors reported no change in levels of HDAC1 and HDAC2 after HSV-1 infection of human cells, but this paper is neither cited nor commented on in this new submission. The experiments are poorly designed. For instance, most of the time points analysed are way beyond the time needed for HSV-1 replication and are therefore not biologically relevant. The infections are done with a dose of virus that does not ensure that all cells are infected synchronously, but rather infection spreads from cell to cell with multiple rounds of replication. Some essential controls are missing. Additionally, this reviewer feels that the data presented do not support the conclusions drawn. Currently, links are not established between a reduction in HDAC1/ 2 and other phenomena such as hyperacetylation of histones, a DDR, and altered virus replication. The paper does not identify which HSV or PRV protein(s) induce reduction in HDACs, nor how the HDACs mediate antiviral activity; what are the HSV-1 or PRV protein targets? Lastly, the paper is not well prepared, and it does not adequately refer to prior literature.

Reviewer #3 (Public review):

Summary:

This paper examines the loss and re-evolution of specific organs during the evolution of ants. The authors show that these organs, the ocelli, disappear and are re-evolved in different ant species and in different ant castes within these species. The authors show that this is linked to dto a conserved GRN discovered in Drosophila, that appears to underlie the development of the ocelli, and demonstrate that this GRN appears to remain active in the developing heads of ants that have no ocelli- implying that it is the evolutionary latency of this GRN that allows loss and subsequent evolution.

Strengths:

This manuscript has outstanding imaging of a very difficult developing organ, and the key data, fluorescence in situ hybridisation, is done well and clearly shows what the authors wish to demonstrate. The methods are well described and underpin the whole work.

The authors convincing demonstatrate that gene expression patterns imply the conservation of the ocellus gene regulatory network from Drosophila to ants. They further show that this network is present even in ants that don't produce an adult ocellus, but do show that in those species, loss of a developing nascent ocellus (which they identify) occurs at the same time as an interruption in the expression of the key genes in the GRN. All of this data is beautifully presented and explained.

Weaknesses:

There is one key weakness in that there are no functional students that indicate that the GRN actually does make the ocellus, though the expression patterns are convincing. This applies to loss of the ocellus as well. It would be nice to see that transient loss of the ocelli GRN might lead to loss of ocelli in ant species that have them. These are very difficult things to achieve, as the key genes have earlier developmental roles, such that CRISPR knockouts would not be interpretable, and transient RNAi in the head capsules of developing pupal ants would be challenging.

Reviewer #3 (Public review):

Summary:

Here, the authors investigate the role of the Trypanosoma brucei polo-like kinase TbPLK in the function of flagellum-associated cellular structures in trypanosomes. They set out to test the hypothesis that a key substrate of TbPLK is the kinesin protein KIN-G, and that TbPLK phosphorylation of KIN-G regulates its functions in cells.

Strengths:

Using in vitro biochemistry with purified proteins, the authors convincingly demonstrate that TbPLK phosphorylates KIN-G at 29 sites. Moreover, they convincingly show that phosphorylation at one site, T301, impairs the binding of purified KIN-G to purified microtubules. Using immunofluorescence-based imaging approaches, they also show that TbPLK colocalizes with KIN-G at centrin arms during the early S-phase of the cell cycle. Centrin arms are structures that are located near the basal body and flagellum and are important for new flagellum biogenesis, Golgi positioning, and cell division. To evaluate the function of KIN-G phosphorylation in cells, they depleted KIN-G by RNAi, simultaneously expressed phospho-mimetic (T301D) and phospho-ablative mutant proteins, and used immunofluorescence to examine the impact on flagellum-associated cellular structures. They show that expression of the phospho-mimetic mutant KIN-G-T301D causes the following defects: reduced cell proliferation, disruption of centrin arm and Golgi biogenesis, impairment of FAZ elongation and flagellum positioning, and misplacement of the cell division plane. The data convincingly support the conclusion that KIN-G phosphorylation on T301 plays an important role in regulating the cellular functions of this kinesin motor protein.

Weaknesses:

Some of the broader conclusions are not directly supported by the data. For example, the title states "Polo-like kinase phosphorylation of the orphan kinesin KIN-G negatively regulates centrin arm biogenesis in Trypanosoma brucei," but the data do not directly address the specific role of TbPLK in phosphorylating KIN-G in cells. Moreover, some of the more specific conclusions in the paper, for example, that "phosphorylation of KIN-G" causes various cellular defects, are a bit of an overstatement. The supporting data rely on the expression of a phospho-mimetic mutant of KIN-G. Presumably, phosphorylation in cells is a normal part of KIN-G regulation, and it is not just phosphorylation, but rather hyperphosphorylation that is being mimicked by the mutant. Some rewording of the specific conclusions is warranted, and the broader conclusion would be better supported with additional experimental evidence.

Reviewer #3 (Public review):

Summary:

In this manuscript, Ndugwa et al. attempt to link specific severe malaria manifestations with particular var gene expression patterns. This is an important question, and the dataset the authors have assembled over decades is impressive. However, greater clarity in the descriptions and statistics would, in my view, help this reviewers, and readers in general develop a more precise understanding of the significance of the findings.

Strengths:

The study addresses a critically important question in malaria pathogenesis, and the dataset is extensive and represents a significant long-term effort by the authors.

Weaknesses:

The Results section often lacks clarity: clinical group definitions (NS, non-IC, non-SMA, mild vs. moderate) are sometimes ambiguous, and key methodological details, including the VEH index calculation, RT-qPCR quantification, antibody detection methods, and rosetting assays, are either missing from the results text or poorly explained in the figure legends. Additionally, figure presentation requires improvement, with inconsistent reporting of sample sizes, undefined colors, and p-values that overlap with data points rather than being clearly displayed above them.

Reviewer #3 (Public review):

Summary:

Qiu et al., present a hierarchical framework that combine AI and molecular dynamic simulation to design α-helical protein with enhanced thermal, chemical and mechanical stability. Strategically chemical modification by incorporating additional α-helix, site-specific salt bridges and metal coordination further enhanced the stability. The experimental validation using single-molecule force spectroscopy and CD melting measurements provide fundamental physical chemical insights into the stabilization of α-helices. Together with the group's prior work on super-stable β strands (https://www.nature.com/articles/s41557-025-01998-3), this research provides a comprehensive toolkit for protein stabilization. This framework has broad implications for designing stable proteins capable of functioning under extreme conditions.

Strengths:

The study represents a complete frame work for stabilizing the fundamental protein elements, α-helices. A key strength of this work is the integration of AI tools with chemical knowledge of protein stability.<br /> The experimental validation in this study is exceptional. The single-molecule AFM analysis provided a high-resolution look at the energy landscape of these designed scaffolds. This approach allows for the direct observation of mechanical unfolding forces (exceeding 200 pN) and the precise contribution of individual chemical modifications to global stability. These measurements offer new, fundamental insights into the physicochemical principles that govern α-helix stabilization.

Weaknesses:

(1) While the initial manuscript lacked a detailed explanation for the stabilizing effect of the additional helix, the revised version now includes a clear structural basis for this improvement. The authors successfully attribute the increased unfolding force threshold to the reinforcement of the hydrophobic core and enhanced cooperative interactions, supported by relevant literature correlations between helix bundle size and stability.

(2) The author analyzed both thermal stability and mechanical stability. It would be helpful for the author to discuss the relationship between these two parameters in the context of their design. Since thermal melting probes equilibrium stability (ΔG), while mechanical stability probes the unfolding energy barriers along pulling coordinate. While the integrative design approach successfully improved both stability types, a deeper exploration of how the specific structural modifications influence the unfolding energy barrier relative to the overall equilibrium stability would further strengthen the mechanistic impact of the work.

(3) While the current study demonstrates a dramatic increase in global stability, the analysis focuses almost exclusively on the unfolding (melting) process. However, thermodynamic stability is a function of both folding (kf) and unfolding (ku) rates. The author have clarified that the observed ultrastability likely originates from a significantly reduced unfolding rates, a hypothesis consistent with the unfolding force. Direct measurements of the kinetics would provide deeper insights.

(4) The authors chose the spectrin repeat R15 as the starting scaffold for their design. R15 is a well-established model known for its "ultra-fast" folding kinetics, with folding rates (kf ~105s), near three orders of magnitude faster than its homologues like R17 (Scott et.al., Journal of molecular biology 344.1 (2004): 195-205). Measuring the folding rates of newly designed proteins would provide additional insights into the design.

Comments on revisions:

I think the author have addressed comments.

Reviewer #3 (Public review):

Summary:

This study provides novel insights into how individuals regulate the speed of their movements both alone and in pairs, highlighting consistent differences in movement vigor across people and showing that these differences can adapt in dyadic contexts. The findings are significant because they reveal stable individual patterns of action that are flexible when interacting with others, and they suggest that multiple factors, beyond reward sensitivity, may contribute to these idiosyncrasies. The evidence is generally strong, supported by careful behavioral measurements and appropriate modeling.

The authors have addressed all of my previous comments. I appreciate the clarification of abbreviations, terminology, and key concepts, the expansion of the discussion, and the adjustments to some of the statistical analyses in response to both my earlier comments and those of Reviewer 1.

Reviewer #3 (Public review):

Summary:

This work is an extension of their previous study (Loewinger et al 2025) describing a statistical framework for the analysis of photometry data using functional linear mixed models with joint confidence intervals, together with an open-source tool implemented in R. The present study extends it by adding the possibility of using 'concurrent' variables (variables that change within a trial) as regressors, for example, capturing the change of speed at each timepoint in the trial. The main claim is that using 'concurrent' regressors can identify associations between signal and behavior that could not be captured by 'non-concurrent' regressors (the value for a regressor on a specific trial is the same for each timepoint), which could lead to misleading conclusions. While the motivation for using time-varying covariates is useful and supported by previous literature (using fixed-effects models, although not cited in this manuscript), the reanalysis of previous studies does not clearly prove the benefit of using concurrent regressors as opposed to non-concurrent, and some of the results are difficult to interpret.

Strengths:

• The motivation for using time-varying covariates is well supported by previous literature using them on fixed-effects models, and here the authors are extending it to mixed-effects models.<br /> • The authors have included this new functionality in their previous open-source R package.

Weaknesses:

• The main weakness of this study is that it is not clear what the conceptual or methodological advance of this work is. As it is written, the manuscript focuses on showing how concurrent regressors offer interpretation advantages over non-concurrent regressors. While the benefit of such time-varying regressors is supported by previous literature (e.g., Engelhard et al., 2020), it is not clear whether the examples provided in the current study clearly support the advantage of one over the other, especially in the reanalysis of Machen et al. (2025), where the choice of regressors is confusing. In this specific example, if the question is about speed and reward type, why variables such as latency to reward or a binary 'reward zone vs corridor' (RZ) regressors are used instead of concurrent velocity (or peak velocity - in the case of the non-concurrent model)? Furthermore, if timing from trial start to reward collection is variable, why not align to reward collection, which would help in the interpretation of the signal and comparison between methods? Furthermore, while for the non-concurrent method, the regressors' coefficients are shown, for the concurrent one, what seems to be plotted are contrasts rather than the coefficients. The authors further acknowledge the interpretational difficulties of their analysis.<br /> • Because the relation between behavioral variables and neuronal signal is not instantaneous, previous literature using fixed effects uses, for example, different temporal lags, splines, and convolutional kernels; however, these are not discussed in the manuscript.<br /> • From the methods, it seems that in the concurrent version of fastFMM, both concurrent and non-concurrent regressors can be included, but this is not discussed in the manuscript.<br /> • The methodological advance is not clearly stated, apart from inputting into fastFMM a 3D matrix of regressors x trial x timepoint, instead of a 2D matrix of regressors x trial.<br /> • This manuscript is neither a clear demonstration of the need for concurrent variables, nor a 'tutorial' of how to use fastFMM with the added extension.

Reviewer #3 (Public review):

Wang et al demonstrate that RNA polymerase II and RNA polymerase III form distinct nuclear foci at the 5S rDNA-SL1 gene cluster in C. elegans. By ChIP, Pol II is highly enriched at the SL1 gene, whereas Pol III is enriched at the 5S rRNA gene. Both polymerase foci are spherical, show rapid exchange in FRAP experiments, and assemble in a cell-cycle-dependent manner, predominantly during S phase. The transcription factors ATTF-6 and SNPC-4 are required for the formation of Pol II foci but are dispensable for Pol III foci. Pol II foci, but not Pol III foci, are temperature-sensitive and dissolve upon heat stress; dissolution correlates with a strong reduction of SL1 transcription, whereas 5S rRNA levels remain largely unaffected.

Overall, this is a clean, well-organized, and well-controlled study, and I only have two comments.

(1) Roundness measurements, FRAP, and sensitivity to 1,6-hexanediol are indicative but not sufficient to show that these foci are condensates. They could, for example, also be scaffolded /chromatin-anchored assemblies (see https://pubmed.ncbi.nlm.nih.gov/36526633/). Please either provide better evidence or rephrase/tone down the condensate statements.

(2) Image quantification is only provided for Figure 5, but should also be reported for Figures 6 and 7. In addition to the foci number, also, e.g., intensity over background (similar to partition coefficient) should be quantified.

Reviewer #3 (Public review):

The study investigates MHC-related mate choice in humans using a sample of couples from a small-scale sub-Saharan society. This is an important endeavour, as the vast majority of previous studies have been based on samples from complex, highly structured societies that are unlikely to reflect most of human evolutionary history. Moreover, the study controls for genome-wide diversity, allowing for a test of the specificity of the MHC region, as theoretically predicted. Finally, the authors examine potential fitness benefits by analysing predicted pathogen-binding affinities. Across all analyses, no deviations from random pairing are detected, suggesting a limited role for MHC-related mate choice in a relatively homogeneous society. Overall, I find the study to be carefully executed, and the paper clearly written. Nevertheless, I believe the paper would benefit if the following points were considered:

(1) The authors claim (p. 2, l. 85) that their study is the first to employ a non-European small-scale society. I believe this claim is incorrect, as Hendrick and Black (1997) investigated MHC similarity among couples from South American indigenous populations.

(2) Regarding the argument that in complex societies, mating with a random individual would already result in sufficient MHC dissimilarity (p. 2, 78), see the paper from Croy et al. 2020, which used the largest sample to date in this research area.

(3) Dataset. As some relationships are parallel, I assume that certain individuals entered the dataset multiple times. This should be explicitly reported in the Methods. If I understand the analyses correctly, this non-independence was addressed by including individual identity as a random effect in the model - the authors should confirm whether this is the case. I am also wondering to what extent so-called "discovered partnerships" may affect the results. Shared offspring may be the outcome of short or transient affairs and could have a different social status compared with other informal relationships. Would the observed patterns change if these partnerships were excluded from the analyses?

