Reviewer #2 (Public review):

Summary:

Wenke et al. investigated the role of vlPAG somatostatin-expressing neurons in the mediation of analgesia during defensive states. A newly developed paradigm of cued fear-conditioned analgesia, which consists of a combination of an auditory fear retrieval session and a pain test, was used to evaluate this cell population's contribution to fear-mediated analgesia. Optogenetic manipulation of vlPAG SST+ neurons modulated the responses to a nociceptive cue (Hot Plate) presented concomitantly with an aversively conditioned tone. At the same time, alterations in the freezing levels could be observed during optogenetic activation of vlPAG SST+ neurons. In order to disentangle the impact of these cells on analgesia from their impact on the expression of defensive behaviors, the authors performed electrophysiological recordings from the dorsal horn in the spinal cord of anesthetized mice. A vlPAG-RVM-DH pathway was identified to trigger nociceptive C-fibers upon optic activation of the RVM. Finally, pathway-specific activation of SST+ vlPAG-RVM neurons could abolish CS-induced analgesia.

Strengths:

The study addresses a relevant topic, that is, brainstem circuits for pain-modulatory mechanisms as part of defensive states evoked by threat. This is important because the circuit mechanisms underlying pain are still not fully understood, and defining molecular markers of cellular circuit substrates may support the identification of potential pharmaceutical targets in treating pain. The authors confirm a previous study in that a somatostatin-positive cellular population presents a crucial vlPAG circuit element mediating anti-nociceptive effects. Key novelty aspects of the present study are the demonstration that these neurons seem to play a role specifically in threat-induced analgesia. This was possible by the elegant design and application of a novel fear analgesia paradigm, combined with cell- and pathway-specific optogenetics.

Weaknesses:

Despite the convincing and rigorous experimental approach, the study leaves some interpretational room when it comes to the proposed circuit mechanism. This could either be addressed by additional experiments or by more discussion of alternative circuit layouts.

Major Comments:

(1) The paper by Zhang et al. (https://pubmed.ncbi.nlm.nih.gov/36641028/), which identified a role for vlPAG SOM+ neurons in mediating anti-nociception in neuropathic pain, needs to be referenced and its results discussed, if not reconciled. While functionally, both studies find an analgetic role of vlPAG SOM+ neurons projecting to the RVM, Zhang et al., using slice physiology, characterize those neurons as glutamatergic. In Figure 4E of Zhang et al. they find general (fear-independent) analgetic effects with PAG-RVM specificity by performing chemogenetic experiments.

It can be argued that in addition to the two functionally distinct inhibitory SOM subtypes hypothesized by Winke et al., there is another, excitatory subpopulation. Also, the different experimental conditions (chronic vs. acute pain, non-threat vs. fearful cues/contexts may recruit different vlPAG SOM+ populations. All of this is conceivable, yet I wonder whether the contrasting findings could more parsimoniously be reconciled. The author's own results presented here in Supplementary Figure 3 suggests that SOM+ vlPAG cells are co-localizing with glutamate and thus could also be excitatory. In addition to this rather complementary piece of evidence, a more extensive characterization of vlPAG neurons using IHC and slice physiology would be needed to justify the unambiguous identification of their inhibitory nature.

In the absence of a direct identification of these cells exclusively releasing GABA, an alternative explanation should be considered. What about looking at vlPAG SOM+ neurons as a putatively mixed bag of local, inhibitory interneurons and long-range, RVM-projecting excitatory cells? This model would then open up interesting questions as to the actual function of somatostatin as a modulator of vlPAG circuit activity and associated function, and from my perspective, would nicely fit into the view of PAG circuits as integrators of complex survival responses.

(2) "Our data indicate that the optogenetic inhibition of SST+ vlPAG cells promotes analgesia irrespective of the animal's defensive state. In contrast, the optogenetic activation of long-range SST+ vlPAG cells that project to the rostral ventromedial medulla (RVM) abolishes the analgesia mediated by fear behavior." (lines 32-35). Consider toning down these conclusions, as contrasting activation with inhibition of two different (though overlapping) populations cannot be fully conclusive. Alternatively, a pathway-specific (vlPAG-RVM) inhibitory experiment could help to fully understand the circuit mechanism and verify the necessity of these neurons.

(3) Despite an overall very thorough reporting style, some information is missing from the manuscript:

a) In Figures 2d and f, what are the freezing levels during optogenetic manipulation? From Figure 3d, one can expect that freezing is inhibited during the hot plate test, which could bias the NC response towards shorter latencies. b) In Figure 5, the histological experiment showing the vlPAG-to-RVM pathway is presented by a qualitative image only. Here, some quantification would strengthen the finding. c) In Figures 6 c and d "Consistently, activation of the SST+ vlPAG-RVM pathway during CFCA had no impact on CS-presentation, whereas the same manipulation performed during CS+ blocked the increase in NC response latency compared to GFP controls." (line 194-196). Is it possible that the NC response cannot be any lower than the one during CS-, thus constituting a floor effect? d) Connected to major point 1- this experiment is important for defining the circuit mode and therefore should be as convincing as possible. However, for the colocalization experiment in Supplementary Figure 3, the methodological description is missing and thus makes it hard to comprehend how this data set was generated (how many data points, etc.). The visual depiction of the results is non-standard and not easily graspable. Consider e.g., a Venn diagram.

Reviewer #2 (Public review):

Summary:

The manuscript by Jia and Chen addresses the structural basis of voltage-activation of BK channels using computational approaches. Although a number of experimental studies using gating current and patch-clamp recording have analyzed voltage-activation in terms of observed charge movements and the apparent energetic coupling between voltage-sensor movement and channel opening, the structural changes that underlie this phenomenon have been unclear. The present studies use a reduced molecular system comprising the transmembrane portion of the BK channel (i.e. the cytosolic domain was deleted), embedded in a POPC membrane, with either 0 or 750 mV applied across the membrane. This system enabled acquisition of long simulations of 10 microseconds, to permit tracking of conformational changes of the channel. The authors principal findings were that the side chains of R210 and R213 rapidly moved toward the extracellular side of the membrane (by 8 - 10 Å), with greater displacements than any of the other charged transmembrane residues. These movements appeared tightly coupled to movement of the pore-lining helix, pore hydration, and ion permeation. The authors estimate that R210 and R213 contribute 0.25 and 0.19 elementary charges per residue to the gating current, which is roughly consistent with estimates based on electrophysiological measurements that used the full-length channel.

Strengths:

The methodologies used in this work are sound, and these studies certainly contribute to our understanding of voltage-gating of BK channels. An intriguing observation is the strongly coupled movement of the S4, S5, and S6 helices that appear to underlie voltage-dependent opening. Based on Fig 2a-d, the substantial movements of the R210 and R213 side chains occur nearly simultaneously to the S6 movement (between 4 - 5 usec of simulation time). This seems to provide support for a "helix-packing" mechanism of voltage gating in the so-called "non-domain-swapped" voltage-gated K channels.

Weaknesses:

The main limitation is that these studies used a truncated version of the BK channel, and there are likely to be differences in VSD-pore coupling in the context of the full-length channels that will not be resolved in the present work. Nonetheless, the authors provide a strong rationale for their use of the truncated channel, and the results presented will provide a good starting point for future computational studies of this channel.

Reviewer #2 (Public review):

Summary:

This study by Sanchez-Vasquez is a very innovative approach to induce aneuploidy and then study the contribution of treated cells to different lineages, including post implantation. It connects well to the authors previous work to induce mosaic aneuploidies. The authors identify sensitivity to HIF1a loss in treated embryos with likely aneuploidy. This work is part of an important line of work with evaluates the consequences of aneuploidy in mammalian embryo.

Weaknesses:

Given that this is a study on the induction of aneuploidy, it would be meaningful to assess aneuploidy immediately after induction, and then again before implantation. This is also applicable to the competition experiments on page 7/8. What is shown is the competitiveness of treated cells. Because the publication centers around aneuploidy, inclusion of such data in the main figure at all relevant points would strengthen it. There is some evaluation of karyotypes only in the supplemental - why? Would be good not to rely on a single assay that the authors appear to not give much importance.

Reviewer #2 (Public review):

This study aims to elucidate the role of fibroblasts in regulating myocardium and vascular development through signaling to cardiomyocytes and endothelial cells. This focus is significant, given that fibroblasts, cardiomyocytes, and vascular endothelial cells are the three primary cell types in the heart. The authors employed a Pdgfra-CreER-controlled diphtheria toxin A (DTA) system to ablate fibroblasts at various embryonic and postnatal stages, characterizing the resulting cardiac defects, particularly in myocardium and vasculature development. Single-cell RNA sequencing (scRNA-seq) analysis of the ablated hearts identified collagen as a crucial signaling molecule from fibroblasts that influences the development of cardiomyocytes and vascular endothelial cells.

This is an interesting manuscript; however, there are several major issues, including an over-reliance on the scRNA-seq data, which shows inconsistencies between replicates.

Some of the major issues are described below.

(1) The CD31 immunostaining data (Figure 3B-G) indicate a reduction in endothelial cell numbers following fibroblast deletion using PdgfraCreER+/-; RosaDTA+/- mice. However, the scRNA-seq data show no percentage change in the endothelial cell population (Figure 4D). Furthermore, while the percentage of Vas_ECs decreased in ablated samples at E16.5, the results at E18.5 were inconsistent, showing an increase in one replicate and a decrease in another, raising concerns about the reliability of the RNA-seq findings.

(2) Similarly, while the percentage of Ven_CMs increased at E18.5, it exhibited differing trends at E16.5 (Fig. 4E), further highlighting the inconsistency of the scRNA-seq analysis with the other data.

(3) Furthermore, the authors noted that the ablated samples had slightly higher percentages of cardiomyocytes in the G1 phase compared to controls (Fig. 4H, S11D), which aligns with the enrichment of pathways related to heart development, sarcomere organization, heart tube morphogenesis, and cell proliferation. However, it is unclear how this correlates with heart development, given that the hearts of ablated mice are significantly smaller than those of controls (Figure 3E). Additionally, the heart sections from ablated samples used for CD31/DAPI staining in Figure 3F appear much larger than those of the controls, raising further inconsistencies in the manuscript.

(4) The manuscript relies heavily on the scRNA-seq dataset, which shows inconsistencies between the two replicates. Furthermore, the morphological and histological analyses do not align with the scRNA-seq findings.

(5) There is a lack of mechanistic insight into how collagen, as a key signaling molecule from fibroblasts, affects the development of cardiomyocytes and vascular endothelial cells.

(6) In Figure 1B, Col1a1 expression is observed in the epicardial cells (Figure 1A, E11.5), but this is not represented in the accompanying cartoon.

(7) Do the PdgfraCreER+/-; RosaDTA+/- mice survive after birth when induced at E15.5, and do they exhibit any cardiac defects?

Comments on Revised Version (from BRE):

The manuscript has greatly improved following the revision, and I have no additional comments to offer.

Reviewer #2 (Public review):

Summary:

In this manuscript the authors investigate how epithelia maintain intercellular barrier function despite and during cellular rearrangements upon e.g. apoptotic extrusion in simple epithelia or regenerative turnover in stratified epithelia like this epidermis. A fundamental question in epithelial biology. Previous literature has shown that Rho mediated local regulation of actomyosin is essential not only for cellular rearrangement itself but also directly controls tight junction barrier function. The molecular mechanics however remained unclear. Here the authors use extensive fluorescence imaging of fixed and live cells together with genetic and drug mediated interference to show that Rho activation is required and sufficient to form de novo tight junctional strands at intercellular contacts in epidermal keratinocytes (HaCat) and mammary epithelial cells. After having confirmed previous literature they then show that Rho activation activates the transmembrane protease matriptase which cleaves EpCAM and TROP2, two claudin binding transmembrane proteins, to release claudins and enable claudin strand formation and therefore tight junction barrier function.