(4) How many pairs were due to relatedness closer than 3rd degree? In addition, why was 4th degree relatedness used as a threshold in some of the other analyses?

(5) I was surprised by the exclusion of HIV, given that Namibia has a very high prevalence of HIV in the general population (e.g., Low et al. 2021).

(6) It appears that age criteria were applied when generating random pairs (p. 8, l. 350). Could the authors please specify what they consider a realistic age gap, and on what basis this threshold was chosen? As these are virtual couples used solely to estimate random variation within the population, it is not entirely clear why age constraints are necessary. Would the observed patterns change if no age criteria were applied?

(7) I think it would be helpful for readers if the Results section explicitly stated that real couples did not differ from randomly generated pairs. At present, only the comparison between chosen and arranged pairs is reported.

(8) I appreciate the separate analyses of pathogen-binding properties for MHC class I and class II, given their functional distinctiveness. For the same reason, I would welcome a parallel analysis of MHC sharing conducted separately for class I and class II loci.

(9) I think the Discussion would benefit from a more detailed comparison with previous studies. In addition, the manuscript does not explicitly address limitations of the current study, including the relatively limited sample size given the extensive polymorphism in the MHC region.

References:

Hedrick, P. W., & Black, F. L. (1997). HLA and mate selection: no evidence in South Amerindians. The American Journal of Human Genetics, 61(3), 505-511.

Croy, I., Ritschel, G., Kreßner-Kiel, D., Schäfer, L., Hummel, T., Havlíček, J., ... & Schmidt, A. H. (2020). Marriage does not relate to major histocompatibility complex: A genetic analysis based on 3691 couples. Proceedings of the Royal Society B, 287(1936), 20201800.

Low, A., Sachathep, K., Rutherford, G., Nitschke, A. M., Wolkon, A., Banda, K., ... & Mutenda, N. (2021). Migration in Namibia and its association with HIV acquisition and treatment outcomes. PLoS One, 16(9), e0256865.

Reviewer #3 (Public review):

Summary:

PIFs play a pivotal role not only in light and temperature signaling pathways, but in many other signaling pathways regulating plant development by modulating transcription of a large number of genes both directly and indirectly. Similarly, alternative splicing (AS) plays a critical role in shaping the splice isoforms of thousands of genes under different environmental conditions to regulate plant development. In fact, AS of PIF6 has been shown to be involved in seed development. PIF4 is a central transcription factor integrating light and temperature signaling pathways. However, AS of PIF4 has not been involved in any pathways. This story first describes how AS of PIF4 is regulated by heat stress, and this regulation is involved in heat stress signaling to regulate plant development. This is an important finding of general interest.

Strengths:

The authors first describe AS of PIF4 is regulated by heat stress, and this regulation is involved in heat stress signaling to regulate plant development.

Weaknesses:

There are many loose ends in this story that need to be tied up.

Major points:

(1) The authors are showing only the AS transcripts by PCR, but no protein data. Given that the hypothesis is that the short form of PIF4 is functioning in a dominant negative fashion, the authors need to show that this short isoform expresses a protein. In addition, they need to show that this form is functioning in a dominant negative fashion with other PIFs, either by showing that this form reduces the DNA binding and/or transcriptional responses of other PIFs.

(2) The two mutant alleles used for this study (pif4-100 and pif4-2) have T-DNA insertion after the AS exon. Do these alleles express any short version of the protein? The previous studies showed no protein production, and thus, they may not function as a dominant negative form. Usually, the T-DNA insertion alleles may express truncated transcripts, but many do not express any protein due to a lack of stop codon and/or degradation of the transcripts. But in this case, the mutants are behaving like WT. The authors need to show that these alleles are expressing a truncated version of the PIF4 protein.

(3) Figure 4 shows phenotypes of independent lines expressing the PIF4 short version. The authors analyzed only the cotyledon and hypocotyl phenotypes, but not Pchlide or bleaching assays. The authors need to do a thorough phenotype analysis, including heat-stress phenotypes of these lines, to test if the data make sense with their hypothesis.

Reviewer #3 (Public review):

Summary:

The manuscript by Shukla and colleagues presents a comprehensive study that addresses a central question in kinesin-1 regulation-how cargo binding to the kinesin light chain (KLC) tetratricopeptide repeat (TPR) domains triggers activation of full-length kinesin-1 (KHC). The authors combine AlphaFold3 modeling, biophysical analysis (fluorescence polarization, hydrogen-deuterium exchange), and electron microscopy to derive a mechanistic model in which the KLC-TPR domains dock onto coiled-coil 1 (CC1) of the KHC to form the "TPR shoulder," stabilizing the autoinhibited (λ-particle) conformation. Binding of a W/Y-acidic cargo motif (KinTag) or deletion of the CC1 docking site (TDS) dislocates this shoulder, liberating the motor domains and enhancing accessibility to cofactors such as MAP7. The results link cargo recognition to allosteric structural transitions and present a unified model of kinesin-1 activation. I recommend acceptance of the manuscript subject to the following additions:

Strengths:

(1) The study addresses a fundamental and long-standing question in kinesin-1 regulation using a multidisciplinary approach that combines structural modeling, quantitative biophysics, and electron microscopy.

(2) The mechanistic model linking cargo-induced dislocation of the TPR shoulder to activation of the motor complex is well supported by both structural and biochemical evidence.

(3) The authors employ elegant protein-engineering strategies (e.g., ElbowLock and ΔTDS constructs) that enable direct testing of model predictions, providing clear mechanistic insight rather than purely correlative data.

(4) The data are internally consistent and align well with previous studies on kinesin-1 regulation and MAP7-mediated activation, strengthening the overall conclusion.

Weaknesses:

(1) While the EM and HDX-MS analyses are informative, the conformational heterogeneity of the complex limits structural resolution, making some aspects of the model (e.g., stoichiometry or symmetry of TPR docking) indirect rather than directly visualized.

(2) The dynamics of KLC-TPR docking and undocking remain incompletely defined; it is unclear whether both TPR domains engage CC1 simultaneously or in an alternating fashion.

(3) The interplay between cargo adaptors and MAP7 is discussed but not experimentally explored, leaving open questions about the sequence and exclusivity of their interactions with CC1.

Comments on revisions:

The authors have addressed my comments satisfactorily.

Reviewer #3 (Public review):

Summary:

In this manuscript, Barré et al utilize the Gp1ba-Cre transgenic mouse model to build upon previous findings in a Pf4-Cre system to investigate the effects of individual and combined Shp1 and Shp2 deletion in megakaryocytes and platelets. They report decreased megakaryocyte maturation, macrothrombocytopenia, and increased bleeding primarily in association with the Shp1/Shp2 double-knockout condition. The authors further show that this phenotype appears to be driven primarily by Shp2 and implicate dysregulation of Mpl signaling and downstream Ras/MAPK pathways, including ERK1/2. Given the key role of these pathways in human diseases such as myeloproliferative neoplasms and the challenges associated with modulating such a central pathway, identification of a specific regulator of Mpl signaling poses intriguing questions for future studies on clinical applicability.

Strengths:

Overall, the experiments combine in vitro, in vivo, and ex vivo approaches and appear to have been carefully designed and carried out, with multiple technical and biological replicates where relevant. The authors make a compelling argument for using the Gp1ba-Cre as opposed to the Pf4-Cre system and demonstrate both the dose- and stage-dependent effects of Shp1 and Shp2 on megakaryopoiesis and thrombopoiesis. They find that Shp1 and Shp2 are required in late-stage megakaryocyte maturation and that even low levels of expression compared to baseline are likely sufficient to yield generally normal megakaryocytes. Their findings also lead to specific future directions, such as the mechanism by which Shp1 regulates megakaryopoiesis and thrombopoiesis that is distinct from TPO-mediated signaling.

Weaknesses:

While the experiments have been thoughtfully designed and carried out, there is limited background explanation on relatively complex or niche pathways/mechanisms, such as the relationship between P-selectin, CRP, and PAR4p; the interactions between SFK, Syk, GPVI, and CLEC-2; and TPO, MPL, ERK1/2, AKT, and STAT3, which, while likely intuitive to experts in their respective fields, may be less obvious to a reader approaching this manuscript with a global interest in megakaryopoiesis/thrombopoiesis and thus detract from the impact of the findings.

With regard to the science itself, some of the conclusions feel premature based on the available data.

(1) The section "Aberrant ITAM signaling in Shp1- and Shp2-deficient platelets" is challenging to follow for those not well-versed in ITAM signaling and associated pathways, and may take additional outside reading to follow the conclusion that Syk-dependent signaling is modulated downstream of GPVI and CLEC-2 based on lack of change in Src p-Tyr418, especially considering that Src p-Tyr418 was previously introduced as a measure of SFK rather than Syk. In the introduction, Shp1 is specifically mentioned as a negative regulator of the ITAM/Syk/phospholipase pathway. However, in Figure 4Ai and Bi, Syk phosphorylation/activation in Shp1 knockout cells did not appear to be different from Shp2 knockout cells, and is lower than the control, which is surprising for a negative regulator. It is also not clear why, in the section (Figure 4A-B), there is reduced Syk activation in Shp1 and Shp2 single knockout cells upon CLEC2 stimulation (but apparently not with CRP) when there was no difference in response to CLEC2 (but a difference in response to CRP) in the previous section (Figure 3A, C).

(2) In the section "Reduced Tpo signaling in Shp1/2-deficient MKs," only Western blot data for (p)ERK1/2, AKT, and STAT3 are presented before concluding that decreased ERK1/2 activity is a mechanistic explanation for thrombocytopenia seen in the Shp1/2 double-knockout condition. Such a statement would benefit from additional experiments, such as protein or transcriptional levels of ERK1/2 targets specifically relevant to megakaryopoiesis, such as ETS, FOS, and JUN, to assess the consequences of decreased phosphorylated ERK1/2.

(3) Suggesting that "inhibiting Shp2 will not hav[e] any bleeding consequence in patients" and that Shp2 may be a therapeutic target in myeloproliferative neoplasms when none of these studies have been carried out in a human model is a bold conclusion. There are no data presented on, for example, whether Shp2 inhibition can help reverse the MPL/JAK/STAT pathway in the setting of gain-of-function mutations specifically associated with myeloproliferative neoplasms.

Reviewer #3 (Public review):

In the manuscript entitled "A Minimal tooth Enhancer Regulates dlx2b Expression During Zebrafish Tooth 1 Formation: Insights into Cis-Regulatory Logic in Organogenesis", the authors explore the cis-regulatory logic of a dlx2b minimal enhancer capable of directing dlx2b gene expression to the developing tooth germs. The study combines (1) CRISPR-mediated GFP knock-in to track endogenous gene expression; (2) a promoter-bashing approach to identify a minimal tooth enhancer (MTE); (3) site-directed mutagenesis coupoled with transgenesis to assess the individual role of conserved TF binding sites; and (4) in vivo deletgion of the MTE to examine the consequences for gene expression. Overall, this is a technically solid study that provides some novel insights into tooth development and extends previous observations by the authors (Jackman & Stock, 2006; PNAS). However, the added value of the manuscript is limited by both the narrow experimental scope and the relatively modest impact of the findings for the broader field of developmental biology.

Main concerns:

(1) My main concern is that the study restricts the search for cis-regulatory information to the 5' region 4kb upstream of the TSS of the gene, rather than encompassing the full genomic locus. This is particularly limiting given that a knock-in allele was generated, which in principle allows interrogation of regulatory elements across the entire locus, and that the authors acknowledge the availability of genome-wide regulatory datasets (e.g. DANIO-CODE) in the Discussion. Despite this, no systematic effort is made to test additional regulatory elements beyond the proximal promoter/enhancers.<br /> This has important implications for the interpretation of the current work as: (a) dlx2b, as many developmental genes, resides in a gene desert enriched in open chromatin regions that may function as distal enhancers, and (b) the deletion of the MTE unmasked a cis-regulatory activity which nature cannot be explained with the information provided, and that may seem relevant for the expression of the gene in the dental mesenchyme.

(2) A second concern is the absence of information on the functional consequences of deleting the gene or the MTE on tooth primordium development. From the description of the KI strategy, it is unclear whether the GFP insertion results in a functional fusion protein. The cytoplasmic GFP distribution and the schematic in Figure S1 instead suggest the presence of a terminal stop codon in the GFP sequence, which would result in a dlx2b loss-of-function allele. If this interpretation is correct, the manuscript does not describe the developmental consequences in homozygous embryos. Similar concerns apply to the MTE deletion: it remains unclear whether loss of this enhancer results in any detectable morphological or developmental defects.

Reviewer #3 (Public review):

Summary:

In this study, Tang, Yu & colleagues investigate the impact of continuous flash suppression (CFS) on the responses of V1 neurons using 2-photon calcium imaging. The report that CFS substantially suppressed V1 orientation responses. This suppression happens in a graded fashion depending on the binocular preference of the neuron: neurons preferring the eye that was presented with the marker stimuli were most suppressed, while the neurons preferring the eye to which the grating stimuli were presented were least suppressed. Binocular neuron exhibited an intermediate level of suppression.

Strengths:

The imaging techniques are cutting-edge.

Weaknesses:

The strength of CFS suppression varies across animals, but the authors attribute this to comparable heterogeneity in the human psychophysics literature.

Comments on revisions:

The authors have addressed my comments from the previous round of review, and I have no further comments

40:20 How about Europe? -- Europe is a dumpster fire

Reviewer #3 (Public review):

Male mice were tested in a classic behavioral "flee the looming stimulus" paradigm. This is a purely behavioral study; no neural analyses were done. Mice were housed socially, but faced the looming stimulus individually, using an elegant automated tunnel (see videos for clarity).

The additional changes made to the paper clarify the work done. While there are some limitations (male mice, weird stimulus), the general results are interesting and a valuable addition to the experimental literature. The main claim of the paper is that the different rewards (none, water, sucrose) did not change the escape properties early in learning, but did late, particularly that in the late (already experienced) conditions, reward value (assuming sucrose > water > no reward) interacted with the salience of the looming stimulus (light gray, dark gray). (Panels 3D, 3G, 3K, 3N).

For readers, I want to note that one of the most interesting results is actually in Figure S2, where they find that a looming stimulus behind the mouse still makes a mouse run to the nest. In these conditions, the mouse runs past the looming stimulus to get to safety! (I also do love the video of the mouse running around the barriers like a snake to get home.)

I have a few minor clarification questions and a few notes that I think would be useful additions for authors and readers to think about.

Dominance: What does the mouse social science literature say about the "test tube" test? What can we conclude from this test? This would be useful when trying to understand what is causing the dominance/submissive difference in responses. Figure 4 shows that the dominant mice are more risk-averse than the submissive mice. Is "dominance" in the test-tube actually a measure of risk-seeking? Is the issue that the submissive mice don't think they can get back to the food-site easily, so they are less willing to sacrifice the current (if dangerous) foraging opportunity? Is the issue that the submissive mice can't get back to the nest? As I understand it, the nest was always available to all the mice, so I suspect inability to get to the nest is an unlikely hypotheses. Is the issue that the submissive mice also don't feel safe in the nest?

Limitations of the study: There is an acknowledged limitation to male mice, and the limitations of the small data sets that are typical of such experiments. In addition, however, it is also worth noting the strangeness of the looming stimulus, which is revealed clearly in the videos. The stimulus is a repeating growing circle, growing in a single location within the environment. The stimulus repeats 10 times, once per second. This is not what an attacking hawk or owl would look like. (I now have this image of an owl diving down, and then teleporting up and diving down again.) Note - I am fine with this stimulus. It produces an interesting experiment and interesting results. I do not think the authors need to change anything in their paper, but readers need to recognize that this is not a "looming predator".

These "limitations" are better seen as "caveats" when folding these results in with the rest of the literature that has gone before and the literature to come. (Generally, I do not believe that science works by studies making discoveries that change how we think about problems - instead, science works by studies adding to the literature that we integrate in with the rest of the literature.) Thus, these caveats should not be taken as problems with the study or as fixes that need to be done. Instead, they are notes for future researchers to notice if differences are found in any future studies.

Thus, my only suggestion is that I think authors could write a more careful paper by using the past and subjunctive tense appropriately. Experimental observations should be in past tense, as in "the influence of reward was context-dependent and emerged in the late phase" instead of "the influence of reward is context-dependent and emerges in the late phase" - it emerged in the late phase this once - it might not in future experiments, not due to any fault in this experiment nor due to replicability problems, but rather due to unexpected differences between this and those future experiments. At which point, it will be up to those future experiments to determine the difference. Similarly, large conclusions should be in the subjunctive tense, as in "these data suggest that threat intensity is likely to be the primary determinant of decision making" rather than "threat intensity is the primary determinant of decision making", because those are hypotheses not facts.

Reviewer #3 (Public review):

Inoshita and Kawaguchi investigated the effects of GPR55 activation on synaptic transmission in vitro. To address this question, they performed direct patch-clamp recordings from axon terminals of cerebellar Purkinje cells and fluorescent imaging of vesicular exocytosis utilizing synapto-pHluorin. They found that exogenous activation of GPR55 suppresses GABA release at Purkinje cell to deep cerebellar nuclei (PC-DCN) synapses by reducing the readily releasable pool (RRP) of vesicles. This mechanism may also operate at other synapses.

Strengths:

The main strength of this study lies in combining patch-clamp recordings from axon terminals with imaging of presynaptic vesicular exocytosis to reveal a novel mechanism by which activation of GPR55 suppresses inhibitory synaptic strength. The results strongly suggest that GPR55 activation reduces the RRP size without altering presynaptic calcium influx.

Weaknesses:

The study relies on the exogenous application of GPR55 agonists. It remains unclear whether endogenous ligands released by physiological or pathological processes would have similar effects. There is also little evidence that GPR55 is expressed in Purkinje cell axon boutons. This study would benefit from the use of GPR55 knockout (KO) mice. The downstream mechanism by which GPR55 mediates the suppression of GABA release remains unknown.

Comments on revisions:

The authors have addressed all my concerns effectively. I have no further comments and want to commend their comprehensive study.

Reviewer #3 (Public review):

Summary:

This work investigates whether human imprecision in numeric perception is a fixed structural constraint or an endogenous property that adapts to environmental statistics and task objectives. By measuring behavioral variability across different uniform prior distributions in both estimation and discrimination tasks, the authors show that perceptual imprecision increases sublinearly with prior width. They demonstrate that the specific exponents of this scaling (1/2 for estimation and 3/4 for discrimination) can be derived from an efficient-coding model, wherein decision-makers optimally balance task-specific expected rewards against the metabolic costs of neural coding. The revised manuscript expands this framework to accommodate logarithmic representations and validates the core model against an independent dataset of risky choices.

Strengths:

The authors have effectively addressed my previous concerns with rigorous additions:

(1) The mathematical formulation has been revised into a discrete signal accumulation framework, making the objective function and resource trade-offs much more transparent and mathematically tractable.

(2) The incorporation of the logarithmic representation resolves prior ambiguities regarding structural constraints.

(3) The new split-half analysis effectively addresses the temporal dynamics of adaptation. The stability of the sublinear scaling across the experiment provides solid evidence that human subjects utilize rapid, top-down modulation to adjust their encoding strategy when explicitly informed about the environment.

(4) Validating the derived scaling exponents on an independent risky-choice dataset robustly supports the generalizability of the theoretical framework beyond a single cognitive domain.

Weaknesses:

The methodological and theoretical issues raised in the first round have been thoroughly resolved, and the evidence supporting the claims regarding response variance is convincing.

There is one remaining theoretical point that warrants discussion to provide a complete picture of the proposed generative model. The manuscript exquisitely models and predicts response variance (imprecision), but it remains largely silent on the closed-form predictions for the mean estimation (i.e., bias). Under the assumption of optimal Bayesian decoding combined with specific encoding schemes (e.g., linear vs. logarithmic), the model implicitly generates mathematical predictions for the subjects' mean estimates. Specifically, varying the scaling exponent (α) and the prior width (w) should systematically alter the predicted bias in different conditions.

While fitting or explicitly explaining this mean bias is not strictly necessary for the core claims regarding variance scaling, acknowledging what the optimal decoder analytically predicts for the mean estimation-and how it aligns or contrasts with typical empirical observations-would strengthen the theoretical transparency of the paper.

Reviewer #3 (Public review):

The study aimed to address a fundamental question in T. cruzi and Chagas disease biology - how much variation is there in gene expression between individual parasites? This is particularly important with respect to the surface protein-encoding genes, which are mainly from massive repetitive gene families with 100s to 1000s of variant sequences in the genome. There is very little direct evidence for how expression of these genes is controlled. The authors conducted a single cell RNAseq experiment of in vitro cultured parasites with a mixture of amastigotes and trypomastigotes. Most of the analysis focused on the heterogeneity of gene expression patterns amongst trypomastigotes. They show that heterogeneity was very high for all gene classes, but surface-protein encoding genes were the most variable. Interestingly, in the case of the trans-sialidase genes, many sequence variants were detected in fewer than 5% of parasites while a subset of 31 others was detected in >40% if parasites, hinting at compartmentalised expression control within the gene family. The biology of the parasite (e.g. extensive post-transcriptional regulation) and potential technical caveats (e.g. high dropout rates across the genome) make it difficult to infer connections to actual protein expression on the parasite surface, but the results are a significant advance for the field.

(1) Limit of detection and gene dropouts.

An average of ~1100 genes are detected per parasite which indicates a dropout rate of over 90%. It appears that RNA for the "average" single copy 'core' gene is only detected in around 3% of the parasites sampled (Figure 2c: ~100 / 3192). While comparable with some other trypanosome scRNAseq studies, this remains a caveat to the interpretation that high cell-to-cell variability in gene expression is explained by biological factors. The argument would be more convincing if the dropout rates and expression heterogeneity were minimal for highly expressed housekeeping genes. The authors are appropriately cautious in their interpretation and acknowledge the need for further validation.

(2) Heterogeneity across the board.

The authors focus on the relative heterogeneity in RNA abundance for surface proteins from the multicopy gene families vs core genes. While multicopy gene sequences do show significantly more cell-to-cell variability, there is still surprisingly high inequality of expression amongst genes in other classes including single copy housekeeping and ribosomal genes. Again the biological relevance of the comparison is uncertain and the authors acknowledge the need for further investigation.

This study provides some tantalising evidence that the expression of surface genes may vary substantially between individual parasites in a single clonal population. The study is also amongst the very first to apply scRNAseq to T. cruzi, so the broader data set will be an important resource for researchers in the field.

Comment on revised version:

The manuscript is significantly improved. The revised explanations and figures make several aspects of the data analysis and interpretation much clearer to me now. Thanks to the authors.

Reviewer #3 (Public review):

Summary:

Maigler & Lin et al present a compelling set of behavioral and electrophysiological experiments exploring how individual differences in taste preference map onto neural responses in the gustatory cortex (GC). They go on to examine how both preferences and neural responses shift following intervening taste experience. Their experiments are strengthened by examining tastes of distinct identities and palatability (sweet, sour, salty, bitter) and corresponding each animal's individual preference to the palatability-related late phase of the neural response.

Strengths:

(1) They demonstrate a relationship between the behavioral expression of taste preference and palatability-related GC neural responses. The direct correlation of expression of taste preference with GC neural responses indicates that taste preference behavior may be less noisy than previously thought, reflecting actual neural activity.

(2) They address the stability of individual taste preference by comparing within and between session expression. This finding indicates that individual preference on any given test session can differ from canonical palatability.

(3) They provide evidence that representational drift in palatability coding may arise from sensory experience rather than from the passive passage of time. The findings are novel and potentially impactful. The results are relatively complete.

Weaknesses:

Experiments require further clarification. The interpretations would be strengthened by reorganizing the experimental design.

(1) Figures 5-6 show shifts in palatability-related GC responses from recording day 1 to recording day 2. The authors propose that this drift is due to the taste experience during recording day 1, but the study, as designed, does not directly test this idea. Without a behavioral measure collected after recording day 1 intraoral exposure, it is not possible to determine whether taste preference was altered by that experience, nor whether the neural responses collected on recording day 2 represent current or most recent palatability expression vs something else. The authors' conclusion would be strengthened by adding an intervening brief access test between recording days 1 and 2. The authors could then determine whether the behavioral preferences changed after intraoral taste exposure on recording day 1, as well as whether the new set of taste-related palatability responses correlates with the updated taste preferences.

(2) The current experimental design exposes animals to 3 distinct sets of substances. These substances differ in identity (some rats never experienced sweet, while others did not experience bitter during the recording sessions) and concentration (ranging from very aversive to slightly aversive or possibly even neutral). Because palatability is known to be comparative depending on the other substances available and concentration-dependent, this introduces challenges to interpretation.

The authors state that "no differences in effects were observed between taste batteries" (Methods), but it is not clear which analyses were performed to determine the lack of difference, especially considering that many of the analyses are within-animal. Without more clarity, it is difficult to evaluate whether the interaction of different tastes within the sets of stimuli biases the main conclusions.

(3) Responses to sweet tastes are not reported in the electrophysiology data. This is seemingly the case because rats given set 1 received no sweet stimulus while rats given set 2 received to 2 distinct sweet tastes. Finally, rats given set 3 did not receive quinine, yet quinine is reported in electrophysiology data.

(4) The choice of reporting average lick cluster size is problematic because the authors use thirsty rats with 10-second-long trials. Thirsty rats are likely to lick in relatively long clusters, especially for neutral and palatable tastes. If the rat is mid-cluster when the trial ends, the final cluster would be cut off prematurely, resulting in shorter overall average lick cluster size, disproportionately affecting neutral and palatable tastes over aversive tastes.

(5) Canonical palatability rankings may not apply to the concentrations selected in every stimulus set. This is particularly true for set 1, which included two concentrations of citric acid and quinine for the behavior. It is also not clear which concentrations are reported in Figures 3A2 and 3B2. Meanwhile, the concentrations of quinine and citric acid used for electrophysiology are quite low.

Reviewer #3 (Public review):

Summary:

The study by Hong, Loer, Hobert, and colleagues is a comprehensive description of monoaminergic neurons in the nematode Pristionchus pacificus. The work used multiple, complementary approaches, including immunostaining and expression of genes involved in neurotransmitter synthesis or transport, to identify neurons that express a monoamine neurotransmitter. Moreover, this study characterized the phenotypes of various mutants to study their organismal function. Extensive comparisons are made to C. elegans, the nematode model that, in a way, anchors the model studied here, and new outgroup species were examined for some features so that the polarity of their evolution could be inferred. Although there is no simple or groundbreaking punchline to distill from the manuscript (i.e., other than some things are the same as in C. elegans, and some things are different), and while the study is basically descriptive in nature, the scope of the project warrants broad attention.

Strengths:

This manuscript offers a tremendous resource for those who use this species as a model, which, based on the author list alone, includes many labs. This study sets the bar for what can be done in a "satellite" model system.

Given the complementarity of approaches used, such as the position of cell bodies, the connectivity and morphology of dendrites, and a previously published atlas of the connectome for this species, the identification of specific neurons (which, as the authors point out, can be easily mistaken) is convincing throughout. Likewise, appropriate caution is observed where neuron identities are ambiguous, e.g., unlabelled cells in Figure 5, or ambiguous identities in other species, as shown in Figure 10. There was a lot of data to unpack in this manuscript, but I could not find any obvious flaws in neuron identification.

Also, the phenotypic assays were straightforward and informative.

Weaknesses:

No serious weaknesses were noted. One minor comment is that in general, I think the Methods could use some additional text to describe what the goal of any given technique was. For example, although there is a description of the HCR protocol in the methods, nowhere does it say what genes this method would be used for. In addition to what is shown in Figure 4, this information should be given in the Methods.

Reviewer #3 (Public review):

Summary:

In this study, Guo and colleagues investigated the effects of stress and retrieval practice on memory inference. In the first experiment, they found that memory inference was significantly worse after induced stress. Conversely, when participants received retrieval practice in the second experiment, they found no significant differences between these conditions. They monitored EEG during the inference phase and applied multivariate decoding analysis to examine evidence of neural reactivation. Complementing the behavioural findings of the first experiment, they found that they were able to decode the stimulus category of the inference item with more fidelity in the no stress condition. Surprisingly, they found the opposite direction when participants had retrieval practice, with stronger evidence of reactivation in the stress condition than in the control condition.

Strengths:

(1) The authors have carefully designed two studies investigating the effects of stress and memory retrieval on memory inference.

(2) The use of multivariate decoding on the inference phase data sheds new light on how stress and retrieval may impact the neural signatures of inference processing.

Weaknesses:

(1) There are some key gaps in the reporting of the data. In particular, data is missing on how many trials were included in the inference phase and how many were retrieved in the direct memory task. This is important to know as the main conclusions are based on inference trials proportional to the direct retrieval trials. Considering that the direct retrieval performance differs significantly between the experiments, there could be issues with floor/ceiling effects (in the behaviour) and statistical power (in the EEG results) that confound the comparisons between experiments. Without the data, it is difficult to draw conclusions.

(2) There are some relatively strong conclusions drawn without the data to support them. An important example is the title suggesting a mechanistic role of memory reactivation for these effects; however, the data instead suggest a relationship between successful inference and evidence of reactivation. Additionally, one-tailed t-tests have been used in follow-up tests, and, as I understand it, no multiple comparisons corrections have been applied to the post-hoc tests, suggesting that these findings should be interpreted with caution.