Strengths:

The presented mechanism is shown to be relevant for epithelial barriers being conserved in simple and stratifying epithelial cells and mainly differs due to tissue specific expression of EpCAM and TROP2. The authors present carefull state of the art imaging and logical experiments that convincingly support the statements and conclusion. The manuscript is well written and easy to follow.

Weaknesses:

Whereas the in vitro evidence of the presented mechanism is strongly supported by the data, the in vivo confirmation is mostly based on the predicted distribution of TROP2. Whereas the causality of Rho mediated matriptase activation has been nicely demonstrated it remains unclear how Rho activates matriptase.

Reviewer #2 (Public review):

Summary:

Here, the authors studied the molecular mechanisms by which the chemoreceptor cluster and flagella motor of Pseudomonas aeruginosa (PA) are spatially organized in the cell. They argue that FlhF is involved in localizing the receptors-motor to the cell pole, and even without FlhF, the two are colocalized. Finally, the authors argue that the functional reason for this colocalization is to insulate chemotactic signaling from other signaling pathways, such as cyclic-di-GMP signaling.

Strength:

The experiments and data are high quality. It is clear that the motor and receptors co-localize, and that elevated CheY levels lead to elevated c-di-GMP.

Weakness:

The explanation for the functional importance of receptor-motor colocalization is plausible but is still not conclusively demonstrated. Colocalization might reduce CheY levels throughout the cell in order to reduce cross-talk with c-di-GMP. This would mean that if physiologically-relevant levels of CheYp near the pole were present throughout the cell, c-di-GMP levels would be elevated to a point that is problematic for the cell. Clearly demonstrating this seems challenging.

Reviewer #2 (Public review):

Summary:

In their study, Cooper et al. investigated the spontaneous fluctuations in extracellular 5-HT release in the CA1 region of the hippocampus using GRAB5-HT3.0. Their findings revealed the presence of ultra-low frequency (less than 0.05 Hz) oscillations in 5-HT levels during both NREM sleep and wakefulness. The phase of these 5-HT oscillations was found to be related to the timing of hippocampal ripples, microarousals, electromyogram (EMG) activity, and hippocampal-cortical coherence. In particular, ripples were observed to occur with greater frequency during the descending phase of 5-HT oscillations, and stronger ripples were noted to occur in proximity to the 5-HT peak during NREM. Microarousal and EMG peaks occurred with greater frequency during the ascending phase of 5-HT oscillations. Additionally, the strongest coherence between the hippocampus and cortex was observed during the ascending phase of 5-HT oscillations. These patterns were observed in both NREM sleep and the awake state, with a greater prevalence in NREM. The authors posit that 5-HT oscillations may temporally segregate internal processing (e.g., memory consolidation) and responsiveness to external stimuli in the brain.

Strengths:

The findings of this research are novel and intriguing. Slow brain oscillations lasting tens of seconds have been suggested to exist, but to my knowledge they have never been analyzed in such a clear way. Furthermore, although it is likely that ultra-slow neuromodulator oscillations exist, this is the first report of such oscillations, and the greatest strength of this study is that it has clarified this phenomenon both statistically and phenomenologically.

Weaknesses:

As with any paper, this one has some limitations. While there is no particular need to pursue them, I will describe ten of them below, including future directions:

Contralateral recordings: 5-HT levels and electrophysiological recordings were obtained from opposite hemispheres due to technical limitations. Ipsilateral simultaneous recordings may show more direct relationships.

Sample size: The number of mice used in the experiments is relatively small (n=6). Validation with a larger sample size would be desirable.

Lack of causality: The observed associations show correlations, not direct causal relationships, between 5-HT oscillations and neural activity patterns.

Limited behavioral states: The study focuses primarily on sleep and quiet wakefulness. Investigation of 5-HT oscillations during a wider range of behavioral states (e.g., exploratory behavior, learning tasks) may provide a more complete understanding.

Generalizability to other brain regions: The study focuses on the CA1 region of the hippocampus. It's unclear whether similar 5-HT oscillation patterns exist in other brain regions.

Long-term effects not assessed: Long-term effects of ultra-low 5-HT oscillations (e.g., on memory consolidation or learning) were not assessed.

Possible species differences: It's uncertain whether the findings in mice apply to other mammals, including humans.

Technical limitations: The temporal resolution and sensitivity of the GRAB5-HT3.0 sensor may not capture faster 5-HT dynamics.

Interactions with other neuromodulators: The study does not explore interactions with other neuromodulators (e.g., norepinephrine, acetylcholine) or their potential ultraslow oscillations.

Limited exploration of functional significance: While the study suggests a potential role for 5-HT oscillations in memory consolidation and arousal, direct tests of these functional implications are not included.

Reviewer #2 (Public review):

Summary:

This study reports on the existence of subpopulations of isogenic E. coli and P. aeruginosa cells that are tolerant to the antimicrobial peptide tachyplesin and are characterized by accumulation of low levels of a fluorescent tachyplesin-NBD conjugate. The authors then set out to address the molecular mechanisms, providing interesting insights even though the mechanism remains incompletely defined: The work demonstrates that increased efflux may cause this phenotype, putatively together with other changes in membrane lipid composition. The authors further demonstrate that pharmacological manipulation can prevent generation of tolerance. The authors are cautious in their interpretation and the claims made are largely justified by the data.

Strengths:

Going beyond the commonly used bulk techniques for studying susceptibility to AMPs, Lee et al. used of fluorescent antibiotic conjugates in combination with flow cytometry analysis to study variability in drug accumulation at the single cell level. This powerful approach enabled the authors to expose bimodal drug accumulation pattern that were condition dependent, but conserved across a variety of E. coli clinical isolates. Using cell sorting in combination with colony-forming unit assays as well as quantitative fluorescence microscopic analysis in a microfludics-setup the authors compellingly demonstrate that low accumulators (where fluorescence signal is mostly restricted to the membrane), can survive antibiotic treatment, whereas high accumulators (with high intracellular fluorescence) were killed.

The relevance of efflux for the ´low accumulator´ phenotype and its survival is convincingly demonstrated by the following lines of evidence: i) A time-course experiment on tachyplesin-NBD pre-loaded cells revealed that all cells initially were high accumulators, before a subpopulation of cells subsequently managed to reduce signal intensity, demonstrating that the ´low accumulator´ phenotype is an induced response and not a pre-existing property. Ii) Double-mutants deficient in the delta acrA delta tolC double-KO, which showed reduced levels of low accumulators´. Interestingly, ´low accumulator´populations were nearly abrogated in bacteria deficient in the qse quorum sensing system, suggesting its centrality for the tachyplesin response. Even though this system may control acrA, the strength of the phenotype may suggest that it may control additional as-of-yet unidenitified factors relevant in the response to tachyplesin. Iii) treatment with efflux pump inhibitor sertraline and verapamil (even though some caution needs to be taken since it is not perfectly selective, see weakness) prevents generation of low accumulators. The observation that sertraline enhances tachyplesin-based killing is an important basis for developing combination therapies.

The study convincingly illustrates how susceptibility to tachyplesin adaptively changes in a heterogeneous way dependent on the growth phases and nutrient availability. This is highly relevant also beyond the presented example of tachyplesin and similar subpopulation-based adaptive changes to the susceptibility towards antimicrobial peptides or other drugs may occur during infections in vivo and they would likely be missed by standardized in vitro susceptibility testing.

Weaknesses:

Some mechanistic questions regarding tachyplesin-accumulation and survival remain. One general shortcoming of the setup of the transcriptomics experiment is that the tachyplesin-NBD probe itself has antibiotic efficacy and induces phenotypes (and eventually cell death) in the ´high accumulator´ cells. As the authors state themselves, this makes it challenging to interpret whether any differences seen between the two groups are causative for the observed accumulation pattern of if they are a consequence of differential accumulation and downstream phenotypic effects.

Reviewer #2 (Public review):

Summary:

In this study, Yagasaki et al. describe an organoid system to study the interactions between smooth muscle cells (SMCs) and interstitial cells of Cajal (ICCs). While these interactions are essential for the control of rhythmic intestinal contractility (i.e., peristalsis), they are poorly understood, largely due to the complexity of and access to the in vivo environment and the inability to co-culture these cell types in vitro for long term under physiological conditions. The "gut contractile organoids" organoids described herein are reconstituted from stromal cells of the fetal chicken hindgut that rapidly reorganize into multilayered spheroids containing an outer layer of smooth muscle cells and an inner core of interstitial cells. The authors demonstrate that they contract cyclically and additionally use calcium imagining to show that these contractions occur concomitantly with calcium transients that initiate in the interstitial cell core and are synchronized within the organoid and between ICCs and SMCs. Furthermore, they use several pharmacological inhibitors to show that these contractions are dependent upon non-muscle myosin activity and, surprisingly, independent of gap junction activity. Finally, they develop a 3D hydrogel for the culturing of multiple organoids and found that they synchronize their contractile activities through interconnecting smooth muscle cells, suggesting that this model can be used to study the emergence of pacemaking activities. Overall, this study provides a relatively easy-to-establish organoid system that will be of use in studies examining the emergence of rhythmic peristaltic smooth muscle contractions and how these are regulated by interstitial cell interactions. However, further validation and quantification will be necessary to conclusively determine show the cellular composition of the organoids and how reproducible their behaviors are.

Strengths:

This work establishes a new self-organizing organoid system that can easily be generated from the muscle layers of the chick fetal hindgut to study the emergence of spontaneous smooth muscle cell contractility. A key strength of this approach is that the organoids seem to contain few cell types (though more validation is needed), namely smooth muscle cells (SMCs) and interstitial cells of Cajal (ICCs). These organoids are amenable to live imaging of calcium dynamics as well as pharmacological perturbations for functional assays, and since they are derived from developing tissues, the emergence of the interactions between cell types can be functionally studied. Thus, the gut contractile organoids represent a reductionist system to study the interactions between SMCs and ICCs in comparison to the more complex in vivo environment, which has made studying these interactions challenging.

Weaknesses:

The study lacks complementary in vivo experiments, but these will be exciting to follow up in future studies.

Reviewer #2 (Public review):

Summary:

The authors analyzed Xenopus oocytes at different stages of meiosis using quantitative phosphoproteomics. Their advanced methods and analyses revealed changes in protein abundances and phosphorylation states to an unprecedented depth and quantitative detail. In the manuscript they provide an excellent interpretation of these findings putting them in the context of past literature in Xenopus as well as in other model systems. The clarity of these explanations improved significantly in the revised version of the manuscript, and several minor imprecisions have been corrected as well.

Strengths:

High-quality data, careful and detailed analysis, and outstanding interpretation in the context of the large body of literature.

Weaknesses:

Merely a resource, none of the findings are tested in functional experiments.

I am very impressed by the quality of the data and the careful and detailed interpretation of the findings. In this form, the manuscript will be an excellent resource to the cell division community in general, and it presents a very large number of hypotheses that can be tested in future experiments. Xenopus has been and still is a popular and powerful model system that led to critical discoveries around countless cellular processes, including the spindle, nuclear envelope, and translational regulation, just to name a few. This also includes a huge body of literature on the cell cycle describing its phosphoregulation. It is indeed somewhat frustrating to see that these earlier studies using phospho-mutants and phospho-antibodies were just scratching the surface. The phosphoproteomics analysis presented here reveals much more extensive and much more dynamic changes in phosphorylation states. Thereby, in my opinion, this manuscript opens a completely new chapter in this line of research, setting the stage for more systematic future studies.

Reviewer #2 (Public review):

Summary:

The investigators tested a workshop intervention to improve knowledge and decrease misconceptions about sex inclusive research. There were important findings that demonstrate the difficulty in changing opinions and knowledge about the importance of studying both males and females. While interventions can improve knowledge and decrease perceived barriers, the impact was small.

Strengths:

The investigators included control groups and replicated the study in a second population of scientists. The results appear to be well substantiated. These are valuable findings that have practical implications for fields where sex is included as a biological variable to improve rigor and reproducibility.

Weaknesses:

I found the figures difficult to understand and would have appreciated more explanation of what is depicted, as well as greater space between the bars representing different categories.