(3) In places, the structure is unclear, making the narrative difficult to follow, often making it necessary for the reader to go back and forth between the sections to understand the study and analyses. I have made some recommendations for how to improve this.

Reviewer #3 (Public review):

Summary

This paper introduces EMUsort, an extension of Kilosort4 designed to sort motor unit action potentials from high-density intramuscular EMG recordings. Using rat and monkey forelimb recordings, the authors generate realistic simulated datasets with known ground truth and demonstrate that EMUsort substantially outperforms Kilosort4 and MUedit, particularly during periods of high motor unit overlap.

Strengths

This is a timely study in light of recent advances in intramuscular muscle recording technologies and the growing interest in automated methods for decoding neural and neuromuscular signals. The work leverages state-of-the-art electrode arrays and combines them with advanced signal processing tools to address a challenging and relevant problem in motor unit analysis.

Weaknesses

There are several aspects of the study that substantially limit the interpretation of the main results and conclusions. The following major points should be carefully considered by the authors.

(1) Choice of experimental model and validation framework: The study aims to validate a new methodology for EMG decomposition, yet the rationale for the chosen experimental models is unclear. Specifically, it is not evident why the authors focused on intramuscular recordings from two animal models performing dynamic tasks. Given the extensive literature on the development and validation of EMG decomposition methods, the choice of an experimental design that substantially deviates from established approaches is insufficiently justified. In particular, it is unclear why the authors did not consider more standard validation paradigms based on (i) isometric contractions, (ii) human data, (iii) surface EMG recordings, or (iv) combinations of their recording technologies with previously validated motor unit identification methods. This methodological divergence makes it difficult to interpret the findings in the context of existing evidence.

(2) Lack of manual EMG decomposition as reference: Related to the previous point, it is unclear why standard manual EMG decomposition methods were not used to generate reference datasets for validation. Manual decomposition has been shown to be reliable under specific conditions (low contraction levels, slow dynamics, etc.) and would have substantially strengthened the validation of the proposed algorithm.

(3) Neglect of muscle deformation effects: While the manuscript discusses several factors that complicate EMG decomposition relative to brain recordings, it does not address the well-known effects of muscle deformation during contractions on motor unit action potential shapes. There is extensive literature demonstrating that dynamic muscle contractions lead to systematic changes in action potential morphology, representing a major challenge for EMG decomposition and a fundamental difference from brain recordings. Additionally, even small relative movements of intramuscular electrodes can produce waveform changes that are large relative to muscle fiber dimensions. These issues are particularly relevant given the highly dynamic tasks studied here (e.g., treadmill walking in rats), yet they are not discussed or incorporated into the analysis.

(4) Exclusive reliance on simulated data for validation: The primary validation of EMUsort is based on simulated data, which represents a major limitation of the study. This reliance should be clearly and explicitly stated in the abstract, introduction, and discussion. Moreover, the simulation approach itself raises concerns. The simulated EMG signals are generated using templates derived from the same sorting framework being validated, which introduces a potential methodological bias. The linear combination of components used to synthesize waveforms constitutes an unjustified modeling assumption that may favor template-based approaches such as EMUsort. Additionally, the spike time generation procedure appears unnecessarily complex and insufficiently justified. Previous validation studies typically modeled motor units as firing at relatively stable levels along their recruitment curves, producing long spike trains with pseudo-random relative timing and diverse overlap conditions. This framework would likely provide a more robust and interpretable validation. If the authors believe their simulation approach is superior, a stronger justification is required. Finally, the limited number of simulated motor units is difficult to reconcile with the expected level of motor unit recruitment during the studied behaviors, and this choice is not adequately justified.

(5) Incomplete reporting and visualization of experimental data: The manuscript would benefit from a clearer description of the number of rats and monkeys used, which should be reported explicitly in the abstract. In addition, visualizations of the raw multichannel EMG data across different task phases and activation levels would substantially improve transparency. Providing comprehensive visualizations of motor unit action potential shapes across all channels and identified units (for both rats and monkeys) would also help readers assess the spatiotemporal features that underpin unit identification and sorting reliability.

(6) Physiological limitations of conduction delay correction: The proposed method for correcting conduction delays across channels is physiologically suboptimal. First, motor unit conduction velocities differ substantially across units, implying that delay correction should be applied at the unit level rather than uniformly across channels. Second, conduction delays depend on fiber orientation and distance relative to electrode geometry; if fibers are oriented at different angles with respect to the array, a single delay correction becomes invalid. Additionally, the schematic in Figure 2A appears to contradict the proposed correction approach: if electrode threads are arranged perpendicular to muscle fibers, conduction delays across channels within a single thread should be minimal.

(7) Clarity issues in Figure 4: Figure 4 (panels A-D) is potentially misleading. It should be clearly stated whether the signals shown are artificial examples or derived from real recordings; ideally, real data should be used to illustrate the advantages of dynamic thresholds. In panels B-D, the depiction of overlapping action potentials is difficult to interpret due to the thickness of the traces, and it is unclear whether overlaps with neighboring action potentials are absent by design or expected to occur in real data. If overlaps are expected, one would also expect to observe contamination in the extracted waveforms, which is not evident in the figure.

(8) Concerns regarding method comparisons: The comparison with existing methods raises methodological concerns. It appears that EMUsort was carefully optimized, whereas the competing algorithms were not equivalently fine-tuned. The literature clearly shows that EMG decomposition performance depends strongly on adapting algorithms to the signal type (intramuscular vs. surface, species, electrode geometry). Furthermore, it is surprising that MUedit is reported to perform particularly poorly during periods of motor unit overlap, as blind source separation methods were explicitly developed to handle convolutive mixtures and overlapping sources, especially in surface EMG (which is an extreme case of motor unit overlapping). This discrepancy requires further explanation.

(9) Insufficient characterization of motor unit firing properties: The study does not provide sufficient information about the firing characteristics of the identified motor units in experimental data. Relevant metrics that should be reported include average, minimum, and maximum firing rates; coefficients of variation of discharge rate; signal-to-noise ratios of motor unit action potentials; potential evidence of motor unit rotation over time; and stability of firing behavior across recording intervals.

(10) Lack of theoretical framing: Given the scope and claims of the paper, it would be valuable to include a more theory-driven introduction explaining why different sorting approaches (e.g., template matching vs. blind source separation) may be more or less suitable depending on the nature of the recorded signals. A clearer conceptual rationale for why the proposed approach is expected to outperform existing methods would substantially strengthen the manuscript.

(11) Limitations of validation metrics: Some of the metrics used to evaluate performance are not ideal. For example, reporting 0% accuracy for a unit is misleading and should instead be described as a failure to identify that unit. Similarly, comparisons of total spike counts are of limited interpretive value and may be misleading, as correct spike counts do not necessarily imply correct spike identities.

(12) Clarification of computational performance claims: Finally, the discussion of computation times should clarify that some existing methods require substantial time for offline estimation of projection vectors but can operate in near real time once these vectors are learned and remain stable. This distinction is important for a fair comparison of practical usability.

Reviewer #3 (Public review):

The manuscript by Fuller et al describes a crosstalk between ARTG2A with components of the early secretory pathway, namely RAB1A and ARFGAP1. They show that ATG2A is recruited to membranes positive for RAB1A, which they also show to interact with ATG2A. In agreement with earlier findings by other groups, silencing RAB1A negatively affects autophagy. While ARFGAP1 was also found on ATG2A positive membranes, silencing ARFGAP1 had no impact autophagy. Notably, these ARFGAP1 positive membranes are not Golgi membranes.

The findings are interesting and the data are in general of good quality. I think the story is good enough to be published in eLife and I have the following questions, which the authors may attend to:

(1) Are the membranes to which ATG2A is recruited a form of ERGIC?

(2) Figure 3A/B: Is it possible to show a better example? The difference is barely detectable by eye. Since Immunoblotting is not really a quantitative method, I think that such a weak effect is prone to be wrong. Is there another tool/assay to validate this result?

(3) Is the curvature-sensitive region of ARFGAP1 required for its co-localization with ATG2A?

(4) What does Rab1A do? What is its effector? Or does the GTPase itself remodel the membrane?

(5) What about Arf1? It appears that this role of ARFGAP1 is unrelated to Arf1 and COPI? Thus, one would predict that Arf1 does not localize to these structures and does not affect ATG2A function

(6) Does ARFGAP1 promote fission of the membrane from its donor compartment?

(7) What are ARFGAP1 and Rab1A recruited to? What is the lipid composition, or protein that recruits these two players to regulate autophagy?

Comments on the latest version:

The revisions carried out by the authors are fine. The new data on ArfGAP1 and about the indirectness of the ATG2A and Rab1A interaction improve both clarity and strength of the manuscript. I have no further comments.

https://www.facebook.com/groups/TypewriterCollectors/posts/10163297970974678/

Reviewer #3 (Public review):

Summary:

Kern et al. critically assess the sensitivity of temporally delayed linear modelling (TDLM), a relatively new method used to detect memory replay in humans via MEG. While TDLM has recently gained traction and been used to report many exciting links between replay and behavior in humans, Kern et al. were unable to detect replay during a post-learning rest period. To determine whether this null result reflected an actual absence of replay or sensitivity of the method, the authors ran a simulation: synthetic replay events were inserted into a control dataset, and TDLM was used to decode them, varying both replay density and its correlation with behavior. The results revealed that TDLM could only reliably detect replay at unrealistically (not-physiological) high replay densities, and the authors were unable to induce strong behavior correlations. These findings highlight important limitations of TDLM, particularly for detecting replay over extended, minutes long time periods.

Strengths:

Overall, I think this is an extremely important paper, given the growing use of TDLM to report exciting relationships between replay and behavior in humans. I found the text clear, the results compelling, and the critique of TDLM quite fair: it is not that this method can never be applied, but just that it has limits in its sensitivity to detect replay during minutes long periods. Further, I greatly appreciated the authors efforts to describe ways to improve TDLM: developing better decoders and applying them to smaller time windows.

The power of this paper comes from the simulation whereby the authors inserted replay events and attempted to detect them using TDLM. Regarding their first study, there are many alternative explanations or possible analysis strategies that the authors do not discuss; however, none of these are relevant if replayed, under conditions where it is synthetically inserted, cannot be detected.

Further, the authors provide a simulation and series of analyses aimed at replicating previous TDLM-based replay studies. They demonstrate methodological flaws, and show that previous simulations greatly overestimated the sensitivity of TDLM. This work emphasizes the need to cast a critical eye over both past and future studies applying TDLM to detect replay.

Finally, the authors are relatively clear about which parameters they chose, why they chose them, and how well they match previous literature (they seem well matched); and provide suggestions for how others can determine the best parameters for TDLM within their own experimental contexts.

Comments on revisions:

The authors thoroughly addressed my previous comments; the added analyses and discussion significantly strengthen the paper's clarity, utility, and impact.

Reviewer #3 (Public review):

Shiqiang Xu and colleagues have examined the importance of ICAM-1 and ALCAM internalization and retrograde transport in cancer cells on formation of a polarized immunological synapse with cytotoxic CD8+ T cells. They find that internalization is mediated by Endophilin A3 (EndoA3) while retrograde transport to the Golgi apparatus is mediated by the retromer complex. Perturbing these trafficking pathways reduces cytokine release, but increases cytolytic killing. The paper is building on previous findings from corresponding author Henri-François Renard showing that ALCAM is an EndoA3 dependent cargo in clathrin-independent endocytosis.

The work is interesting as it describes a novel mechanism by which cancer cells might influence CD8+ T cell activation and immunological synapse formation, and the authors have used a variety of cell biology and immunology methods to study this. The authors have also made substantial efforts to address the reviewers comments to the first version of the paper. However, there are still some points which could be further improved to underpin their conclusions:

The movies and the related micrographs of EndoA3-mediated ICAM-1 endocytosis could be more convincing. Is the invagination of large membrane patches visible by volumetric imaging (e.g. confocal z-stacks) or brightfield microscopy?

There is still a lack of quantitative evidence for polarized transport of ICAM-1 positive vesicles towards the immunological synapse. Only one example is shown and the authors state that the data is from a single movie representative of two independent experiments. If there are multiple cells per experiment, the number of cells should be stated and more examples should be included.

Reviewer #3 (Public review):

Summary of work:

Cheng, Liu, Dong, et al. demonstrate that anterior endoderm cells can arise from prechordal plate progenitors, which is suggested by pseudotime reanalysis of published scRNAseq data, pseudotime analysis of new scRNAseq data generated from Nodal-stimulated explants, live imaging from sox17:DsRed and gsc:eGFP transgenics, fluorescent in situ hybridization, and a Cre/Lox system. Early fate mapping studies already suggested that progenitors at the dorsal margin give rise to both of these cell types (Warga), and live imaging from the Heisenberg lab (Sako 2016, Barone 2017) convincingly showed this previously. However, the data presented for this point are very nice and further cement this result. Though better demonstrated by previous work (Alexander 1999, Gritsman 1999, Gritsman 2000, Sako 2016, Rogers 2017, others), the manuscript presents confirmatory data that high Nodal signaling is required for both cell types. The manuscript generates new single-cell RNAseq data from Nodal-stimulated explants with increased (lft1 KO) or decreased (ndr1 KD) Nodal signaling and multi-omic ATAC+scRNAseq data from wild-type 6 hpf embryos, which can be used as a resource, though few new conclusions are drawn from it in this manuscript. Lastly, the manuscript presents suggests that SWI/SNF remodelers and Ripply1 may be involved in the anterior endoderm - prechordal plate decision, but these data are less convincing. The SWI/SNF remodeler experiments are unconvincing because the demonstration that these factors are differentially expressed or active between the two cell types are weak. The Ripply1 gain-of function experiments are unconvincing because they are based on incredibly high overexpression of ripply1 (500 pg or 1000 pg) that generates a phenotype that is not in line with previously demonstrated overexpression studies (with phenotypes from 10-20x lower expression). Similarly, the cut-and-tag data seems low quality and is based on high overexpression, so may not support direct binding of ripply1 to these loci.

During revision, the authors addressed some comments, including eliminating references to "lineage" when referring to pseudotime trajectories, eliminating conclusions drawn from locations of cells on UMAP plots, and reducing use of the term "cooperative" which may have been confusing in this context, as well as increasing the number of embryos analyzed for some experiments. The authors also point out that whole-embryo transcriptional trajectories typically do not associate endodermal cells with prechordal plate cells, despite classical evidence that they are related. This is most likely because endodermal cells arise from several different previous transcriptional states in different regions of the embryonic margin and are, as the authors point out, difficult to computationally sort into dorsal, lateral, and ventral populations. Thus, there is value in generating data to more specifically look at the relationship between dorsal mesodermal and endodermal populations. However, the decision to use an artificial Nodal-treated explant system, rather than isolating the relevant population from whole embryos (such as by dissection prior to dissociation) remains a weakness of the manuscript, since it is unclear whether endodermal specification has been altered in this system (there seem to be few endodermal cells produced and the system involves manipulating one of the signals under study in this work). Concerns about the rigor of experiments concerning ripply1 and SWI/SNF experiments remains. While the authors improved peak calling in their ripply1 cut-and-tag, it is still based on massive overexpression of ripply1 that may drive binding outside of its endogenous loci.