Reviewer #2 (Public review):

Summary:

I am not familiar with cancer biology, so my review mainly focuses on the infection part of the manuscript. Wang et al identified an RNA-binding protein RBMX2 that links the Mycobacterium bovis infection to the epithelial-Mesenchymal transition and lung cancer progression. Upon mycobacterium infection, the expression of RBMX2 was moderately increased in multiple bovine and human cell lines, as well as bovine lung and liver tissues. Using global approaches, including RNA-seq and proteomics, the authors identified differential gene expression caused by the RBMX2 knockout during M. bovis infection. Knockout of RBMX2 led to significant upregulations of tight-junction related genes such as CLDN-5, OCLN, ZO-1, whereas M. bovis infection affects the integrity of epithelial cell tight junctions and inflammatory responses. This study establishes that RBMX2 is an important host factor that modulates the infection process of M. bovis.

Strengths:

(1) This study tested multiple types of bovine and human cells, including macrophages, epithelial cells, and clinical tissues at multiple timepoints, and firmly confirmed the induced expression of RBMX2 upon M. bovis infection.

(2) The authors have generated the monoclonal RBMX2 knockout cell lines and comprehensively characterized the RBMX2-dependent gene expression changes using a combination of global omics approaches. The study has validated the impact of RBMX2 knockout on the tight-junction pathway and on the M. bovis infection, establishing RBMX2 as a crucial host factor.

Weaknesses:

(1) The RBMX2 was only moderately induced (less than 2-fold) upon M. bovis infection, arguing its contribution may be small. Its value as a therapeutic target is not justified. How RBMX2 was activated by M. bovis infection was unclear.

(2) Although multiple time points have been included in the study, most analyses lack temporal resolution. It is difficult to appreciate the impact/consequence of M. bovis infection on the analyzed pathways and processes.

Reviewer #2 (Public review):

The authors constructed an in-frame deletion of moeA gene, which is involved in molybdopterin cofactor (MoCo) biosynthesis, and investigated its role in structural colors in Flavobacterium IR1. The deletion of moeA shifted colony color from green to blue, reduced colony spreading, and increased starch degradation, which was attributed to the upregulation of various proteins in polysaccharide utilization loci. This study lays the ground for developing new colorants by modifying genes involved in structural colors.

Overall, this is a well-written paper in which the authors effectively address their research questions through proper experimentation. This work will help us understand the genetic basis of structural colors in Flavobacterium and open new avenues to study the roles of additional genes and proteins in structural colors.

Reviewer #2 (Public review):

Summary:

In the article Goyal et al. investigate how protein/polymer phase transition behavior is modulated by different binding partners-specifically, DNA and polyphosphate (PolyP). The authors show that while both DNA and PolyP can induce metastable condensates, only PolyP drives unique phase transition behaviors by effectively discriminating among initial protein ensembles with varying degrees of conformational heterogeneity, compactness, and plasticity. This selectivity is attributed to PolyP's ability to unfold the enzyme during condensate formation, supported by the observation of polyproline II-rich structures in two tested variants (CytR WT and DM). Overall, this work offers valuable insights into the mechanistic factors underlying condensation assembly and advances our understanding of how molecular interactions influence phase behavior.

Strengths:

The authors employed a well-designed and technically sound experimental approach to investigate how the initial protein conformational ensemble influences phase transition behavior in the presence of two charged polymers. Specifically, they examined phase transitions of CytR and FruR variants in the context of either polyphosphate (PolyP) or DNA, enabling a direct comparison that effectively highlights key differences. This study provides mechanistic insights into the role of PolyP in driving condensation and may contribute to a broader understanding of assembly processes involving PolyP, particularly in the context of bacterial stress responses.

Weaknesses:

The primary weakness of this manuscript lies in the lack of a consistent trend linking the unique phase transitions observed in protein/PolyP systems to the initial protein conformational ensemble. The observed differences in assembly and maturation behavior do not consistently correlate with conformational heterogeneity, plasticity, or compactness of the starting ensemble. This is particularly evident in the divergent outcomes between the CytR/PolyP and FruR/PolyP systems. Consequently, the phase behavior of protein/PolyP condensates does not reliably reflect the composition of the initial conformational ensemble, limiting its effectiveness as a probe for conformational state characterization.

Reviewer #2 (Public review):

Summary:

An initial screening of pretreatment with different stress treatments of K. pneumonia allowed the identification of heat stress as a protection factor against the infection of the lytic phage Kp11. Then experiments prove that this is mediated not by an increase of phage resistant bacteria but due to an increase in phage transient tolerant population, that the authors identified as bacteriophage persistence in analogy to antibiotic persistence. Then they proved that phage persistence mediated by heath shock enhanced the evolution of bacterial resistance against the phage. The same trait was observed using other lytic phages, their combinations and two clinical strains, as well as E. coli and two T phages, hence the phenomenon may be widespread in enterobacteria.

Next, the elucidation of heat induced phage persistence was done, determining that phage adsorption was not affected but phage DNA internalization was impaired by the heat pretreatment, likely to alterations in the bacterial envelope, including the downregulation of envelope proteins and of LPS; furthermore, heat treated bacteria were less sensitive to polymyxins due to the decrease in LPS.

Finally, cyclic exposure to heat stress allowed the isolation of a mutant that was both resistant to heat treatment, polymyxins and lytic phage, that mutant had alterations in PspA protein that allowed a gain of function and that promoted the reduction of capsule production and loss of its structure; nevertheless this mutant was severely impaired in immune evasion as it was easily cleared from mice blood, evidencing the trade-off's between phage/heat and antibiotic resistance and the ability to counteract the immune response.

Strengths:

The experimental design and the sequence in which they are presented is ideal for the understanding of their study and the conclusions are supported by the findings, also the discussion points out the relevance of their work particularly in the effectiveness of phage therapy and allow the design of strategies to improve their effectiveness.

Weaknesses:

In its present form it lacks the incorporation of some relevant previous work that explored the role of heat stress in phage susceptibility, antibiotic susceptibility, trade offs between phage resistance and resistance against other kinds of stress, virulence, etc. and the fact that exposure to lytic phages induces antibiotic persistence.

Comments on revised version:

Thanks for addressing most of my comments; however, although you replied this in the rebuttal:

"Thank you for highlighting these important studies. We have incorporated the work by Majkowska-Skrobek et al. (2021), Gordillo Altamirano et al. (2021), and García-Cruz et al. (2024) into the discussion "

I was not able to find the new section in the discussion of the manuscript.

Reviewer #3 (Public review):

The Mutational Hazard Hypothesis (MHH) suggests that lineages with smaller effective population sizes should accumulate slightly deleterious transposable elements leading to larger genome size. Marino and colleagues tested the MHH using a set of 807 vertebrate, mollusc and insect species. The authors mined repeats de novo and estimated dN/dS for each genome. Then, they used dN/dS and life history traits as reliable proxies for effective population size and tested for correlations between these proxies and repeat content while accounting for phylogenetic nonindependence. The results suggest that overall, lineages with lower effective population sizes do not exhibit increases in repeat content or genome size. This contrasts with expectations from the MHH. The authors speculate that changes in genome size may be driven by lineage-specific host-TE conflicts rather than effective population size.

Strengths:

The general conclusions of this paper are supported by a powerful dataset of phylogenetically diverse species. Furthermore, the hypothesis tested is important and has proved challenging to test in the past due to technical challenges and confounding factors. The use of C-values rather than assembly size for many species (when available) helps to mitigate the challenges associated with underrepresentation of repetitive regions in short-read based genome assemblies. Overall, both the phylogenetic breadth of species considered and the approaches employed make the results highly convincing.

Weaknesses:

My primary concerns were related to possible biases in the author's data due to their approach to TE annotation. The authors have sufficiently acknowledged and addressed these concerns in their revised manuscript. I note no further weaknesses.

Reviewer #2 (Public review):

This is an interesting paper that delves into the post-translational modifications of the yeast Srs2 helicase and proteins with which it interacts in coping with DNA damage. The authors use mutants in some interaction domains with RPA and Srs2 to argue for a model in which there is a balance between RPA binding to ssDNA and Srs2's removal of RPA.

The manuscript mostly addresses previous concerns by doubling down on the model without providing additional direct evidence of interactions between Srs2 and PCNA, and that "precise sites of Srs2 actions in the genome remain to be determined." One additional Srs2 allele has been examined, showing some effect in combination with rfa1-zm2.

Reviewer #2 (Public review):

Summary:

This study aimed to uncover the protein composition and evolutionary conservation of electrical synapses in retinal neurons. The authors employed two complementary BioID approaches: expressing a Cx35b-TurboID fusion protein in zebrafish photoreceptors and using GFP-directed TurboID in Cx36-EGFP-labeled mouse AII amacrine cells. They identified conserved ZO proteins and endocytosis components in both species, along with over 50 novel proteins related to adhesion, cytoskeleton remodeling, membrane trafficking, and chemical synapses. Through a series of validation studies¬-including immunohistochemistry, in vitro interaction assays, and immunoprecipitation-they demonstrate that novel scaffold protein SIPA1L3 interacts with both Cx36 and ZO proteins at electrical synapse. Furthermore, they identify and localize proteins ZO-1, ZO-2, CGN, SIPA1L3, Syt4, SJ2BP, and BAI1 at AII/cone bipolar cell gap junctions.

Strengths:

The study demonstrates several significant strengths in both experimental design and validation approaches. First, the dual-species approach provides valuable insights into the evolutionary conservation of electrical synapse components across vertebrates. Second, the authors compare two different TurboID strategies in mice and demonstrate that the HKamac promoter and GFP-directed approach can successfully target the electrical synapse proteome of mouse AII amacrine cells. Third, they employed multiple complementary validation approaches-including retinal section immunohistochemistry, in vitro interaction assays, and immunoprecipitation-providing evidence supporting the presence and interaction of these proteins at electrical synapses.

Weaknesses:

The major weakness of this paper is the insufficient number of replicates in the proteomics datasets. The zebrafish datasets include only two biological replicates, while the mouse dataset has only one high-quality replicate. Due to the limited number of replicates, it is not possible to determine which enriched proteins are statistically significant.

Additionally, the Neutravidin staining in the TurboID condition is not restricted to where Cx35 is expressed but is broadly distributed throughout the INL and IPL in the zebrafish retina (Figure 1B, bottom). Therefore, it is necessary to include NeutrAvidin staining in non-labeled retinas to verify whether the biotinylated proteins are specifically associated with Cx35 expression. Although the western blot results showed increased protein enrichment in the TurboID condition compared to non-labeled retinas, this does not confirm that the streptavidin pull-down proteins are associated with Cx35.

Similarly, it is important to include NeutrAvidin staining in both TurboID and non-labeled conditions in the mouse retina to verify that the biotinylated proteins are specifically associated with gap junctions.

Reviewer #2 (Public review):

This study presents a comprehensive investigation of remote memory deficits in the APP/PS1 mouse model of Alzheimer's disease. The authors convincingly show that these deficits emerge progressively and are paralleled by selective hyperexcitability of PV interneurons in the mPFC. Using viral-TRAP labeling and patch-clamp electrophysiology, they demonstrate that inhibitory input onto labeled engram cells is selectively increased in APP/PS1 mice, despite unaltered engram size or reactivation. These findings support the idea that alterations in inhibitory microcircuits may contribute to cognitive decline in AD.

However, several aspects of the study merit further clarification. Most critically, the central paradox, i.e., increased inhibitory input without an apparent change in engram reactivation, remains unresolved. The authors propose possible mechanisms involving altered synchrony or impaired output of engram cells, but these hypotheses require further empirical support. Additionally, the study employs multiple crossed transgenic lines without reporting the progression of amyloid pathology in the mPFC, which is important for interpreting the relationship between circuit dysfunction and disease stage. Finally, the potential contribution of broader network dysfunction, such as spontaneous epileptiform activity reported in APP/PS1 mice, is also not addressed.

Reviewer #2 (Public review):

Summary:

In this article, the authors investigate enhancing the therapeutic and regenerative properties of mesenchymal stem cells (MSCs) through genetic modification, specifically by overexpressing genes involved in the glycogen synthesis pathway. By creating a non-phosphorylatable mutant form of glycogen synthase (GYSmut), the authors successfully increased glycogen accumulation in MSCs, leading to significantly improved cell survival under starvation conditions. The study highlights the potential of glycogen engineering to improve MSC function, especially in inflammatory or energy-deficient environments. However, critical gaps in the study's design, including the lack of validation of key findings, limited differentiation assessments, and missing data on MSC-GYSmut resistance to reactive oxygen species (ROS), necessitate further exploration.

Strengths:

(1) Novel Approach: The study introduces an innovative method of enhancing MSC function by manipulating glycogen metabolism.

(2) Increased Glycogen Storage: The genetic modification of GYS1, resulting in GYSmut, significantly increased glycogen accumulation, leading to improved MSC survival under starvation, which has strong implications for enhancing MSC therapeutic properties in energy-deficient environments.

(3) Potential Therapeutic Impact: The findings suggest significant therapeutic potential for MSCs in conditions that require improved survival, persistence, and immunomodulation, especially in inflammatory or energy-limited settings.

(4) In Vivo Validation: The in vivo murine model of pulmonary fibrosis demonstrated the improved survival and persistence of MSC-GYSmut, supporting the translational potential of the approach.

Weaknesses:

(1) Lack of Differentiation Assessments: The study did not evaluate key MSC differentiation pathways, including chondrogenic and osteogenic differentiation. The absence of analysis of classical MSC surface markers and multipotency limits the understanding of the full potential of MSC-GYSmut.

(2) Missing Validation of RNA Sequencing Data: Although RNA sequencing data revealed promising transcriptomic changes in chondrogenesis and metabolic pathways, these findings were not experimentally validated, limiting confidence.

(3) Lack of ROS Resistance Analysis: Resistance to reactive oxygen species (ROS), an important feature for MSCs under regenerative conditions, was not assessed, leaving out a critical aspect of MSC function.

(4) Inconsistencies in In Vivo Data: There is a discrepancy between the number of animals shown in the figures and the graph (three individuals vs. five animals), as well as missing details on how luciferase signal intensity was quantified, requiring further clarification.

(5) Limited Exploration of Immunosuppressive Properties: The study did not address the immunosuppressive functions of MSC-GYSmut, which are critical for MSC-based therapies in clinical settings.

Conclusion:

The study presents an exciting new direction for enhancing MSC function through glycogen metabolism engineering. While the results show promise, key experiments and validations are missing, and several areas, such as differentiation capacity, ROS resistance, and immunosuppressive properties, require further investigation. Addressing these gaps would solidify the conclusions and strengthen the potential clinical applications of MSC-GYSmut in regenerative medicine.

Reviewer #3 (Public review):

Valencia et al. aim to elucidate the biochemical and cellular mechanisms through which the human formin FHOD3 drives sarcomere assembly in cardiomyocytes. To do so, they combined rigorous in vitro biochemical assays with comprehensive in vivo characterizations, evaluating two wild type FHOD3 isoforms and two function-separating mutants. Surprisingly, they found that both wild type FHOD3 isoforms can nucleate new actin filaments, as well as elongate existing actin filaments in conjunction with profilin following barbed-end capping. This is in addition to FHOD3's proposed role as an actin bundler. Next, the authors focused on the longer isoform FHOD3L due to its essential role in sarcomere assembly in cardiomyocytes. They asked whether FHOD3L promote sarcomere assembly through its activity in actin nucleation or rather elongation. To do so, the authors designed two function-separating mutants: the K1193L mutation in the FH2 domain, known for its importance in actin nucleation, and the glycine-serine linker substitution in the FH1 domain ("GS-FH1",) known for its requirement in actin elongation. They demonstrated that while K1193L maintains its elongation activity and greatly diminishes nucleation and bundling, in GS-FH1 keeps its nucleation activity while lose its capacity to drive elongation. Armed with these tools, the authors attempted to rescue FHOD3L siRNA-treated neonatal rat ventricular myocytes (NRVM) with transgenes carrying wild type, K1193L, or GS-FH1 mutant forms of human FHOD3. In each condition, they evaluated the numbers and morphology of sarcomeres, as well as their ability to beat and generate cardiac rhythm. The authors found that while the wild type FHOD3L and the K1193L mutant can rescue sarcomere morphology and physiology, the GS-FH1 mutant fails to do so. Given that in GS-FH1 mainly elongation activity is compromised, the authors concluded that the elongation activity of FHOD3 is essential for its role in sarcomere assembly in cardiomyocytes, while its nucleator activity is dispensable. Overall, this important study provided a broadened view on the biochemical activities of FHOD3, and a pioneering view on a possible cellular mechanism of how FHOD3L drives sarcomere assembly. If further validated, this can lead to new mechanistic models of sarcomere assembly and potentially new therapeutic targets of cardiomyopathy.

The conclusions of this paper are mostly well supported by the comprehensive biochemical analyses performed by the authors. In my original assessment, I raised the point that the extreme low level of GS-FH1 signal in transfected cells in Figure 6A may reflect a failure of actin-binding by this construct in vivo, rather than its inability of driving elongation. The authors have thoroughly addressed this concern by: 1) providing new images of the GS-FH1 rescue condition with HA-FHOD3L signal intensities matching that of the K1193L rescue condition, and 2) quantitatively demonstrating that the expression levels in the GS-FH1 rescue condition are comparable with that of wild type FHOD3L rescue condition. This is nicely complemented by the new phalloidin staining of the GS-FH1 rescue condition, which showcased additional details of actin puncta reminiscent of that present in muscle stress fibers or premyofibrils. Overall, I am now convinced that the GS-FH1 cannot rescue sarcomere formation even when expressed at comparable levels. Given that GS-FH1 demonstrates actin elongation defects in vitro, it is reasonable to conclude that the actin elongation function of FHOD3L is essential for sarcomere formation in vivo.

Reviewer #2 (Public review):

Summary:

The authors present Altair-LSFM (Light Sheet Fluorescence Microscope), a high-resolution, open-source microscope, that is relatively easy to align and construct and achieves sub-cellular resolution. The authors developed this microscope to fill a perceived need that current open-source systems are primarily designed for large specimens and lack sub-cellular resolution or are difficult to construct and align, and are not stable. While commercial alternatives exist that offer sub-cellular resolution, they are expensive. The authors' manuscript centers around comparisons to the highly successful lattice light-sheet microscope, including the choice of detection and excitation objectives. The authors thus claim that there remains a critical need for high-resolution, economical, and easy-to-implement LSFM systems.

Strengths:

The authors succeed in their goals of implementing a relatively low-cost (~ USD 150K) open-source microscope that is easy to align. The ease of alignment rests on using custom-designed baseplates with dowel pins for precise positioning of optics based on computer analysis of opto-mechanical tolerances, as well as the optical path design. They simplify the excitation optics over Lattice light-sheet microscopes by using a Gaussian beam for illumination while maintaining lateral and axial resolutions of 235 and 350 nm across a 260-um field of view after deconvolution. In doing so they rest on foundational principles of optical microscopy that what matters for lateral resolution is the numerical aperture of the detection objective and proper sampling of the image field on to the detection, and the axial resolution depends on the thickness of the light-sheet when it is thinner than the depth of field of the detection objective. This concept has unfortunately not been completely clear to users of high-resolution light-sheet microscopes and is thus a valuable demonstration. The microscope is controlled by an open-source software, Navigate, developed by the authors, and it is thus foreseeable that different versions of this system could be implemented depending on experimental needs while maintaining easy alignment and low cost. They demonstrate system performance successfully by characterizing their sheet, point-spread function, and visualization of sub-cellular structures in mammalian cells, including microtubules, actin filaments, nuclei, and the Golgi apparatus.

Weaknesses:

There is a fixation on comparison to the first-generation lattice light-sheet microscope, which has evolved significantly since then:

(1) The authors claim that commercial lattice light-sheet microscopes (LLSM) are "complex, expensive, and alignment intensive", I believe this sentence applies to the open-source version of LLSM, which was made available for wide dissemination. Since then, a commercial solution has been provided by 3i, which is now being used in multiple cores and labs but does require routine alignments. However, Zeiss has also released a commercial turn-key system, which, while expensive, is stable, and the complexity does not interfere with the experience of the user. Though in general, statements on ease of use and stability might be considered anecdotal and may not belong in a scientific article, unreferenced or without data.

(2) One of the major limitations of the first generation LLSM was the use of a 5 mm coverslip, which was a hinderance for many users. However, the Zeiss system elegantly solves this problem, and so does Oblique Plane Microscopy (OPM), while the Altair-LSFM retains this feature, which may dissuade widespread adoption. This limitation and how it may be overcome in future iterations is not discussed.

(3) Further, on the point of sample flexibility, all generations of the LLSM, and by the nature of its design, the OPM, can accommodate live-cell imaging with temperature, gas, and humidity control. It is unclear how this would be implemented with the current sample chamber. This limitation would severely limit use cases for cell biologists, for which this microscope is designed. There is no discussion on this limitation or how it may be overcome in future iterations.

(4) The authors' comparison to LLSM is constrained to the "square" lattice, which, as they point out, is the most used optical lattice (though this also might be considered anecdotal). The LLSM original design, however, goes far beyond the square lattice, including hexagonal lattices, the ability to do structured illumination, and greater flexibility in general in terms of light-sheet tuning for different experimental needs, as well as not being limited to just sample scanning. Thus, the Alstair-LSFM cannot compare to the original LLSM in terms of versatility, even if comparisons to the resolution provided by the square lattice are fair.

(5) There is no demonstration of the system's live-imaging capabilities or temporal resolution, which is the main advantage of existing light-sheet systems.

While the microscope is well designed and completely open source, it will require experience with optics, electronics, and microscopy to implement and align properly. Experience with custom machining or soliciting a machine shop is also necessary. Thus, in my opinion, it is unlikely to be implemented by a lab that has zero prior experience with custom optics or can hire someone who does. Altair-LSFM may not be as easily adaptable or implementable as the authors describe or perceive in any lab that is interested, even if they can afford it. The authors indicate they will offer "workshops," but this does not necessarily remove the barrier to entry or lower it, perhaps as significantly as the authors describe.

There is a claim that this design is easily adaptable. However, the requirement of custom-machined baseplates and in silico optimization of the optical path basically means that each new instrument is a new design, even if the Navigate software can be used. It is unclear how Altair-LSFM demonstrates a modular design that reduces times from conception to optimization compared to previous implementations.

Reviewer #2 (Public review):

In this study, the authors show that alterations in the lipid composition of the inner mitochondrial membrane, particularly changes in cardiolipin (CL) content, lead to defects in electron transport, supercomplex formation, and oxidative stress. Using liver-specific CLS knockout mice, which are characterized by dysfunctional capacity for cardiolipin synthesis, the authors highlight an underappreciated role for CL in MASH pathology. Overall, this is an interesting study highlighting the importance of functional/physiological electron transport (and in this context, electron leakage) in MASH pathophysiology. Despite that, this manuscript has several weaknesses that require attention.

(1) For all LKO studies, it is stated that the decrease in hepatic CL is causal for the observed phenotype. However, it is evident that many other lipids are impacted by CLS KO, including a marked increase in hepatic PG. In this respect, the authors show no evidence that the observed metabolic phenotype is indeed due to the reduction in CL and not to other accompanying changes.

(2) In the results, the authors highlight that 'MASLD has been shown to alter the total cellular lipidome in liver.' Given that this study focused on CL, it would be useful to include specific studies that pointed to changes in hepatic CL content in MASLD/MASH/fibrosis.

(3) The initial human mitochondrial lipidomics studies show a reduction in mitochondrial CL and PG content. What was the content/expression of CL synthase and PGP synthase in these samples? If this cannot be assessed, is there any association of CLS or PGPS expression and MASLD/fibrosis (etc) in publicly available databases (e.g, GEP liver) that may explain the reduction in mitochondrial PG and CL content?