In the end, this study provides some additional details in the cell fate decision between the prechordal plate and anterior endoderm and generates new data that may be useful for reanalysis by other experts in the field. However, this work does not make clear how Nodal signaling, FGF signaling, and elements of the gene regulatory network (including gsc, possibly ripply1, and other factors) interact to make the decision. I suggest that this manuscript is of interest to Nodal signaling or zebrafish germ layer patterning afficionados, but may not be of interest to a broad audience. While it provides new datasets and observations, it does not weave these into a convincing story that advances our understanding of the specification of these cell types.

Reviewer #3 (Public review):

In this revised report, De Franceschi et al. purify components of the Cdv machinery in archaeon M. sedula and probe their interactions with membrane and with one-another in vitro using two main assays - liposome flotation and fluorescent imaging of encapsulated proteins. This has the potential to add to the field by showing how the order of protein recruitment seen in cells is related to the differential capacity of individual proteins to bind membranes when alone or when combined.

Using the floatation assay, they demonstrate that CdvA, CdvB, and CdvB1 bind liposomes. CdvB2 lacking its C-terminus is not efficiently recruited to membranes unless CdvAB or CdvB1 are present. The authors then employ a clever liposome assay that generates chained spherical liposomes connected by thin membrane necks, which allows them to accurately control the buffer composition inside and outside of the liposome. With this, they show that all four proteins accumulate in necks of dumbbell-shaped liposomes that mimic the shape of constricting necks in cell division, possibly indicating a sensing of catenoid membrane geometry. Taken altogether, these data lead them to propose that Cdv proteins are sequentially recruited to the membrane as has also been suggested by in vivo studies of ESCRT-III dependent cell division in crenarchaea.

In their revision, the authors have addressed the vast majority of our previous concerns. The paper is much improved as a result. The Figures are improved and the authors have added appropriate controls and additional experiments, strengthening their conclusions.

There are still some discrepancies between these results and what is know about Sulfolobus division. Since the initial submission, other work has shown that in S. acidocaldarius, CdvA is the first component to assemble a ring (in absence of CdvB , doi.org/10.1073/pnas.2513939122) and that CdvB2 is able to bind membranes in vitro (doi.org/10.1073/pnas.2525941123). This might reflect differences between Sulfolobus and Metallosphaera, but probably should be discussed.

Reviewer #3 (Public review):

Summary:

The authors of this manuscript have addressed a key concept in T cell development: how early thymus gd T cells subsets are specified and the elements that govern gd T17 versus other gd T cell subset or ab T cell subsets are specified. They show that the transcriptional regulator HEB/Tcf12 plays a critical role in specifying the gd T17 lineage and, intriguingly that it up regulates the inhibitor Id3 which is later required for further gd T17 maturation.

Strengths:

The conclusions drawn by the authors are amply supported by a detailed analysis of various stages of T cell maturation in WT and KO mouse strains at the single cell level both phenotypically, by flow cytometry for various diagnostic surface markers, and transcriptionally, by single cell sequencing. Their conclusions are balanced and well supported by the data and citations of previous literature.

Weaknesses:

I actually found this work to be quite comprehensive.

Comments on revisions:

Nothing to add here. The authors were very thorough in their original submission, and all minor issues identified have been addressed to my satisfaction.

Reviewer #3 (Public review):

Summary:

The constraints limiting the usage of especially repetitive amino acid sequences in proteins remain enigmatic. In their manuscript, Murase et al. analyse the impact of polyamino acid homorepeats (PolyX) on the expression of EGFP-variants with PolyX modifications. Introducing a new measure, relative neutrality, allows us to rate beneficial versus harmful sequences. The authors find that especially hydrophobic repeats (I, V, W, F, Y) show harmful effects on the respective proteins, enhancing their aggregation. Hydrophilic repeats (E, S, N, Q), on the other hand, show beneficial properties but suppress proteotoxic stress. Interestingly, these observations correlate with the occurrence of such PolyX in natural proteins across the proteomes of different organisms.

Strengths:

The manuscript seems especially valuable in the context of rational or de novo protein design. The observations on the one hand should allow for enhancing the solubility of proteins by using beneficial PolyX. On the other hand, they explain very well why some PolyX do not occur in natural proteins. The authors present a sound, broad and well-analysed dataset. The study is well designed, the manuscript is very well written, and the conclusions drawn are overall valid.

Weaknesses:

The whole data set relies on the definition of the newly introduced "relative neutrality" score. Besides being a well-chosen tool, this score is limited and biased as it does not directly include a measure for "solubility" but relies on "fluorescence emission" derived from the respective EGFP-fusion-proteins.

A second major weakness is that the influence of PolyX-modifications on secondary structure is neither analysed nor discussed.

Reviewer #3 (Public review):

Summary:

In the present study, through international gene-matching efforts, the authors present 29 individuals with rare, heterozygous ARID5B variants and find that these individuals have a newly recognizable neurodevelopmental syndrome. A recurring clinical syndrome of developmental delay/intellectual disability, behavioral difficulties, renal malformation, and recurrent infections is described. 19 of these variants were confirmed to be de novo, and only one was inherited from an unaffected parent. 24/29 of these variants introduce premature termination codons in the final exon and are predicted to escape nonsense-mediated decay. The ARID5B p.Q522Ter variant was studied in a mouse heterozygous knock-in model, found to be associated with behavioral abnormalities. The well-described genetic and phenotypic data for this cohort provide convincing clinical evidence for a novel neurodevelopmental syndrome. The functional evidence provided is preliminary, and further studies are needed to understand disease mechanisms.

Strengths:

(1) The authors give a good description of a novel clinical syndrome manifesting as developmental delay/intellectual disability, facial dysmorphism, and behavioral challenges.

(2) The authors create a mouse model harboring an Arid5b(Q522*/+) variant and identify subtle behavioral changes.

(3) Attempts are made to functionally characterize a subset of ARID5B variants in human cell lines.

Weaknesses:

(1) The title - "ARID5B mutations cause a neurodevelopmental syndrome with neuroinflammation episodes" - should be revised. 2/29 individuals (7%) had CNS inflammation; this does not appear to be a core feature of the disease and should not be highlighted as such. If this is going to be a feature that is highlighted, then more details are needed. MRI images of cerebellitis and/or ADEM would be helpful, as well as lumbar puncture results and supplemental information detailing the treatment course.

(2) The abstract states that "Remarkably, 19 of 29 variants (66%) cluster within the first quarter of exon 10, are de novo, and escape nonsense-mediated mRNA decay (NMD), which we confirmed for two variants affecting seven individuals." The authors state in the Results that they "indeed found no signs of NMD". In Figure 3f, when assessing for transcript amount, there appears to be a great deal of variability. Three ARID5B variant lines are tested. Transcript amounts in two lines appear to be near control levels, but one LCL ARID5B Ile497AsnfsTer31 line appears to demonstrate significantly lower levels of transcript. The control lines also show a great deal of variability. No explanation is given for this large difference between LCL ARID5B Ile497AsnfsTer31 lines and for the variability in control lines, making these data uninterpretable. A major theme of the paper is that early truncating variants in exon 10 escape NMD and lead to the described phenotypes, so this is an important point that needs to be resolved, either by testing more patient-derived lines or knocking in these variants into cell lines.

(3) The Arid5b(Q522*/+) mice are not sufficiently molecularly characterized. Does the variant transcript escape NMD? What happens at the protein level? Is there mislocalization of the protein?

(4) For the HEK293T cell experiments, variants are overexpressed and compared to a control. These experiments appear to leave endogenous ARID5B intact. What might the authors expect to see if these variants were knocked in?

(5) The functional consequences of the missense variants are not tested. The authors suggest that missense variants may be more associated with macrocephaly and possibly ASD. Are these missense variants causing loss-of-function or gain-of-function? Is there preserved protein function?

(6) There are a number of functional assays performed, but it remains unclear if the tested variants are operating through a loss- or gain-of-function. Are truncating variants early in exon 10 leading to a partial loss-of-function? Or do they prevent the functioning of the other allele through a dominant negative mechanism? These possibilities are not directly tested.

Reviewer #3 (Public review):

Summary:

In this paper, the authors identified dual inhibitory mechanisms, an intrinsic juxtamembrane (JM) region and an extrinsic cytoplasmic tail (CCT) domain in the binding protein PAN-1, that suppress MYRF-1 cleavage in C. elegans. The authors showed that MYRF-1 cleavage oscillates across larval stages, peaking in mid-to-late phases and being suppressed during molts. This oscillatory pattern is consistent with MYRF-1's role in promoting transitions of larval stages, particularly in late-L1 involving lin-4 activation and DD neuron remodeling.

Strengths:

This work generated several knock-in strains of fluorescent tags and mutations in the endogenous myrf-1 and pan-1gene loci, which will provide resources for future identification and characterization of the underlying molecular mechanisms regulating MYRF-1 cleavage inhibition.

The results presented in the paper are solid enough to support the paper's main conclusions.

This study is valuable for establishing MYRF-1 cleavage as a key gatekeeper of the C. elegans developmental timing. Findings from C. elegans MYRF-1 may provide insight into the regulation and function of mammalian MYRF.

Weaknesses:

The following points should be discussed to further support the authors' model that MYRF-1 cleavage is a key gatekeeper of developmental timing.

(1) Recent findings by Helge Großhans and Jordan Ward groups showed that KIN-20 (CK1δ) and LIN-42 (PERIOD) are required for proper molt timing in C. elegans, and that loss of LIN-42 binding or of the phosphorylated LIN-42 tail impairs nuclear accumulation of KIN-20, resulting in arrhythmic molts (EMBO J. 44, 6368-6396, 2025). In this paper, the authors concluded that PAN-1 promotes MYRF trafficking to the cell membrane, where MYRF-1 cleavage and nuclear translocation occur, and that oscillates with developmental molting cycles in C. elegans. It is unclear whether MYRF-1 and KIN-20 interact in the nucleus and, if so, how this interaction controls developmental timing.

(2) Separately, it was previously shown that the let-7 primary transcript (pri-let-7) exhibits oscillating, pulse-like expression that peaks during each larval stage, rather than a steady increase, and directly correlates with developmental molting cycles. It is unclear whether the nuclear-localized MYRF-1 fragment regulates the oscillatory primary let-7 expression during larval transition (McCulloch and Rougvie, 2014; Van Wynsberghe et al., 2011).

DOI: 10.7554/eLife.106720

Resource: None

Curator: @evieth

SciCrunch record: RRID:ZFIN_ZDB-ALT-130816-3

What is this?

Reviewer #3 (Public review):

Summary:

The study by Schönmann et al. presents compelling analyses based on two MEG datasets, offering strong evidence that the pre-onset response observed in a highly influential study (Goldstein et al., 2022) can be attributed to stimulus dependencies-specifically, the auto-correlation in the stimuli-rather than to predictive processing in the brain. Given that both the pre-onset response and the encoding model are central to the landmark study, and that similar approaches have been adopted in several influential works, this manuscript is likely to be of high interest to the field. Overall, this study encourages more cautious interpretation of pre-onset responses in neural data, and the paper is well written and clearly structured.

Strengths:

• The authors provide clear and convincing evidence that inherent dependencies in word embeddings can lead to pre-activation of upcoming words, previously interpreted as neural predictive processing in many influential studies.

• They demonstrate that dependencies across representational domains (word embeddings and acoustic features) can explain the pre-onset response, and that these effects are not eliminated by regressing out neighboring word embeddings-an approach used in prior work.

• The study is based on two large MEG datasets and one ECoG dataset, showing that results previously observed in ECoG data can be replicated in MEG. Moreover, the stimulus dependencies appear to be consistent across the three datasets.

Weaknesses:

• While this study shows that stimulus dependency can account for pre-onset responses, it remains unclear whether this fully explains them, or whether predictive processing still plays a role. The more important question is whether pre-activation remains after accounting for these confounds.

Comments on revisions:

I appreciate the added analyses. This study raises an important methodological concern regarding an influential paper and will certainly have a high impact on our field.

Reviewer #4 (Public review):

Summary:

Short photoperiod is an important experimental manipulation in neurobiology, endocrinology, and metabolism studies. However, the molecular mechanisms by which short photoperiod gives rise to behavioral phenotypes that are seen in seasonal affective disorders remain unknown. Using the classic circadian model organism Drosophila, this study examines short photoperiod-induced hypersomnolence and identifies the circadian photoreceptor cryptochrome as a regulator of GABAergic tone within the clock neural circuit to promote wakefulness under short photoperiod conditions. The discovery has broad implications for understanding how short photoperiod modulates neural inhibition in circadian circuits in regulating sleep.

Strengths:

The Drosophila model provided a powerful platform to dissect the molecular mechanisms underlying short photoperiod-induced hypersomnolence. A battery of behavioral, imaging, circuit-manipulation approaches was employed to test the novel hypothesis that the circadian photoreceptor cryptochrome modulates GABAergic tone within the clock neural circuit to promote wakefulness under short photoperiod conditions.

Weaknesses:

The current model proposed by the authors suggests that the small ventral lateral neurons of the Drosophila clock circuit are GABAergic; however, this remains unclear. At present, the field lacks sufficient data and validated reagents to definitively establish the GABAergic identity of these neuropeptidergic neurons.

Reviewer #3 (Public review):

Summary:

The manuscript by Zilinskas et al seeks to understand the mechanisms underlying the ability of Mtb to suppress Th17 differentiation. As Th17 responses are needed for protective immunity against TB, this is an important topic of investigation. They use Mtb mutants that lack eccC1 (from the ESX-1 locus) and fadD28 (encoding PDIM) and implicate a Tbet-dependent pathway by which Mtb modulates Th17 differentiation. The mechanism by which ESX-1/PDIM function to impact Th17 differentiation is, however, unclear, which limits the novelty of the results.

Strengths:

Understanding how Mtb limits Th17 differentiation has implications for vaccine development. Comparative study of KO mice and Mtb mutants is a strength.

Weaknesses:

(1) The authors should acknowledge and reference key findings from the literature that have identified suppression of Th17 differentiation as an Mtb virulence mechanism, e.g., the role of the Hip1 protease and CD40 signaling (Madan-Lala JI 2014, Sia Plos Path 2017, Enriquez iScience 2022) and Khader JI 2005, showing the requirement of IL-23 for Th17 responses in vivo in a TB mouse model.