(4) The validation of MASH in patients (Figure 1B) is not convincing (ie., no quantification/scoring provided). NAS /fibrosis scoring (according to Kleiner) would help to define if all patients have indeed MASH, and what subset has fibrosis. Could the reduction in CL/PG content be (also) associated with fibrosis? In addition, Masson's Trichrome should be added to Figure 1B.

(5) In human lipidomics, the authors suggest that reductions are observed in tetralinoleoyl CL (Figure 1C). However, Figure 1C only shows the combined FA acyl chain length + unsaturation, therefore not allowing for FA-specific ID (unless such data are available from the LC/MS analysis).

(6) Figures 1 J/K/I. It is obvious that the background in all murine immunoblotting analysis has been altered. The authors should provide unaltered images for these immunoblots.

(7) For Figure 1, it is unclear what is meant by 'we performed all mitochondrial lipidomic analyses by quantifying lipids per mg of mitochondrial proteins'. Was the murine lipidomics carried out on fractionated mitochondria or whole liver? If whole liver, then how were the data corrected, particularly given that PG is not a mitochondria-specific lipid?

(8) While total CL content seems indeed decreased across the different mouse models, this is mostly due to 1-2 CL species showing a pronounced reduction, with the remainder being unaltered. This should at least be acknowledged in the results. This is similarly the case in the LKO livers.

(9) Figure 2. A secondary biochemical analysis of changes in lipid content should be provided, e.g., total triglyceride content, particularly given that the histology analysis does not show any major changes in hepatic lipid droplets/steatosis. In addition, the Masson's Trichrome staining shows almost no collagen deposition.

(10) Figure 3. 'CLS deletion modestly reduced glucose handling' should be reworded. The LKO mice show improved glucose tolerance (despite the MASH phenotype), which is not evident from the above wording.

(11) Looking at the mechanism behind the increase in hepatic steatosis, the authors state that lipid accumulation can occur due to increased lipogenesis, or dysfunctional VLDL secretion or beta oxidation, and subsequently assessed the relevant proteins/pathways. What about fatty acid uptake, which is also one of the four major pathways impacted in MASLD? This should be included in this assessment in Figure 3.

(12) For Figure 5A, it is simply stated 'CLS deletion promotes liver fibrosis in standard chow-fed condition', and it is unclear what is highlighted within the selected EM images and what the arrows refer to. The authors should clarify this within the text.

Reviewer #2 (Public review):

Summary:

The manuscript by Zheng et al reports the structural and biochemical study of novel effectors from the bacterial pathogen Legionella pneumophila. The authors continued from results from their earlier screening for L. pneumophila proteins that affect host F-actin dynamics to show that Llfat1 (Lpg1387) interacts with actin via a novel actin-binding domain (ABD). The authors also determined the structure of the Lfat1 ABD-F-actin complex, which allowed them to develop this ABD as a probe for F-actin. Finally, the authors demonstrated that Llfat1 is a lysine fatty acyltransferase that targets several small GTPases in host cells.

Strengths:

This is a very complete work that shows the structure of a novel bacterial actin-binding protein in complex with F-actin, and the biochemical activity of the protein was also revealed. Overall, this is a very exciting study and should be of great interest to scientists in both bacterial pathogenesis and the actin cytoskeleton of eukaryotic cells.

Weaknesses:

(1) The authors should use biochemical reactions to analyze the KFAT of Llfat1 on one or two small GTPases shown to be modified by this effector in cellulo. Such reactions may allow them to determine the role of actin binding in its biochemical activity. This notion is particularly relevant in light of recent studies that actin is a co-factor for the activity of LnaB and Ceg14 (PMID: 39009586; PMID: 38776962; PMID: 40394005). In addition, the study should be discussed in the context of these recent findings on the role of actin in the activity of L. pneumophila effectors.

(2) The development of the ABD domain of Llfat1 as an F-actin domain is a nice extension of the biochemical and structural experiments. The authors need to compare the new probe to those currently commonly used ones, such as Lifeact, in labeling of the actin cytoskeleton structure.

Reviewer #2 (Public review):

Summary:

In this manuscript, Monziani et al. identified long noncoding RNAs (lncRNAs) that act in cis and are coregulated with their target genes located in close genomic proximity. The authors mined the GeneHancer database, and this analysis led to the identification of four lncRNA-target pairs. The authors decided to focus on lncRNA EPB41L4A-AS1.

They thoroughly characterised this lncRNA, demonstrating that it is located in the cytoplasm and the nuclei, and that its expression is altered in response to different stimuli. Furthermore, the authors showed that EPB41L4A-AS1 regulates EPB41L4A transcription, leading to a mild reduction in EPB41L4A protein levels. This was not recapitulated with siRNA-mediated depletion of EPB41L4AAS1. RNA-seq in EPB41L4A-AS1-depleted cells with single LNA revealed 2364 DEGs linked to pathways including the cell cycle, cell adhesion, and inflammatory response. To understand the mechanism of action of EPB41L4A-AS1, the authors mined the ENCODE eCLIP data and identified SUB1 as an lncRNA interactor. The authors also found that the loss of EPB41L4A-AS1 and SUB1 leads to the accumulation of snoRNAs, and that SUB1 localisation changes upon the loss of EPB41L4A-AS1. Finally, the authors showed that EPB41L4A-AS1 deficiency did not change the steady-state levels of SNORA13 nor RNA modification driven by this RNA. The phenotype associated with the loss of EPB41L4A-AS1 is linked to increased invasion and EMT gene signature.

Overall, this is an interesting and nicely done study on the versatile role of EPB41L4A-AS1 and the multifaceted interplay between SUB1 and this lncRNA, but some conclusions and claims need to be supported with additional experiments. My primary concerns are using a single LNA gapmer for critical experiments, increased invasion, and nucleolar distribution of SUB1- in EPB41L4A-AS1-depleted cells. These experiments need to be validated with orthogonal methods.

Strengths:

The authors used complementary tools to dissect the complex role of lncRNA EPB41L4A-AS1 in regulating EPB41L4A, which is highly commendable. There are few papers in the literature on lncRNAs at this standard. They employed LNA gapmers, siRNAs, CRISPRi/a, and exogenous overexpression of EPB41L4A-AS1 to demonstrate that the transcription of EPB41L4A-AS1 acts in cis to promote the expression of EPB41L4A by ensuring spatial proximity between the TAD boundary and the EPB41L4A promoter. At the same time, this lncRNA binds to SUB1 and regulates snoRNA expression and nucleolar biology. Overall, the manuscript is easy to read, and the figures are well presented. The methods are sound, and the expected standards are met.

Weaknesses:

The authors should clarify how many lncRNA-target pairs were included in the initial computational screen for cis-acting lncRNAs and why MCF7 was chosen as the cell line of choice. Most of the data uses a single LNA gapmer targeting EPB41L4A-AS1 lncRNA (eg, Fig. 2c, 3B, and RNA-seq), and the critical experiments should be using at least 2 LNA gapmers. The specificity of SUB1 CUT&RUN is lacking, as well as direct binding of SUB1 to lncRNA EPB41L4A-AS1, which should be confirmed by CLIP qPCR in MCF7 cells. Finally, the role of EPB41L4A-AS1 in SUB1 distribution (Figure 5) and cell invasion (Figure 8) needs to be complemented with additional experiments, which should finally demonstrate the role of this lncRNA in nucleolus and cancer-associated pathways. The use of MCF7 as a single cancer cell line is not ideal.

Reviewer #2:

The study of Rollenhagen et al examines the ultrastructural features of Layer 1 of human temporal cortex. The tissue was derived from drug-resistant epileptic patients undergoing surgery, and was selected as further from the epilepsy focus, and as such considered to be non-epileptic. The analyses has included 4 patients with different age, sex, medication and onset of epilepsy. The manuscript is a follow-on study with 3 previous publications from the same authors on different layers of the temporal cortex:

Layer 4 - Yakoubi et al 2019 eLife

Layer 5 - Yakoubi et al 2019 Cerebral Cortex,

Layer 6 - Schmuhl-Giesen et al 2022 Cerebral Cortex

They find, the L1 synaptic boutons mainly have single active zone a very large pool of synaptic vesicles and are mostly devoid of astrocytic coverage.

Strengths:

The MS is well written easy to read. Result section gives a detailed set of figures showing many morphological parameters of synaptic boutons and surrounding glial elements. The authors provide comparative data of all the layers examined by them so far in the Discussion. Given that anatomical data in human brain are still very limited, the current MS has substantial relevance. The work appears to be generally well done, the EM and EM tomography images are of very good quality. The analyses is clear and precise.

Weaknesses:

The authors made all the corrections required and answered all of my concerns, included additional data sets, and clarified statements where needed.

Reviewer #2 (Public review):

Summary:

A core task of the brain is processing sensory cues from the environment. The neural mechanisms of how sensory information is transmitted from peripheral sense organs to subsequent being processing in defined brain centers remains an important topic in neuroscience. The taste system hereby assesses the palatability of food by evaluating the chemical composition and nutrient content while integrating the current need of energy by assessing the satiation level of the organism. The current manuscript provides insights into the early circuits gustatory coding using the fruit fly as model. By combining trans-tango and FACS-based bulk RNAseq to assess the target neurons of sweet sensing (using by Gr64f-Gal4) and bitter sensing (using Gr66a-Gal4) in a first set of experiments the authors investigate genes that are differentially expressed or co-expressed in normal and starved conditions. With a focus on neuropeptides and neurotransmitters differential expression in the different conditions were assessed resulting in the identification of Leucokinin as potentially interesting gene. The notion is further supported by RNAseq of Lk-Gal4>mCD8:GFP sorted cells and immunostainings. GRASP and BacTrace experiments further supports that the two Lk expressing cells in the SEZ should indeed be postsynaptic to both type of sensors. Using EM-based connectomics data (based on a previous publication by Engert et al.), the authors also look for downstream targets of the bitter versus sweet gustatory neurons to identify the Lk-neurons. Based on morphology they identify candidates and further depict the potential downstream neurons in the connectome, which appears largely in agreement with GRASP experiments. Finally silencing the Lk-neurons shows an increased PER response in starved flies (when combined with bitter compounds) as well as increased feeding in a FlyPad assay.

Strengths:

Overall this is an intriguing manuscript, which provides insight into the organization of 2nd order gustatory neurons. It specifically provides strong evidence for the Lk-neurons as target of sweet and bitter GRNs and provides evidence for their role in regulating sweet vs bitter based behavioral responses. Particularly the integration of different techniques and datasets in an elegant fashion is a strong side of the manuscript. Moreover to put the known LK-neurons into the context of 2nd order gustatory signalling is strengthening the knowledge about this pathway.

Weaknesses:

I do not see any major weakness in the current manuscript. Novelty is to some degree lessened by the fact, that the RNAseq approach did not identify new neurons but rather put the known LK-neurons as major finding. Similarly the final behavioral section is not very deep and to some degree corroborates the previous publication by the Keene and Nässel labs- that said, the model they propose is indeed novel (but lacks depth in analyses, e.g. there is no physiology that would support the modulation of Lk neurons by either type of GRN). The connectomic section appears a bit out of place and after reading it it's not really clear what one should make of the potential downstream neurons (particularly since the Lk-receptor expression has been previously analyzed); here it might have been interesting to address if/how Lk-neurons may signal directly via a classical neurotransmitter (an information that might be found easily in the adult brain single-cell data).

Comments on the latest version:

I feel all points have been included to a satisfactory degree.

Reviewer #2 (Public review):

In this manuscript, Ridout et al. present an intriguing extension of beta cell mass-focused models for diabetes. Their model incorporates reversible glucose-dependent inactivation of beta cell mass, which can trigger sudden-onset hyperglycemia due to bistability in beta cell mass dynamics. Notably, this hyperglycemia can be reversed with insulin treatment. The model is simple, elegant, and thought-provoking.