(2) Addressing several questions related to the Tbet KO mouse experiments would strengthen the study. Do the Tbet KO mice have elevated IL-4/5/13 (which has been previously reported in non-TB studies) in addition to IL-17? The lack of Th17 cells in the IFNg KO compared to the Tbet KO may be due to a difference in timing, since only 3-week data are shown; earlier and later time points would provide better interpretation. The authors do not present any data on neutrophil infiltration in WT vs Tbet KO vs IFNg KO mice. Since IL-17 is known to be important for recruiting neutrophils to the lung, data on neutrophils are important for clarifying the mechanism for the CFU outcomes.

(3) While IL-23 is important for sustaining IL-17 production, IL-6, TGF-b and/or IL-1β are necessary for Th17 polarization. What were the levels of these cytokines in DCs in the lung? (Figure 5). Additionally, Tbet-deficient DCs exhibit impaired activation of antigen-specific Th1 cells and have reduced IL-12 production. Given the data showing higher IL-17 levels in Tbet KO mice, the authors should provide information on the DC phenotype (IL-23, IL-6, etc.) in the Tbet KO experiments.

(4) The mechanism by which ESX-1/PDIM function to impact Th17 differentiation is not clear. While data showing a role for ESX-1 and PDIMs in inhibiting Th17 responses is interesting, there is no insight into the potential mechanism of action. Figure 3 showing reduction in IFNg+ CD4 T cells after infection with eccC1 and fadD28 mutants suggests that this outcome is due to a lower bacterial load relative to WT Mtb at the 3-week time point. Since IFNg is known to suppress IL-17, the higher levels of Th17 cells could be due to the reduction in IFNg due to the attenuated growth of the mutants. Additionally, what was the level of Type I IFNs elicited by these mutants?

(5) Since macrophages have been implicated in the reduced cytokines seen in the ESX-1 mutant, IL-23 and other cytokine data on lung macrophages would complement the DC data.

(6) Figure 5. There are many fewer DCs overall in the eccC1 and fadD28 mutant groups, which could account for the increased % IL-23p19 in DCs (5D). What were the levels of IL-23 in DC1s?

Reviewer #3 (Public review):

In this work, the authors wanted to evaluate repurposed small molecule inhibitors for the treatment of envenomation by snakes of the Bothrops genus; one of the most medically relevant in the Americas. I believe the objectives of the research were clearly achieved, and compelling evidence for the ability of these molecules to neutralize enzymatic and toxic activities of metalloproteinases and phospholipases in all the tested venoms is provided. Furthermore, the work highlights the limited efficacy of the tested serineprotease inhibitor, suggesting a need for drug discovery campaigns to address toxicity caused by this protein family. The methods are well designed and performed, and the use of both in vitro and in vivo methodologies makes this a thorough and robust work.

These results are extremely relevant, since they take us one step further to a potential orally administered snakebite treatment. The existence of such a treatment could improve the outcomes for thousands of snakebite victims worldwide. I have a few comments and questions that I hope will be useful to the authors:

During the introduction, the authors mention that small-molecule inhibitors can neutralize the localized tissue damage via cytotoxicity of some venoms, and cite PLA2s, SVMPs and/or cytotoxic 3FTxs as the main causing agents of this pathology. I am not aware of any direct effect described by small molecule inhibitors on cytotoxic 3FTxs alone. Has this been observed at all? Or is it more likely that the small molecule inhibitors act on the enzymatic toxins only, preventing synergistic effects with 3FTxs?

I think it would be relevant to address the effects of non-enzymatic PLA2s, such as myotoxin II, which have been described in detail within Bothrops venoms. I believe there is some evidence of Varespladib also having a neutralizing effect on the myotoxicity caused by these non-enzymatic PLA2s. I suggest adding a comment about the contribution of these toxins in the discussion or in the section where PLA2 activity of the venoms is compared. In my opinion, right now it seems like these were overlooked.

Regarding Marimastat and the other MP inhibitors, are there any studies showing that they don't have an effect on endogenous MPs? I understand they have been approved for human use before, but is there any indication that they would not have an effect at the doses that would be required to treat envenomation?

Regarding the quenched fluorescence substrate used for enzymatic activity. Is there a possibility that some of the SVMPs would not act on this substrate, and therefore their activity or neutralization is not observed? Would it be relevant to test other substrates, such as gelatin, collagen, or even specific clotting factors?

Finally, could the authors comment or provide some bibliography regarding the translatability of the chicken embryo model in the context of envenomation?

Reviewer #3 (Public review):

Summary:

This manuscript analyzes two independent datasets collected at different sites. Using the same willed-attention paradigm (instructional vs. choice cues) and combining fMRI and EEG analyses, the authors investigate how attentional direction is selected when no external instruction is provided. Their main claims are that the dorsal attention network is engaged by both cue types, whereas the choice cue additionally involves a frontoparietal decision network. Moreover, left-versus-right attentional decisions can be decoded in this decision network only on choice trials, and multichannel pre-stimulus alpha patterns predict the subsequent attentional choice. Finally, individuals with more predictive alpha patterns show greater neural efficiency in the decision network, i.e., higher decoding with lower BOLD activation.

The question is worthwhile and the two-site design is a genuine strength. At the same time, several central inferences rely on decoding analyses for which the statistical testing and cross-validation structure are not described in enough detail to assess robustness. In addition, using a ratio-based neural-efficiency measure make the interpretation more fragile than it needs to be. With a focused revision that tightens inference around MVPA and clarifies a few methodological points, I think the paper could become substantially more convincing.

Strengths:

The work extends previous willed attention studies by attempting to link pre-stimulus alpha pattern predictability to post-cue frontoparietal representations, and by testing reproducibility across two datasets. The conceptual advance beyond previous studies, e.g., Bengson et al. (2015), however, depends on how solid the decoding-based evidence is and whether alternative explanations are convincingly excluded. At present, the strength of support is limited mainly by incomplete reporting and/or controls for MVPA significance testing, as well as potential inflation of decoding estimates if folds are not independent of run structure. Concerns about statistical assessment of decoding accuracy are well documented in the literature (Combrisson & Jerbi, 2015).

Weaknesses:

(1) The manuscript describes the decoding pipeline for both fMRI and EEG MVPA. However, it does not clearly specify how "significantly above chance" is determined for the fMRI ROI decoding, nor how multiple comparisons across ROIs are handled, even though p-values are reported. The same issue applies to the time-resolved EEG analysis across many time points. For each decoding analysis, please specify the inferential test (e.g., permutation test within participant, group-level test on subject accuracies, binomial test, etc.) and report effect sizes with confidence intervals (e.g., Combrisson & Jerbi, 2015). Further, for EEG decoding over time, it would be preferable to control family-wise error, e.g., cluster-based permutation, rather than thresholding pointwise p-values. A standard approach here is the nonparametric cluster framework (e.g., Maris & Oostenveld, 2007).

(2) The cross-validation approach used here is appreciated and appropriate in principle. However, random 10-fold splits across trials can inflate accuracy if training and test folds share run-specific noise, scanner drift, or autocorrelated structure. The manuscript should indicate whether folds were blocked by run or randomized across the entire session. In addition, please report the number of trials per condition after artifact rejection and after removing short ITIs for the long prestimulus epochs (−2500 ms to 0 ms) for each dataset in the section of EEG preprocessing. Similarly, please report how often participants chose left vs. right on choice trials, and whether balanced folds (or an equivalent balancing procedure) were used if needed.

(3) Moreover, ROI definition is not sufficiently specified and independence should be clarified. The ROIs are defined based on peaks from the choice-instructed univariate contrast (Table 2) and then used for MVPA. First, are these ROIs defined as spheres around peaks or using anatomical masks? What radius or voxel count was used? This needs to be explicit. Second, I am concerned about circularity risk. Although choice-vs-instructed selection is not identical to left-vs-right decoding, ROI selection from the same dataset can still bias descriptive estimates and encourages overinterpretation if not carefully justified (Kriegeskorte et al., 2009). At minimum, the authors should explain why their selection criterion is independent of the decoded contrast under the null, and ideally provide a robustness check using either anatomical ROIs or independently defined ROIs, e.g., from prior literature or an atlas.

(4) Using an index of neural efficiency is conceptually interesting. However, if the denominator, computed as the activation difference between choice and instructional conditions, is near zero or noisy, the ratio can become unstable. I would rather see a multivariate model that treats activation and decoding as separate dependent measures, or a latent-variable approach, than a single ratio.

This is surprising because it treats speech as a cost instead of a connection. It turns silence into a form of discipline that shapes the whole family.

This is interesting because it shows identity as a messy mix, not a clean category. The question forces you to notice how culture and personal history can blur into each other.

The sentence is troubling because it turns violence into a close physical detail. It makes the threat feel intimate and unavoidable inside the home. The image also suggests that cruelty is being displayed on purpose rather than hidden.

https://www.facebook.com/groups/TypewriterCollectors/posts/10163311006014678/

Some interesting 3-D printing advice for replacement typewriter feet here.

Zach Hubbird recommends using a filament dryer and Shore A 85 (or lower) TPU from Siraya Tech as 95A isn't great. Many feel that 95A isn't as "grippy".

Erik Bruchez also recommends 85A while Marty Morren suggests 70A and is contemplating sourcing 60A in the future.

Reviewer #3 (Public Review):

Summary:

It has been proposed that the FOI is a method of using parasite genetics to determine changes in transmission in areas with high asymptomatic infection. The manuscript attempts to use queuing theory to convert multiplicity of infection estimates (MOI) into estimates of the force of infection (FOI), which they define as the number of genetically distinct blood-stage strains. They look to validate the method by applying it to simulated results from a previously published agent-based model. They then apply these queuing theory methods to previously published and analysed genetic data from Ghana. They then compare their results to previous estimates of FOI.

Strengths:

It would be great to be able to infer FOI from cross-sectional surveys which are easier and cheaper than current FOI estimates which require longitudinal studies. This work proposes a method to convert MOI to FOI for cross-sectional studies. They attempt to validate this process using a previously published agent-based model which helps us understand the complexity of parasite population genetics.

Weaknesses:

(1) I fear that the work could be easily over-interpreted as no true validation was done, as no field estimates of FOI (I think considered true validation) were measured. The authors have developed a method of estimating FOI from MOI which makes a number of biological and structural assumptions. I would not call being able to recreate model results that were generated using a model that makes its own (probably similar) defined set of biological and structural assumptions a validation of what is going on in the field. The authors claim this at times (for example, Line 153 ) and I feel it would be appropriate to differentiate this in the discussion.

(2) Another aspect of the paper is adding greater realism to the previous agent-based model, by including assumptions on missing data and under-sampling. This takes prominence in the figures and results section, but I would imagine is generally not as interesting to the less specialised reader. The apparent lack of impact of drug treatment on MOI is interesting and counterintuitive, though it is not really mentioned in the results or discussion sufficiently to allay my confusion. I would have been interested in understanding the relationship between MOI and FOI as generated by your queuing theory method and the model. It isn't clear to me why these more standard results are not presented, as I would imagine they are outputs of the model (though happy to stand corrected - it isn't entirely clear to me what the model is doing in this manuscript alone).

(3) I would suggest that outside of malaria geneticists, the force of infection is considered to be the entomological inoculation rate, not the number of genetically distinct blood-stage strains. I appreciate that FOI has been used to explain the latter before by others, though the authors could avoid confusion by stating this clearly throughout the manuscript. For example, the abstract says FOI is "the number of new infections acquired by an individual host over a given time interval" which suggests the former, please consider clarifying.

(4) Line 319 says "Nevertheless, overall, our paired EIR (directly measured by the entomological team in Ghana (Tiedje et al., 2022)) and FOI values are reasonably consistent with the data points from previous studies, suggesting the robustness of our proposed methods". I would agree that the results are consistent, given that there is huge variation in Figure 4 despite the transformed scales, but I would not say this suggests a robustness of the method.

(5) The text is a little difficult to follow at times and sometimes requires multiple reads to understand. Greater precision is needed with the language in a few situations and some of the assumptions made in the modelling process are not referenced, making it unclear whether it is a true representation of the biology.

Reviewer #3 (Public review):

Summary:

This manuscript by Earp et al reports cryoEM structures of the hexameric (MmpS4)₃-(MmpL4)₃ complex from Mycobacterium tuberculosis, which belongs to the RND family of transporters and is known to have a role in the export of siderophores and contribute to drug resistance. The experimental workflow showcased involves the design of disulfide pairs using distance constraints obtained from the AlphaFold predicted structure of the hexameric complex. One such disulfide pair was used to determine the ~3.0 Å structures. The structure reveals density for the previously unresolved coiled-coil domain (CCD), a tilted CCD arrangement, and a cavity within the periplasmic domain, which the authors assert is occupied by detergent. Comparison of this complex with the monomer structure of MmpL4 shows conformational variations interpreted to implicate different domains and conserved residues involved in proton coupling, which might be related to the transport mechanism. While the methodological aspects of the manuscript are solid, enthusiasm for the overall advance/significance is less so, with doubts about the relevance of the tilted CCD structure, considering disulfide trapping and an incomplete validation of the claim that the titled CCD represents a stable intermediate conformation. A clear, updated transport mechanism is largely missing from the manuscript.

Strengths:

Beautiful structures, AF prediction-experimental validation nexus that could be fine-tuned for different systems/difficult to target complexes.

Weaknesses:

Physiological relevance of the tilted CCD conformation. No clear mechanistic model for the transport. While the CCD may indeed be a stable intermediate, the fact that the rest of the trimeric arrangement is unaffected does not fully rule out disulfide trapping as a factor in promoting this. The findings would be strengthened if the same tilted conformation is seen using a different set of disulfides. The significance of the detergent molecule and the new cavity observed could also be better discussed in terms of an updated transport model.

Reviewer #3 (Public review):

The paper begins by analyzing the drift in individual behavior over time. Specifically, it quantifies the circling direction of freely walking flies in an arena. The main takeaway from this dataset is that while flies exhibit an individual turning bias (when averaged over time), yet their preferences fluctuate over slow timescales.

To understand whether genetic or neuromodulatory mechanisms influence the drift in individual preference, the authors test different fly strains in a Y maze concluding that both genetic background and the neuromodulator serotonin contribute to the degree of drift (although with some contrasting results). The use of a different assay for this different dataset (Y maze istead of wide arena) is justified by previous observation of similar behavioral biases in these assay. Yet the conceptual link between the spectral power analysis used for the first dataset and the autoregressive model used for the second remains unclear.

Finally, the authors use theoretical approaches to show the potential advantage of individual drift for survival in unpredictable, fluctuating environments. They demonstrate that while bet-hedging provides an advantage over timescales matching the generation time (since reproduction is required), it offers less benefit on shorter timescales, where an increased individual drift could be advantageous.