Reviewer #2 (Public review):

Summary:

Argunsah and colleagues demonstrate that SST-expressing interneurons are concentrated in the mouse septa and differentially respond to repetitive multi-whisker inputs. Identifying how a specific neuronal phenotype impacts responses is an advance.

Strengths:

(1) Careful physiological and imaging studies.

(2) Novel result showing the role of SST+ neurons in shaping responses.

(3) Good use of a knockout animal to further the main hypothesis.

(4) Clear analytical techniques.

Weaknesses:

No major weaknesses were identified by this reviewer. Overall I appreciated the paper but feel it overlooked a few issues and had some recommendations on how additional clarifications could strengthen the paper. These include:

(1) Significant work from Jerry Chen on how S1 neurons that project to M1 versus S2 respond in a variety of behavioral tasks should be included (e.g. PMID: 26098757). Similarly, work from Barry Connor's lab on intracortical versus thalamocortical inputs to SST neurons, as well as excitatory inputs onto these neurons (e.g. PMID: 12815025) should be included.

(2) Using Layer 2/3 as a proxy to what is happening in layer 4 (~line 234). Given that layer 2/3 cells integrate information from multiple barrels, as well as receiving direct VPm thalamocortical input, and given the time window that is being looked at can receive input from other cortical locations, it is not clear that layer 2/3 is a proxy for what is happening in layer 4.

(3) Line 267, when discussing distinct temporal response, it is not well defined what this is referring to. Are the neurons no longer showing peaks to whisker stimulation, or are the responses lasting a longer time? It is unclear why PV+ interneurons which may not be impacted by the Elfn1 KO and receive strong thalamocortical inputs, are not constraining activity.

(4) Line 585 "the earliest CSD sink was identified as layer 4..." were post-hoc measurements made to determine where the different shank leads were based on the post-hoc histology?

(5) For the retrograde tracing studies, how were the M1 and S2 injections targeted (stereotaxically or physiologically)? How was it determined that the injections were in the whisker region (or not)?

(6) Were there any baseline differences in spontaneous activity in the speta versus barrel regions, and did this change in the KO animals?

Reviewer #2 (Public review):

Summary

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

Strengths

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

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

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

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

Weaknesses

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

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

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

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

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

Reviewer #2 (Public review):

This is an interesting study in Drosophila comparing potentially differential requirements for subsets of Kenyon Cells (KCs) and Dopaminergic neurons (DANS) in olfactory dishabituation driven by either a novel odor ("homosensory") or footshock ("heterosensory). The authors measure olfactory aversion to Octanol (OCT) in a T-maze, induce olfactory habituation with a 4-minute prior exposure to OCT, and use either brief yeast odor (YO) or footshock (FS) to achieve dishabituation. The major observation that YO-mediated dishabituation is mediated by reward-activated DANs (PAM cluster), while FS-mediated dishabituation is mediated by punishment-activated PPL-DANs is generally solid and convincing. Also convincing are experiments showing the involvement of KCs in the pathway for YO and FS-induced dishabituation, and the argument that KCs drive DAN activation that causes dishabituation, though not experimentally shown, is more than reasonable. The work is significant because, as the authors take pains to point out, circuits and pathways for dishabituation have been very lightly studied, and clear identification of dopaminergic neuron subsets in dishabituation achieved by different means represents unique and interesting progress.

However, the claim that this represents a fundamental difference between homosensory and heterosensory pathways for dishabituation is overstated. The introductory section does not adequately present current broad models for habituation and dishabituation. There are many different time scales, even for Drosophila olfactory habituation. These, as well as potential underlying mechanistic differences, need to be acknowledged; any claim should be specifically qualified for the time scales being studied here. Additionally, there are several unclear, vague, and inaccurate sections and statements. A more careful, precise, and considered presentation of current views, as well as more measured claims of the impact of the findings, would substantially enhance my enthusiasm.

Reviewer #2 (Public review):

Summary:

In this paper, entitled "SpikeMAP: An unsupervised spike sorting pipeline for cortical excitatory and inhibitory 2 neurons in high-density multielectrode arrays with ground-truth validation", the authors present spikeMAP, a pipeline for the analysis of large-scale recordings of in vitro cortical activity. According to the authors, spikeMAP not only allows for the detection of spikes produced by single neurons (spike sorting), but also allows for the reliable distinction between genetically determined cell types by utilizing viral and optogenetic strategies as ground-truth validation. While I find that the paper is nicely written and easy to follow, I find that the algorithmic part of the paper is not really new and should have been more carefully compared to existing solutions. While the GT recordings to assess the possibilities of a spike sorting tool to distinguish properly between excitatory and inhibitory neurons are interesting, spikeMAP does not seem to bring anything new to state-of-the-art solutions, and/or, at least, it would deserve to be properly benchmarked. I would suggest that the authors perform a more intensive comparison with existing spike sorters.

Strengths:

The GT recordings with optogenetic activation of the cells, based on the opsins, is interesting and might provide useful data to quantify how good spike sorting pipelines are, in vitro, to discriminate between excitatory and inhibitory neurons. Such an approach can be quite complementary to artificially generated ground truth.

Weaknesses:

(1) The global workflow of spikeMAP, described in Figure 1, seems to be very similar to that of Hilgen et al. 2020 (10.1016/j.celrep.2017.02.038). Therefore, the first question is what is the rationale of reinventing the wheel, and not using tools that are doing something very similar (as mentioned by the authors themselves). I have a hard time, in general, believing that spikeMAP has something particularly special, given its Methods, compared to state-of-the-art spike sorters. This is why, at the very least, the title of the paper is misleading, because it lets the reader think that the core of the paper will be about a new spike sorting pipeline. If this is the main message the authors want to convey, then I think that numerous validations/benchmarks are missing to assess first how good spikeMAP is, with reference to spike sorting in general, before deciding if this is indeed the right tool to discriminate excitatory vs inhibitory cells. The GT validation, while interesting, is not enough to entirely validate the paper. The details are a bit too scarce for me, or would deserve to be better explained (see other comments after).

(2) Regarding the putative location of the spikes, it has been shown that the center of mass, while easy to compute, is not the most accurate solution [Scopin et al, 2024, 10.1016/j.jneumeth.2024.110297]. For example, it has an intrinsic bias for finding positions within the boundaries of the electrodes, while some other methods, such as monopolar triangulation or grid-based convolution,n might have better performances. Can the authors comment on the choice of the Center of Mass as a unique way to triangulate the sources?

(3) Still in Figure 1, I am not sure I really see the point of Spline Interpolation. I see the point of such a smoothing, but the authors should demonstrate that it has a key impact on the distinction of Excitatory vs. Inhibitory cells. What is special about the value of 90kHz for a signal recorded at 18kHz? What is the gain with spline enhancement compared to without? Does such a value depend on the sampling rate, or is it a global optimum found by the authors?

(4) Figure 2 is not really clear, especially panel B. The choice of the time scale for the B panel might not be the most appropriate, and the legend filtered/unfiltered with a dot is not clear to me in Bii. In panel E, the authors are making two clusters with PCA projections on single waveforms. Does this mean that the PCA is only applied to the main waveforms, i.e. the ones obtained where the amplitudes are peaking the most? This is not really clear from the methods, but if this is the case, then this approach is a bit simplistic and does not really match state-of-the-art solutions. Spike waveforms are quite often, especially with such high-density arrays, covering multiple channels at once, and thus the extracellular patterns triggered by the single units on the MEA are spatio-temporal motifs occurring on several channels. This is why, in modern spike sorters, the information in a local neighbourhood is often kept to be projected, via PCA, on the lower-dimensional space before clustering. Information on a single channel only might not be informative enough to disambiguate sources. Can the authors comment on that, and what is the exact spatial resolution of the 3Brain device? The way the authors are performing the SVD should be clarified in the methods section. Is it on a single channel, and/or on multiple channels in a local neighbourhood?

(5) About the isolation of the single units, here again, I think the manuscript lacks some technical details. The authors are saying that they are using a k-means cluster analysis with k=2. This means that the authors are explicitly looking for 2 clusters per electrode? If so, this is a really strong assumption that should not be held in the context of spike sorting, because, since it is a blind source separation technique, one can not pre-determine in advance how many sources are present in the vicinity of a given electrode. While the illustration in Figure 2E is ok, there is no guarantee that one can not find more clusters, so why this choice of k=2? Again, this is why most modern spike sorting pipelines do not rely on k-means, to avoid any hard-coded number of clusters. Can the authors comment on that?

(6) I'm surprised by the linear decay of the maximal amplitude as a function of the distance from the soma, as shown in Figure 2H. Is it really what should be expected? Based on the properties of the extracellular media, shouldn't we expect a power law for the decay of the amplitude? This is strange that up to 100um away from the soma, the max amplitude only dropped from 260 to 240 uV. Can the authors comment on that? It would be interesting to plot that for all neurons recorded, in a normed manner V/max(V) as function of distances, to see what the curve looks like.

(7) In Figure 3A, it seems that the total number of cells is rather low for such a large number of electrodes. What are the quality criteria that are used to keep these cells? Did the authors exclude some cells from the analysis, and if yes, what are the quality criteria that are used to keep cells? If no criteria are used (because none are mentioned in the Methods), then how come so few cells are detected, and can the authors convince us that these neurons are indeed "clean" units (RPVs, SNRs, ...)?

(8) Still in Figure 3A, it looks like there is a bias to find inhibitory cells at the borders, since they do not appear to be uniformly distributed over the MEA. Can the authors comment on that? What would be the explanation for such a behaviour? It would be interesting to see some macroscopic quantities on Excitatory/Inhibitory cells, such as mean firing rates, averaged SNRs... Because again, in Figure 3C, it is not clear to me that the firing rates of inhibitory cells are higher than Excitatory ones, whilst they should be in theory.

(9) For Figure 3 in general, I would have performed an exhaustive comparison of putative cells found by spikeMAP and other sorters. More precisely, I think that to prove the point that spikeMAP is indeed bringing something new to the field of spike sorting, the authors should have compared the performances of various spike sorters to discriminate Exc vs Inh cells based on their ground truth recordings. For example, either using Kilosort [Pachitariu et al, 2024, 10.1038/s41592-024-02232-7], or some other sorters that might be working with such large high-density data [Yger et al, 2018, 10.7554/eLife.34518].

(10) Figure 4 has a big issue, and I guess the panels A and B should be redrawn. I don't understand what the red rectangle is displaying.

(11) I understand that Figure 4 is only one example, but I have a hard time understanding from the manuscript how many slices/mices were used to obtain the GT data? I guess the manuscript could be enhanced by turning the data into an open-access dataset, but then some clarification is needed. How many flashes/animals/slices are we talking about? Maybe this should be illustrated in Figure 4, if this figure is devoted to the introduction of the GT data.

(12) While there is no doubt that GT data as the ones recorded here by the authors are the most interesting data from a validation point of view, the pretty low yield of such experiments should not discourage the use of artificially generated recordings such as the ones made in [Buccino et al, 2020, 10.1007/s12021-020-09467-7] or even recently in [Laquitaine et al, 2024, 10.1101/2024.12.04.626805v1]. In these papers, the authors have putative waveforms/firing rate patterns for excitatory and inhibitory cells, and thus, the authors could test how good they are in discriminating the two subtypes.

Reviewer #2 (Public review):

Summary:

This work seeks to establish the Open Field Test (OFT) as a paradigm to measure emotion-like states in the fruit fly Drosophila. To do this, the authors first applied various stressors and aversive stimuli to wild-type flies and tracked their locomotion. By measuring wall-following (WAFO) and total walking (TOWA), they showed that these behaviors are generally increased by stressors, but return to baseline levels after their removal. Then, they used the same approach to analyze the effects of pharmacological, genetic, and neuronal activity manipulations, showing that diazepam, serotonin, dopamine, and neuropeptide F affect locomotion in the OFT in largely expected ways that are consistent with their functions in rodents. Finally, the authors demonstrate that wild-type fly strains from the laboratory or caught in the wild differ significantly in their OFT behavior, with wild-caught flies generally behaving as if more 'stressed'. Given the numerous advantages of Drosophila, this study can form the foundation for using the OFT in conjunction with this animal model to elucidate the molecular and neuronal mechanisms that underlie emotion primitives.

Strengths:

The main strength of the paper is the rigorous use of several stressful or aversive treatments and their subsequent removal to show that WAFO is a robust proxy for stress-like emotional primitives across multiple stimuli. The pharmacological, molecular, and neuronal activity manipulations, although more limited in scope, lend further credence to the authors' central claim.

Weaknesses:

The conceptual advance of this research is unclear, as previous work (Mohammad et al., 2016, Curr Biol.) carried out similar treatments and manipulations and reached largely similar conclusions. Moreover, while WAFO is a good proxy for 'stress', I am not convinced that TOWA necessarily represents an emotional state in all cases. Indeed, as the authors themselves acknowledge, changes in total walking may be associated with other factors, such as starvation-induced hyperactivity, physical exhaustion after sleep deprivation, increased sex drive after mating, alcohol sedation, etc. Another unclear point is the interpretation of some unexpected results, such as the finding that both serotonin transporter overexpression and its knockdown give the same phenotype. Finally, there are some issues with the use of the OFT in rodent research (e.g., inconsistent effects of anxiolytic drugs; see Rosso et al., 2022, Neurosci Biobehav Rev., for a meta-analysis). These should be explained to place the Drosophila findings in their appropriate context.

Reviewer #2 (Public review):

Summary

Roiuk et al describe a work in which they have investigated the role of eIF2A in translation initiation in mammals without much success. Thus, the manuscript focuses on negative results. Further, the results, while original, are generally not novel, but confirmatory, since related claims have been made before independently in different systems with Haikwad et al study recently published in eLife being the most relevant.

Despite this, we find this work highly important. This is because of a massive wealth of unreliable information and speculations regarding eIF2A role in translation arising from series of artifacts that began at the moment of eIF2A discovery. This, in combination with its misfortunate naming (eIF2A is often mixed up with alpha subunit of eIF2, eIF2S1) has generated a widespread confusion among researchers who are not experts in eukaryotic translation initiation. Given this, it is not only justifiable but critical to make independent efforts to clear up this confusion and I very much appreciate the authors' efforts in this regard.

Strengths

The experimental investigation described in this manuscript is thorough, appropriate and convincing.

Weaknesses

No major weaknesses as the authors have improved their presentation.

Reviewer #2 (Public review):

Sourmpis et al. present a study in which the importance of including certain inductive biases in the fitting of recurrent networks is evaluated with respect to the generalization ability of the networks when exposed to untrained perturbations.

The work proceeds in three stages:<br /> (1) a simple illustration of the problem is made. Two reference (ground-truth) networks with qualitatively different connectivity, but similar observable network dynamics, are constructed, and recurrent networks with varying aspects of design similarity to the reference networks are trained to reproduce the reference dynamics. The activity of these trained networks during untrained perturbations is then compared to the activity of the perturbed reference networks. It is shown that, of the design characteristics that were varied, the enforced sign (Dale's law) and locality (spatial extent) of efference were especially important.<br /> (2) The intuition from the constructed example is then extended to networks that have been trained to reproduce certain aspects of multi-region neural activity recorded from mice during a detection task with a working-memory component. A similar pattern is demonstrated, in which enforcing the sign and locality of efference in the fitted networks has an influence on the ability of the trained networks to predict aspects of neural activity during unseen (untrained) perturbations.<br /> (3) The authors then illustrate the relationship between the gradient of the motor readout of trained networks with respect to the net inputs to the network units, and the sensitivity of the motor readout to small perturbations of the input currents to the units, which (in vivo) could be controlled optogenetically. The paper is concluded with a proposed use for trained networks, in which the models could be analyzed to determine the most sensitive directions of the network and, during online monitoring, inform a targeted optogenetic perturbation to bias behavior.

The authors do not overstate their claims, and in general, I find that I agree with their conclusions. A couple of points to be made:

(1) Some aspects of the methods are unclear. For comparisons between recurrent networks trained from randomly initialized weights, I would expect that many initializations were made for each model variant to be compared, and that the performance characteristics are constructed by aggregating over networks trained from multiple random initializations. I could not tell from the methods whether this was done or how many models were aggregated.

2) It is possible that including perturbation trials in the training sets would improve model performance across conditions, including held-out (untrained) perturbations (for instance, to units that had not been perturbed during training). It could be noted that if perturbations are available, their use may alleviate some of the design decisions that are evaluated here.

Reviewer #2 (Public review):

Summary:

The manuscript presents valuable new datasets from two ancient maize seeds that contribute to our growing understanding of the maize evolution and biodiversity landscape in pre-colonial South America. Some of the analyses are robust, but the selection elements are not supported.

Strengths:

The data collection is robust, and the data appear to beof sufficiently high quality to carry out some interesting analytical procedures. The central finding that aBM maize is closely related to maize from the core Inca region is well supported, although the directionality of dispersal is not supported.

Weaknesses:

The selection results are not justified, see examples in the detailed comments below.

(1) The manuscript mentions cultural and natural selection (line 76), but then only gives a couple of examples of selecting for culinary/use traits. There are many examples of selection to tolerate diverse environments that could be relevant for this discussion, if desired.

(2) I would be extremely cautious about interpreting the observations of a Spanish colonizer (lines 95-99) without very significant caveats. Indigenous agriculture and foodways would have been far more nuanced than what could be captured in this context, and the genocidal activities of the Europeans would have impacted food production activities to a degree, and any contemporaneous accounts need to be understood through that lens.

(3) The f3 stats presented in Figure 2 are not set up to test any specific admixture scenarios, so it is unsupported to conclude that the aBM maize is not admixed on this basis (lines 201-202). The original f3 publication (Patterson et al, 2012) describes some scenarios where f3 characteristics associate with admixture, but in general, there are many caveats to this approach, and it's not the ideal tool for admixture testing, compared with e.g., f4 and D (abba-baba) statistics.

(4) I'm a little bit skeptical that the Locator method adds value here, given the small training sample size and the wide geographic spread and genetic diversity of the ancient samples that include Central America. The paper describing that method (Battey et al 2020 eLife) uses much larger datasets, and while the authors do not specifically advise on sample sizes, they caution about small sample size issues. We have already seen that the ancient Peruvian maize has the most shared drift with aBM maize on the basis of the f3 stats, and the Locator analysis seems to just be reiterating that. I would advise against putting any additional weight on the Locator results as far as geographic origins, and personally I would skip this analysis in this case.

(5) The overlap in PCA should not be used to confirm that aBM is authentically ancient, because with proper data handling, PCA placement should be agnostic to modern/ancient status (see lines 224-226). It is somewhat unexpected that the ancient Tehuacan maize (with a major teosinte genomic component) falls near the ancient South American maize, but this could be an artifact of sampling throughout the PCA and the lack of teosinte samples that might attract that individual.

(6) What has been established (lines 250-251) is genetic similarity to the Inca core area, not necessarily the directionality. Might aBM have been part of a cultural region supplying maize to the Inca core region, for example? Without a specific test of dispersal directionality, which I don't think is possible with the data at hand, this is somewhat speculative.

(7) Singleton SNPs are not a typical criterion for identifying selection; this method needs some citations supporting the exact approach and validation against neutral expectations (line 278). Without Datasets S2 and S3, which are not included with this submission, it is difficult to assess this result further. However, it is very unexpected that ~18,000 out of ~49,000 SNPs would be unique to the aBM lineage. This most likely reflects some data artifact (unaccounted damage, paralogs not treated for high coverage, which are extremely prevalent in maize, etc). I'm confused about unique SNPs in this context. How can they be unique to the aBM lineage if the SNPs used overlap the Grzybowski set? The GO results do not include any details of the exact method used or a statistical assessment of the results. It is not clear if the GO terms noted are statistically enriched.

(8) The use of XP-EHH with pseudohaplotype variant calls is not viable (line 293). It is not clear what exact implementation of XP-EHH was used, but this method generally relies on phased or sometimes unphased diploid genotype calls to observe shared haplotypes, and some minimum population size to derive statistical power. No implementation of XP-EHH to my knowledge is appropriate for application to this kind of dataset.

Reviewer #2 (Public review):

Summary:

In this article, Assimopoulos et al. expand the FSL-XTRACT software to include new protocols for identifying cortical-subcortical tracts with diffusion MRI, with a focus on tracts connecting to the amygdala and striatum. They show that the amygdalofugal pathway and divisions of the striatal bundle/external capsule can be successfully reconstructed in both macaques and humans while preserving large-scale topographic features previously defined in tract tracing studies. The authors set out to create an automated subcortical tractography protocol, and they accomplished this for a subset of specific subcortical connections for users of the FSL ecosystem.

Strengths:

A main strength of the current study is the translation of established anatomical knowledge to a tractography protocol for delineating cortical-subcortical tracts that are difficult to reconstruct. Diffusion MRI-based tractography is highly prone to false positives; thus, constraining tractography outputs by known anatomical priors is important. Key additional strengths include 1) the creation of a protocol that can be applied to both macaque and human data; 2) demonstration that the protocol can be applied to be high quality data (3 shells, > 250 directions, 1.25 mm isotropic, 55 minutes) and lower quality data (2 shells, 100 directions, 2 mm isotropic, 6.5 minutes); and 3) validation that the anatomy of cortical-subcortical tracts derived from the new method are more similar in monozygotic twins than in siblings and unrelated individuals.

Weaknesses:

Although this work validates the general organizational location and topographic organization of tractography-derived cortical-subcortical tracts against prior tract tracing studies (a clear strength), the validation is purely visual and thus only qualitative. Furthermore, it is difficult to assess how the current XTRACT method may compare to currently available tractography approaches to delineating similar cortical-subcortical connections. Finally, it appears that the cortical-subcortical tractography protocols developed here can only be used via FSL-XTRACT (yet not with other dMRI software), somewhat limiting the overall accessibility of the method.

Overall Appraisal:

This new method will accelerate research on anatomically validated cortical-subcortical white matter pathways. The work has utility for diffusion MRI researchers across fields.

Reviewer #2 (Public review):

The authors have revised their manuscript in response to reviewer feedback, incorporating several modifications to improve clarity and provide additional supporting information. To address concerns about confusing terminology, they have standardized the reference to PRDM16 overexpressing cells as Prdm16_OE, clarifying its expression from a constitutive promoter. They also revised the text to resolve seemingly contradictory statements about ChP development in the mutant. New bioinformatic analysis comparing PRDM16 binding in E12.5 ChP cells to co-repressed versus BMP-only-repressed genes has been performed and included in Supplementary Figure 5C, providing a statistical assessment of PRDM16's regulatory role on co-repressed genes. Several figures were updated, including adding an illustration of the Prdm16 cGT allele to Figure 1B, providing a zoomed-in inset for Figure 1E, and including individual channels for Wnt2b and marking boundaries in Figure 7A. Full-view images and examples of spot segmentation for SCRINSHOT analysis are now available in a new supplementary figure, and the presentation of RT-qPCR data in Supplementary Figure 2B was improved by using separate graphs for overexpression samples to avoid a broken Y-axis. Furthermore, the authors have added more references to introductory statements, annotated structures like the ChP, CH, and fourth ventricle in figures, and clarified that the beta-Gal signal was used as a marker for mutant ChP cells in Figure 1D. Finally, the manuscript now includes a discussion of the recently published, related study by Hurwitz et al. (2023) in the discussion section, highlighting similarities and differences. Overall, the authors have satisfactorily addressed the reviewers' comments.

Reviewer #2 (Public review):

The revised manuscript by Genzoni et al. reports the striking discovery of a regulatory role for trophic eggs. Prior to this study, trophic eggs were widely assumed to play a nutritional role in the colony, but this study shows that trophic eggs can suppress queen development, and therefore, can play a role in regulating caste determination in specific social contexts. In this revised version of the manuscript, the authors have addressed many of the concerns raised in the first version regarding the lack of sufficient information and context in the Introduction and Discussion.