Reviewer #3 (Public review):

Summary:

This manuscript is focused on studying the spatial frequency selectivity of individual neurons in the mouse primary visual cortex (V1) in the anesthetized and awake brain states using 2-photon calcium imaging. Although previous studies have demonstrated that anesthesia decreases both size tuning and spatial selectivity in V1 neurons, the strength of this study is its focus on characterization of the same neurons in awake and anesthetized states in combination with transgenic mouse lines selectively labeling pan-inhibitory neurons and also more specific neuronal subtypes, including parvalbumin-positive (PV+) or somatostatin-positive (SOM+) interneurons. A combination of these methodologies allows for a more in-depth mechanistic study of the properties of different types of neurons. The main findings suggest that in excitatory neurons, anesthesia leads to a shift in preferred SF and broadening of SF tuning, with no changes in orientation and direction selectivity. Downward shift in preferred SF was more pronounced in both SOM+ and PV+ interneurons.

Strengths:

(1) 2-photon calcium imaging with single-cell resolution.

(2) Characterization of excitatory and two types of inhibitory neurons.

Weaknesses:

(1) VIP interneurons are critical to the neural circuit, and their characterization would be critical to the mechanistic understanding of this process, but is missing.

(2) Unfortunately, the manuscript does not lead to an additional insight into the nature of this anesthesia-induced shift in SF preference.

(3) Furthermore, it also doesn't help understand how SF preference is encoded in V1.

(4) Finally, some critical histological controls are missing.

Reviewer #3 (Public review):

Summary:

There is a threat of mirror life bacteria, which could possibly evade immunity and cause problems for human/animal hosts. This paper evaluates enantiomeric antibiotics and vaccines as a means to understand how this could be combatted in the future.

Strengths:

It is valuable to collect such information, as it is not always clear how an antibiotic in its enantiomeric form would interact with a bacterium in terms of its MIC or towards toxicity. The paper is scientifically sound with regard to assays and statistical methods.

Weaknesses:

The beginning of the paper could be described as hyperbolic. For a paper that demonstrates that mirror-image molecules have (expected) lower MICs and toxicity, some of the claims in the beginning that they are going to cause a pandemic of evading the immune system seem to be a bit overstated. If they are mirror images, how are these bacteria going to generate virulence factors or mediate pathogenesis mechanisms? It seems like the lack of adaptation would go both ways - supported by the empirical data gathered in this manuscript. There is also the issue of only relatively simple and accessible mirror-image antibiotics being available. This is a limitation that - to their credit - the authors do discuss in the discussion section.

Reviewer #3 (Public review):

Summary:

In the present study, the authors examined pupillary responses to uncolored stimuli (number graphemes) among number-color synesthetes and non-synesthetes. After seeing a digit, the synesthetes and active control participants were asked to indicate which color they perceived using three dimensions of hue, saturation, and lightness. The lightness values were the primary independent variable for follow-up analyses. To see how the pupil responded to psychologically "bright" and "dark" digits, the authors split the reported lightness values at the median and plotted them. The synesthetes showed a pupillary constriction to digits they perceived as bright and dilation to digits they perceived as dark. Active control participants did not show that effect. In a subsequent block, only the synesthetes were shown the colors they reported perceiving as colored discs. Their pupillary responses were similar. The authors also found that the differences in pupillary responses between light and dark perceptions (with digits) were only slightly delayed in their onset to the perception of a colored disc, and therefore, the color perception accompanying a digit is unlikely to be effortful or a retrieved association, but occurs rather automatically.

Strengths:

The authors employed a well-controlled and designed quasi-experiment comparing color-grapheme synesthetes to non-synesthetes and showed convincingly that the color perceptions accompanying graphemes alter the physical perception of brightness. They also made a reasoned attempt to rule out the possibility that color associations are occurring effortfully via retrieved associations.

Weaknesses:

There are some areas in which the implications of these findings could be elaborated upon. I had the following questions:

(1) Are the pupillary responses among synesthetes, which objectively do not seem to match the degree of physical stimulation entering the retina, in any way maladaptive for eye functioning? I understand the constriction/dilation of the pupil to not only benefit visual acuity but also to protect the retina from damage. Are synesthetes at any risk of retinal damage due to over-dilation of the pupil to brighter stimuli? Or are these effects of a magnitude that is too small to matter? As reported in arbitrary units, it was hard to know how large these effects were in terms of measurable changes in dilation (e.g., millimeters).

(2) Likewise, is the automatic synesthetic merging of two percepts something that could be learned such that natural synesthetes and "artificial" synesthetes would look similar? For example, if a group of non-synesthetic participants were to learn a color-grapheme association to automaticity, would you expect their pupillary responses to the graphemes look similar to the synesthetes'? If so (or if not), what would this tell us anything about the phenomenology of synesthesia?

(3) Do the synesthetic perceptions of digit graphemes merge in a sensible way? For example, if a synesthete sees a particular color with the digit 1, and a different color with the digit 9, what do they perceive when they see 19? or 1-9, or 1 9? Is there color blending, or an altogether different color perception?

Reviewer #3 (Public review):

This study investigated the in vitro amplification of donor fecal virus using chemostat culturing technology, aiming to reduce eukaryotic virus load while preserving bacteriophage community diversity, thereby optimizing the safety and efficacy of FVT. The research employed a preterm pig model to evaluate the effects of chemostat-propagated viromes (CVT) in preventing necrotizing enterocolitis (NEC) and mitigating adverse effects such as diarrhea.

Strengths:

(1) Enhanced Safety Profile:<br /> Chemostat cultivation effectively reduced eukaryotic virus load, thereby minimizing the potential infection risks associated with virome transplantation and offering a safer virome preparation method for clinical applications.

(2) Process Reproducibility:<br /> The chemostat system achieved stable amplification of bacteriophage communities (Bray-Curtis similarity >70%), mitigating the impact of donor fecal variability on therapeutic efficacy.

Comments on revision:

The authors have satisfactorily addressed all comments and concerns raised during the review process. The revised manuscript is clear, complete, and meets the standards of the journal.

Reviewer #3 (Public review):

Summary:

This work proposes DASM, a new transformer-based approach to learning the distribution of antibody sequences which outperforms current foundational models at the task of predicting mutation propensities under selected phenotypes, such as protein expression levels and target binding affinity. The key ingredient is the disentanglement, by construction, of selection-induced mutational effects and biases intrinsic to the somatic hypermutation process (which are embedded in a pre-trained model).

Strengths:

The approach is benchmarked on a variety of available datasets and for two different phenotypes (expression and binding affinity). The biologically informed logic for model construction implemented is compelling and the advantage, in terms of mutational effects prediction as well as computational efficiency, is clearly demonstrated via comparisons to state-of-the-art models.

Weaknesses:

While all the main points are well addressed and supported, it could have been interesting to strengthen the claim of gain in interpretability by investigating it explicitly in relation to the functional effects studied in this paper.

Comments on revisions:

I thank the authors for clarifying a few points I had flagged up and I appreciate much better that the content of the companion paper was precisely covering model selection and structural interpretability.

Regarding my first point (references for language models for antibodies), I feel that the parenthetical citation format shouldn't be a problem (but the editors might advise here). Antiberta2 is this paper: https://www.biorxiv.org/content/10.1101/2023.12.12.569610v1.full.pdf (yet, I understand if the authors want to focus on models purely sequence-based). A couple of additional references could be: https://academic.oup.com/bioinformatics/article/40/11/btae659/7888884; https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1012646; https://www.pnas.org/doi/10.1073/pnas.2418918121; https://arxiv.org/abs/2506.13006.

A very minor comment: could one add some p-value (it could be a supplementary table) for the Pearson correlation coefficients? The comparison between methods is rather clear, but for some correlations it's a bit unclear whether they should be considered significant. It would be important to understand the extent to which in different datasets one might expect functional prediction power based on an evolutionary objective function alone.

Reviewer #3 (Public review):

Summary:

The researchers performed a genetic screen to identify a protein, ZNF-236, which belongs to the zinc finger family, and is required for repression of heat shock inducible genes. The researchers applied a new method to map the binding sites of ZNF-236, and based on the data, suggested that the protein does not repress genes by directly binding to their regulatory regions targeted by HSF1. Insertion of a reporter in multiple genomic regions indicates that repression is not needed in repetitive genomic contexts. Together, this work identifies ZNF-236, a protein that is important to repress heat-shock-responsive genes in the absence of heat shock.

Strengths:

A hit from a productive genetic screen was validated, and followed up by a series of well-designed experiments to characterize how the repression occurs. The evidence that the identified protein is required for the repression of heat shock response genes is strong.

Weaknesses:

The researchers propose and discuss one model of repression based on protein binding data, which depends on a new technique and data that are not fully characterized.

Major Comments:

(1) The phrase "results from a shift in genome organization" in the abstract lacks strong evidence. This interpretation heavily relies on the protein binding technique, using ELT-2 as a positive and an imperfect negative control. If we assume that the binding is a red herring, the interpretation would require some other indirect regulation mechanism. Is it possible that ZNF-236 binds to the RNA of a protein that is required to limit HSF-1 and potentially other transcription factors' activation function? In the extrachromosomal array/rDNA context, perhaps other repressive mechanisms are redundant, and thus active repression by ZNF-236 is not required. This possibility is mentioned in one sentence in the discussion, but most of the other interpretations rely on the ZNF-236 binding data to be correct. Given that there is other evidence for a transcriptional role for ZNF-236, and no negative control (e.g. deletion of the zinc fingers, or a control akin to those done for ChIP-seq (like a null mutant or knockdown), a stronger foundation is needed for the presented model for genome organization.

(2) Continuing along the same line, the study assumes that ZNF-236 function is transcriptional. Is it possible to tag a protein and look at localization? If it is in the nucleus, it could be additional evidence that this is true.

(3) I suggest that the authors analyze the genomic data further. A MEME analysis for ZNF-236 can be done to test if the motif occurrences are enriched at the binding sites. Binding site locations in the genome with respect to genes (exon, intron, promoter, enhancer?) can be analyzed and compared to existing data, such as ATAC-seq. The authors also propose that this protein could be similar to CTCF. There are numerous high-quality and high-resolution Hi-C data in C. elegans larvae, and so the authors can readily compare their binding peak locations to the insulation scores to test their hypothesis.

(4) The researchers suggest that ZNF-236 is important for some genomic context. Based on the transcriptomic data, can they find a clue for what that context may be? Are the ZNF-236 repressed genes enriched for not expressed genes in regions surrounded by highly expressed genes?

Reviewer #3 (Public review):

Summary:

Sethi and Zou present a new neural network to study the importance of epistatic interactions in pairs and groups of amino acids to the function of proteins. Their new model is validated on a small simulated data set, and then applied to 10 empirical data sets. Results show that epistatic interactions in groups of amino acids can be important to predict the phenotype of a protein, especially for sequences that are not very similar to the training data.

Strengths:

The manuscript relies on a novel neural network architecture that makes it easy to study specifically the contribution of interactions between 2, 3, 4 or more amino acids. The novel network architecture achieves such a level of interpretability without noticeable performance penalty. The study of 10 different protein families shows that there is variation among protein families in the importance of these interactions, and that higher order interactions are particularly important to predict the phenotypes of distant proteins.

Weaknesses:

The Github repository provides a README file to run a standard pipeline, but a user will need to go through the code to actually know what that pipeline is doing.

Reviewer #3 (Public review):

The SET1C/COMPASS complex is the histone H3K4 methyltransferase in Saccharomyces cerevisiae, where it plays pivotal roles in transcriptional regulation, DNA repair, and chromatin dynamics. While its canonical function in histone methylation is well-established, its full interactome remains poorly defined. Moreover, whether SET1C methylates non-histone substrates has been an open question.

In this study, Luciano et al. employ systematic yeast two-hybrid (Y2H) screening to uncover novel interactors and functions of SET1C. Their findings reveal potential functional connections to RNA biogenesis, chromatin remodeling, and non-histone methylation.

The authors performed multiple Y2H screens using Set1 (full-length, N-terminal, and C-terminal fragments) and each of its seven subunits as baits. They identified high-confidence interactors that link SET1C to diverse cellular processes, including chromatin regulation (e.g., the SWI/SNF complex via Snf2), DNA replication (e.g., Mcm2, Orc6), RNA biogenesis (e.g., spliceosome components Prp8 and Prp22; polyadenylation factors Pta1 and Ref2), tRNA processing (e.g., Trm1, Trm732), and nuclear import/export (e.g., importins Kap104 and Kap123). Some of these interactions were further validated by immunoprecipitation or in vitro assays.

Given the interaction of Set1 with Slx5 and Wss1 - proteins involved in SUMO-dependent processes - the authors investigated and convincingly demonstrated that Set1 is sumoylated. This modification may influence the function and regulation of the SET1C complex.

Finally, the authors provide evidence that SET1C methylates proteins beyond histone H3K4, notably Nrm1, a transcriptional corepressor, and Snf2, the catalytic subunit of the SWI/SNF chromatin remodeling complex. Although Nrm1 contains a domain resembling the H3K4-methylated sequence (H3K4-like domain), this region does not appear to be required for its methylation. The search for other proteins containing similar domains as potential methylation candidates (p.12, first paragraph) seems less justified, given the lack of evidence supporting the requirement for the H3K4-like domain in methylation.

This study offers valuable insights into the interactome of SET1C, suggesting potential links between the complex and a wide range of cellular processes. However, the functional implications of the Y2H interactions remain to be explored further. Additionally, the study provides intriguing information on the possible regulation of Set1 by sumoylation. The discovery of Nrm1 and Snf2 as methylation substrates could significantly expand the known targets and functions of SET1C.

The results are supported by high-quality data.

Reviewer #3 (Public review):

Summary:

The authors performed single-cell RNA sequencing of adult zebrafish hearts and identified markers for distinct cardiomyocyte subpopulations. One marker, phlda2, marks primordial cardiomyocytes. They generated transgenic reporter lines to characterize phlda2 expression patterns and a phlda2-NTR ablation line to determine the functional requirement of primordial cardiomyocytes during heart regeneration. They found that phlda2+ primordial cardiomyocytes are essential for myocardial morphogenesis and coronary vessel development. Interestingly, when phlda2+ primordial cardiomyocytes are ablated during heart regeneration, gata4+ cortical cardiomyocytes, coronary vessel revascularization, and scar tissue formation are not affected.

Strengths:

The authors identified a new primordial cardiomyocyte marker, phlda2. They further demonstrated that primordial cardiomyocytes are important for heart morphogenesis but dispensable for heart regeneration. Their findings reveal a potential difference between heart development and regeneration programs.

Weakness:

Despite the interesting findings, the authors did not provide supplemental data for their scRNAseq to demonstrate the data quality and support their conclusions, and some results are not well described.

Reviewer #3 (Public review):

Summary:

The primary objective of this study is to develop high-throughput screening assays utilizing homogeneous 3D cell cultures that more accurately replicate the intricate architecture and cellular communication found in tissues. The authors have chosen pancreatic islet β-cells as a model system to evaluate agents that modulate insulin release, which is particularly relevant given the increasing prevalence of diabetes mellitus-a significant global health concern. Moreover, the incorporation of human-based 3D spheroids, organoids, or organ-on-chip technologies into drug discovery protocols is essential for enhancing clinical translation, as candidate compounds identified using animal models have often demonstrated limited success in clinical settings.

Strengths:

This study was thoughtfully planned and skillfully carried out. The use of micropatterned hydrogels to observe 19 spheroids at once is an ingenious aspect, which has been effectively validated with Ca microfluorography. Overall, I found this investigation to be exceptionally well-executed and free from notable flaws, as the results clearly back up the conclusions. Additionally, the developed method achieved the proposed aims, providing a high-throughput format with 3D cultures. I believe this study deserves publication.

Weaknesses:

For an HTS assay, authors should incorporate the Z-factor.

Reviewer #3 (Public review):

Summary:

Sheidaei et al., report how chromosomes are brought to positions that facilitate kinetochore-microtubule interactions during mitosis. The study focusses on an important early step of the highly orchestrated chromosome segregation process. Studying kinetochore capture during early prophase is extremely difficult due to kinetochore crowding but the team has taken up the challenge by classifying the types of kinetochore movements, carefully marking kinetochore positions in early mitosis and linking these to map their fate/next-positions over time. The work is an excellent addition to the field as most of the literature has thus far focussed on tracking kinetochore in slightly later stages of mitosis. The authors show that the PANEM facilitates chromosome positioning towards the interior of the newly forming spindle, which in turn facilitates chromosome congression - in the absence of PANEM chromosomes end up in unfavourable locations, and they fail to form proper kinetochore-microtubule interactions. The work highlights the perinuclear actomyosin network in early mitosis (PANEM) as a key spatial and temporal element of chromosome congression which precedes the segregation process.

Major Comments:

(1) The complexity of tracking has been managed by classifying kinetochore movements into 4 categories, considering motions towards or away from the spindle mid-plane. While this is a very creative solution in most cases, there may be some difficult phases that involve movement in both directions or no dominant direction (e.g. Phase3-like). It is unclear if all kinetochores go through phase1, 2, 3 and 4 in a sequential or a few deviate from this pattern. A comment on this would be helpful. Also, it may be interesting to compare those that deviate from the sequence and ask how they recover in the presence and absence of azBB.

(2) Would peripheral kinetochore close to poles behave differently compared to peripheral kinetochore close to the midplane (figure S4) ?In figure 3D, are they separated? If not, would it look different?

(3) Uncongressed polar chromosomes (e.g., CENPE inhibited cells) are known to promote tumbling of the spindle. In figure 5B with polar chromosomes, it will be helpful to indicate how the authors decouple spindle pole movements from individual kinetochore movements.

(4) The work has high quality manual tracking of objects in early mitosis- if this would be made available to the field, it can help build AI models for tracking. The authors could consider depositing the tracking data and increasing the impact of their work.

Significance:

The current work builds upon their previous work, in which the authors demonstrated that an actomyosin network forms on the cytoplasmic side of the nuclear envelope during prophase. This work explains how the network facilitates chromosome capture and congression by tracking motions of individual kinetochores during early mitosis. The findings can be broadly useful for cell division and the cytoskeletal fields.

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.

Comments on revised version:

The authors have done a great job addressing reviewer concerns with the addition of updated figures, new experiments, and changes to the manuscript. I am supportive of this version and agree with the updated assessment.

she feels that being with her boyfriend makes her feel valued and become important to someone.

The speaker has hope and believes that she and her boyfriend can change their lives by saving money and work hard.

The speaker begins to doubt whether she can really escape this kind of life, or they will be stuck in the same life forever.

Reviewer #3 (Public review):

Summary:

In this work, the authors aim to improve neural encoding models for naturalistic video stimuli by integrating temporally aligned multimodal features derived from a deep learning model (VALOR) to predict fMRI responses during movie viewing.

Strengths:

The major strength of the study lies in its systematic comparison across unimodal and multimodal models using large-scale, high-resolution fMRI datasets. The VALOR model demonstrates improved predictive accuracy and cross-dataset generalization. The model also reveals inherent semantic dimensions of cortical organization and can be used to evaluate the integration timescale of predictive coding.

This study demonstrates the utility of modern multimodal pretrained models for improving brain encoding in naturalistic contexts. While not conceptually novel, the application is technically sound, and the data and modeling pipeline may serve as a valuable benchmark for future studies.

Weaknesses:

The overall framework of using data-driven features derived from pretrained AI models to predict neural response has been well studied and accepted by the field of neuroAI for over a decade. The demonstrated improvements in prediction accuracy, generalization, and semantic mapping are largely attributable to the richer temporal and multimodal representations provided by the VALOR model, not a novel neural modeling framework per se. As such, the work may be viewed as an incremental application of recent advances in multimodal AI to a well-established neural encoding pipeline, rather than a conceptual advance in modeling neural mechanisms.

Within this setup, the finding that VALOR outperforms CLIP, AlexNet, and WordNet is somewhat expected. VALOR encodes rich spatiotemporal information from videos, making it more aligned with movie-based neural responses. CLIP and AlexNet are static image-based models and thus lack temporal context, while WordNet only provides coarse categorical labels with no stimulus-specific detail. Therefore, the results primarily reflect the advantage of temporally-aware features in capturing shared neural dynamics, rather than revealing surprising model generalization. A direct comparison to pure video-based models, such as Video Swin Transformers or other more recent video models, would help strengthen the argument.

Moreover, while WordNet-based encoding models perform reasonably well within-subject in the HCP dataset, their generalization to group-level responses in the Short Fun Movies (SFM) dataset is markedly poorer. This could indicate that these models capture a considerable amount of subject-specific variance, which fails to translate to consistent group-level activity. This observation highlights the importance of distinguishing between encoding models that capture stimulus-driven representations and those that overfit to individual heterogeneities.

Reviewer #3 (Public review):

Summary:

The authors record from the ACC during a task in which animals must switch contexts to avoid shock as instructed by a cue. As expected, they find neurons that encode context, with some encoding of actions prior to the context, and encoding of neurons post-action. The primary novelty is dynamic encoding of action-outcome in a discrimination-avoidance domain, while this is traditionally done using operant methods.

Comments on revised version:

I appreciate the considerable work done on review, and additional details added throughout. I also noted the additional sessions included in analyses, and additional behavioral data in response to R1 and R2's insightful comments.

The only remaining comment that was not addressed pertains to anatomy and recording details. Some electrodes appear to be clearly in M2 (Fig 2A), and the tetrodes were driven each day. I would strongly suggest that this be included as a further limitation, particularly given the statement on line 178.

Reviewer #3 (Public review):

Summary:

The manuscript addresses an important, yet unresolved and long-debated, question: whether atypical protein kinase C is required for the maintenance of late-long-term synaptic potentiation (L-LTP) and long-term memory (LTM). The authors confirm previous findings that persistent activity of PKMζ is required for hippocampal L-LTP and spatial memory. They demonstrate that genetically deleting PKCι/λ and PKMζ individually induces compensatory upregulation, whereas deleting both atypical PKCs abolishes hippocampal L-LTP spatial long-term memory. The study uses an elegant combination of immunoblots, electrophysiology, and behavioral assays. The use of Cre-recombinase to target specific hippocampal regions and neurons adds to the rigor of the findings.

Strengths:

The manuscript addresses an important, yet unresolved and long-debated, question; whether PKMζ is required for the maintenance of L-LTP and LTM. The study demonstrates that PKCι/λ, which was previously shown to be critical for the initial generation of the early phase of LTP and short-term memory, becomes persistently active in L-LTP and LTM in a PKMζ knock-out model, compensating for the loss of PKMζ. Furthermore, when the compensation mechanisms are eliminated by simultaneous deletion of both PKMζ and PKCι/λ, maintenance of LTP and long-term spatial memory, but not of short-term memory, is diminished. The strength of this study is that the authors used a double-knockout strategy to directly address the controversy concerning the roles of PKMζ in memory formation. By showing that PKCι/λ compensates when PKMζ is deleted, the authors provided a compelling explanation for previous contradictory findings.

Weaknesses:

(1) The authors should provide the numerical values for all data.

(2) It appears that blind procedures were only used for the behavioral experiments. Some explanation is warranted.

(3) The description of the immunoblotting procedures lacks sufficient detail. The authors state that immunoblots were stained with multiple antisera to visualize multiple PKCs on the same immunoblot. To conserve antisera, the immunoblots were cut to isolate the relevant proteins based on molecular weight. Isoforms with similar molecular weights were either stained with antisera of different species or on separate blots. Despite this explanation, it is unclear how immunoblotting was performed in practice. For example, in Figure 1B, the authors compared the changes of four conventional PKC isoforms. Because all four antibodies are mouse monoclonal antibodies recognizing proteins of similar molecular weights, each probing should presumably have its own actin loading controls. However, these controls are missing from the figure. Some clarification is warranted.

(4) The statement in the legend to Figure 4B, that the increases of maximum avoidance time from pretraining to trial 1 are not different, indicates both groups of mice successfully established short-term memory, which is not correct. The analysis only reveals that there is no difference between the two groups. No differences could be due to both groups learning the same, as the authors suggest, or alternatively to no learning in either group.

(5) The labeling on some of the illustrations (e.g., Figure 2B) is unreadable.

(6) In Figure 4B, only the single statistical comparison between "pretaining" and "1 trial" is shown. The other comparisons described in the legend should also be illustrated.

(7) There is no documentation to support the statement that "The prevailing textbook mechanism for how memory is retained asserts that stable structural changes at synapses, the result of initial protein synthesis and growth, sustain memory without the need for ongoing biochemical activity dedicated to storing information" or for the statement in the Discussion that the structural model of memory storage is the standard account.

Reviewer #3 (Public review):

Summary:

TDP-43 proteinopathy is broadly found in neurodegenerative diseases. This manuscript investigates how nuclear export influences the biophysical properties of TDP-43. The authors use a combination of chemical screening and genome-wide siRNA screening to identify pathways that modulate TDP-43 liquid-to-solid transitions. Overall, the study employs a broad array of approaches and addresses an important question in TDP-43 pathobiology. The identification of nuclear export as a central regulator is compelling and conceptually aligns with the emerging view that TDP-43 nucleocytoplasmic trafficking is a major defect in neurodegeneration.

Strengths:

This work integrates chemical and genetic screening to identify novel modifiers. The candidates were validated in both reporter cell lines and iPS-differentiated organoids. The findings support the nucleocytoplasmic transport is important for the biophysical properties of TDP-43.

Weaknesses:

The mechanisms underlying the connection between nuclear export and phase transition need further clarification. Broader consequences of XPO1 inhibition are not addressed.

This is surprising because I thought research papers should mostly focus on sources. I didn’t realize the writer’s own ideas are actually the most important part.

This part is interesting because it shows that many of the people who were detained were normal community members. It makes the situation feel more real and personal.

This is troubling because only a small number of accounts created most of the hate. It shows that a few people or bots can make the internet feel negative.

This is interesting because it shows how a “small” decision can shape ten years. The details make her life feel realistic, not dramatic. It also shows how money and exhaustion can block steady progress, even when she keeps trying.

This is surprising because she says it in a very direct way. It shows a sudden moment when she stops believing what media has shown her. The line turns everyday TV stories into proof of unfair access.

This line feels troubling because it exposes an unequal bargain. The program is framed as a benefit for the privileged students’ awareness. It quietly implies the public school kids are there as a lesson, not as equal partners.

Reviewer #3 (Public review):

Summary:

In this paper, Weber and colleagues develop a generalization of the HGF, a widely used modeling tool. The generalization allows coupling between latent variables that was not possible in the original HGF. The resulting inference algorithm invites a predictive coding interpretation. The modular structure allows the construction of complex models out of simpler building blocks.

Strengths:

Overall, I think this is a valuable technical contribution, which will have applications to neuroscience, behavior, and psychiatry. It is mathematically rigorous, and the exposition is, for the most part, clear. It also comes with open-source software, so it should be a valuable resource to the modeling community.

Weaknesses:

My main concern is that the way that this paper is written will only be accessible and interesting to a niche audience interested in particular kinds of approximate inference schemes. The paper doesn't draw out the implications until the very end, so it's hard for readers to understand the motivation for certain modeling choices. It also requires readers to work through many pages of math before getting to applications. The applications themselves are very abstract.

Reviewer #3 (Public review):

Summary:

The manuscript is interesting, and it is clearly written. While the experiments are well executed, a general flaw is that the LppA/B analyses are done in the E. coli K12 host as surrogate for Salmonella enterica. For the mechanistic and molecular analyses of LppB a surrogate host is certainly adequate, yet it limits extrapolation of the physiological implications of LppB in the natural context.

Strengths:

The work convincingly demonstrates that LppB forms disulfide-based dimers and that it is crosslinked to PG via LdtB in E. coli. Moreover, dimerisation is required for LppB abundance in E. coli and LppB can inhibit crosslinking of Lpp/A to PG in E. coli.

Weaknesses:

Regarding the key conclusion of the work: while it is shown that LppB is oxidized in E. coli, whether envelope integrity (or OMV production) changes arise from switches in oxidation of the LppB cysteines remains to be shown, for E. coli let alone in the native host Salmonella. Does expression of LppB influence Lpp/A activity or OM tethering in E. coli? Since the inhibition of the Lpp/A linking to PG is not affected by the oxidation state of LppB, the abstract/title implies redox-control of envelope integrity which is a bit misleading and an overstatement. Both are features of LppB: i.e. it dimerizes through disulfide bond formation and it reduces PG binding of Lpp/A through trimerisation. However, no link between the two is shown.

Reviewer #3 (Public review):

Summary:

This paper uses a single Bayesian modelling framework to derive specific predictions for making inference, either with assumptions of a change-point structure or a gradually changing structure across tasks.

Strengths:

The paper nicely summarizes the slightly different subliteratures that have studied human behavior with models that only assume a single underlying task structure. The diagnostic predictions from the models are presented clearly, and the human data are nicely consistent with the model predictions.

As the authors discuss themselves, this work opens the door to many questions on the structured learning of inferring (from experience or verbal instructions) which meta-model is most appropriate to use.

Weaknesses:

Alignment between models and human behavior is mostly qualitative; the models are not fit to individual data (which could, for instance, uncover interesting differences between individuals.

There is no consideration of the possibility that individuals may not fully use one or the other meta-model (of gradual change vs changepoints), but instead a hybrid. Fits of the models to data may help uncover if some people (e.g., the 10% in experiment 2 that were best matched by the CP model?) use a slightly different mix of strategies than the one suggested by the verbal instructions received (which may cause the pattern in Figure 6d, which looks to have featured both models).