Reviewer #3 (Public review):

Zhao et al. provide new insights into the mechanism by which a high-fat diet (HFD) induces cardiac arrhythmia employing Drosophila as a model. HFD induces cardiac arrhythmia in both mammals and Drosophila. Both glucagon and its functional equivalent in Drosophila Akh are known to induce arrhythmia. The study demonstrates that Akh mRNA levels are increased by HFD and both Akh and its receptor are necessary for high-fat diet-induced cardiac arrhythmia, elucidating a novel link. Notably, Zhao et al. identify a pair of AKH receptor-expressing neurons located at the posterior of the heart tube. Interestingly, these neurons innervate the heart muscle and form synaptic connections, implying their roles in controlling the heart muscle. The study presented by Zhao et al. is intriguing, and the rigorous characterization of the AKH receptor-expressing neurons would significantly enhance our understanding of the molecular mechanism underlying HFD-induced cardiac arrhythmia.

Many experiments presented in the manuscript are appropriate for supporting the conclusions while additional controls and precise quantifications should help strengthen the authors' arguments. The key results obtained by loss of Akh (or AkhR) and genetic elimination of the identified AkhR-expressing cardiac neurons do not reconcile, complicating the overall interpretation.

The most exciting result is the identification of AkhR-expressing neurons located at the posterior part of the heart tube (ACNs). The authors attempted to determine the function of ACNs by expressing rpr with AkhR-GAL4, which would induce cell death in all AkhR-expressing cells, including ACNs. The experiments presented in Figure 6 are not straightforward to interpret. Moreover, the conclusion contradicts the main hypothesis that elevated Akh is the basis of HFD-induced arrhythmia. The results suggest the importance of AkhR-expressing cells for normal heartbeat. However, elimination of Akh or AkhR restores normal rhythm in HFD-fed animals, suggesting that Akh and AkhR are not important for maintaining normal rhythms. If Akh signaling in ACNs is key for HFD-induced arrhythmia, genetic elimination of ACNs should unalter rhythm and rescue the HFD-induced arrhythmia. An important caveat is that the experiments do not test the specific role of ACNs. ACNs should be just a small part of the cells expressing AkhR. Specific manipulation of ACNs will significantly improve the study. Moreover, the main hypothesis suggests that HFD may alter the activity of ACNs in a manner dependent on Akh and AkhR. Testing how HFD changes calcium, possibly by CaLexA (Figure 2) and/or GCaMP, in wild-type and AkhR mutant could be a way to connect ACNs to HFD-induced arrhythmia. Moreover, optogenetic manipulation of ACNs may allow for specific manipulation of ACNs.

Interestingly, expressing rpr with AkhR-GAL4 was insufficient to eliminate both ACNs. It is not clear why it didn't eliminate both ACNs. Given the incomplete penetrance, appropriate quantifications should be helpful. Additionally, the impact on other AhkR-expressing cells should be assessed. Adding more copies of UAS-rpr, AkhR-GAL4, or both may eliminate all ACNs and other AkhR-expressing cells. The authors could also try UAS-hid instead of UAS-rpr.

Reviewer #2 (Public review):

This study investigates the role of arginase-II (Arg-II) in cardiac aging. The authors challenge previous assumptions by demonstrating that Arg-II is not expressed in aged cardiomyocytes, but is upregulated in non-myocyte cells, specifically macrophages, fibroblasts, and endothelial cells. Using Arg-II knockout mice, they show protection against age-associated cardiac inflammation, fibrosis, apoptosis, endothelial-to-mesenchymal transition (EndMT), and ischemic injury. Mechanistically, Arg-II promotes IL-1β release from macrophages and increases mitochondrial ROS in fibroblasts, contributing to cardiac aging through both cell-autonomous and non-cell-autonomous mechanisms.

The study is well-structured and combines genetic models, molecular assays, and histological analyses to support its conclusions. Including both human and mouse samples strengthens the translational relevance of the findings. The authors have addressed most of the reviewers' comments and have made efforts to improve the manuscript by adding experimental data, explanations, and further discussion.

The data convincingly support their conclusions. This work provides valuable insights into the mechanisms of cardiac aging, aligns with growing evidence of non-cell-autonomous contributions to aging-related pathologies, and highlights the importance of intercellular signaling in maintaining cardiac health during aging.

Although the use of cell-specific knockout mouse models would enhance the depth and translational potential of the findings, it is understandable that such an approach would be beyond the scope of a single study. This work lays the groundwork for future investigations into conditional Arg-II knockouts in specific cell types to elucidate the cell-specific roles of Arg-II in cardiac aging.

Overall, this is a solid and impactful study with strong experimental support

Reviewer #2 (Public review):

In this manuscript by Wolfson et al., various adeno-associated viruses (AAVs) were delivered to mice to assess the cardiac-specificity, injury border-zone cardiomyocyte transduction rate, and temporal dynamics, with the goal of finding better AAVs for gene therapies targeting the heart. The authors delivered tissue regeneration enhancer elements (TREEs) controlling luciferase expression and used IVIS imaging to examine transduction in the heart and other organs. They found that luciferase expression increased in the first week after injury when using AAV9-TREE-Hsp68 promoter, waning to baseline levels by 7 weeks. However, AAV9 vectors transduced the liver, which was significantly reduced by using an AAV.cc84 liver de-targeting capsid. The authors then performed in vivo screening of AAV9 capsids and found AAV-IR41 to preferentially transduce injured myocardium when compared to AAV9. Finally, the authors combined TREEs with AAV-IR41 to show improved luciferase expression compared to AAV9-TREE at 7, 14, and 21 days after injury.

Overall, this manuscript provides insights into TREE expression dynamics when paired with various heart-targeting capsids, which can be useful for researchers studying ischemic injury of murine hearts. While the authors have shown the success of using AAV9-TREEs in porcine hearts, it is unknown whether the expression dynamics would be similar in pigs or humans, as mentioned in the limitations.

The following questions and concerns can be addressed to improve the manuscript:

(1) From the IVIS data, it seems that the Hsp68 promoter might not be "normally silent in mouse tissues," specifically in the liver (Figure S1B). Are there any other promoters that can be combined with TREEs to induce cardiac-injury specific expression while minimizing liver expression? This could simplify capsid design to focus on delivery to injured areas.

(2) Why is it that AAV9-TREE-Hsp68-Luc wane in expression (Figure 1C and 1D), whereas AAV.cc84-TREE-Hsp68-Luc expresses stably for over 2 months (3E)? This has important implications for the goal of transience in gene delivery.

(3) AAV-IR41 was found to transduce cardiomyocytes in the injured zone. However, this capsid also shows a very strong off-target liver expression. From a capsid design perspective, is it possible to combine AAV-cc84 and AAV-IR41?

(4) It would be helpful to see immunostaining for the various time points in Figure 5. Is it possible to use an anti-luciferase antibody (or AAV-TREE-Hsp68-eGFP) to compare the two TREE capsids?

Reviewer #2 (Public review):

Summary:

The manuscript "Independent validation of transgenerational inheritance of learned pathogen avoidance in C. elegans" by Akinosho and Vidal-Gadea offers evidence that learned avoidance of the pathogen PA14 can be inherited for at least two generations. In spite of initial preference for the pathogen when exposed in a 'training session', 24 hours of feeding on this pathogen evoked avoidance. The data are robust, replicated in 4 trials, and the authors note that diminished avoidance is inherited in generations F1 and F2.

Strengths:

These results contrast with those reported by Gainey et al, who only observed intergenerational inheritance for a single generation. Although the authors' study does not explain why Gainey et el fail to reproduce the Murphy lab results, one possibility is that a difference in a media ingredient could be responsible.

Weaknesses:

The authors do not list the sources of their media ingredients, which might be important with regard to reproducibility.

Reviewer #2 (Public review):

Summary:

This study presents a useful finding that the high susceptibility to CLP sepsis of Kit-mutant mice is not due to mast cell deficiency, but to dysbiosis.

However, the present data are insufficient and incomplete to support the conclusion, and would benefit from more rigorous approaches. With the mechanism part strengthened, this paper would be of interest to researchers on mast cell biology and mucosal immunology.

Recommendations:

(1) The authors showed that E. coli increases in the cecum of Kit-mutant mice, which causes high CLP susceptibility. However, they did not provide any evidence E. coli is responsible for the high susceptibility. In the Figure 3 experiments, the authors administered the same number of cecal bacteria and did not show the number of E. coli after the administration. The authors should provide evidence showing that depletion of E. coli decreases susceptibility.

(2) The author should provide direct evidence of dysbiosis by, for example, shotgun sequencing of cecal and fecal contents.

(3) In case the authors find dysbiosis, they should analyze the mechanisms by which Kit mutation causes dysbiosis.

Reviewer #2 (Public review):

The major conclusion of the manuscript is expressed in the title: "NR2F2 is required in the embryonic testis for Fetal Leydig Cell development" and also at the end of the introduction and all along the result part. All the authors' assertions are supported by very clear and statistically validated results from ISH, IHC, precise cell counting and gene expression levels by qPCR. The authors used two different conditional Nr2f2 gene ablation systems that demonstrate the same effects at the FLC level. They also showed that the haplo-insufficiency of Wt1 in the first system (knock-in Wt1-cre-ERT2) aggravated the situation in FLC differentiation by disturbing the differentiation of Sertoli cells and their secretion of pro-FLC factors, which had a confounding effect and encouraged them to use the second system. This demonstrates the great rigor with which the authors interpreted the results. In conclusion, all authors' claims and conclusions are justified by their high-quality results.

Comments on revised version:

In their revised version, the authors have taken full account of all my suggestions, and I congratulate them on this. I have no further comments to make on this new version.

Reviewer #2 (Public review):

Summary:

The authors tested the efficiency of a model combining Pavlovian fear valuation and instrumental valuation. This model is amenable to many behavioral decision and learning setups - some of which have been or will be designed to test differences in patients with mental disorders (e.g., anxiety disorder, OCD, etc.).

Strengths:

(1) Simplicity of the model which can at the same time model rather complex environments.

(2) Introduction of a flexible omega parameter.

(3) Direct application to a rather advanced VR task.

(4) The paper is extremely well written. It was a joy to read.

Weaknesses:

Almost none! In very few cases, the explanations could be a bit better.

Comments on revised version:

No further comments.

Reviewer #1 (Public review):

Summary:

In this manuscript, Azlan et al. identified a novel maternal factor called Sakura that is required for proper oogenesis in Drosophila. They showed that Sakura is specifically expressed in the female germline cells. Consistent with its expression pattern, Sakura functioned autonomously in germline cells to ensure proper oogenesis. In sakura KO flies, germline cells were lost during early oogenesis and often became tumorous before degenerating by apoptosis. In these tumorous germ cells, piRNA production was defective and many transposons were derepressed. Interestingly, Smad signaling, a critical signaling pathway for the GSC maintenance, was abolished in sakura KO germline stem cells, resulting in ectopic expression of Bam in whole germline cells in the tumorous germline. A recent study reported that Bam acts together with the deubiquitinase Otu to stabilize Cyc A. In the absence of sakura, Cyc A was upregulated in tumorous germline cells in the germarium. Furthermore, the authors showed that Sakura co-immunoprecipitated Otu in ovarian extracts. A series of in vitro assays suggested that the Otu (1-339 aa) and Sakura (1-49 aa) are sufficient for their direct interaction. Finally, the authors demonstrated that the loss of otu phenocopies the loss of sakura, supporting their idea that Sakura plays a role in germ cell maintenance and differentiation through interaction with Otu during oogenesis.

Strengths:

To my knowledge, this is the first characterization of the role of CG14545 genes. Each experiment seems to be well-designed and adequately controlled

Weaknesses:

However, the conclusions from each experiment are somewhat separate, and the functional relationships between Sakura's functions are not well established. In other words, although the loss of Sakura in the germline causes pleiotropic effects, the cause-and-effect relationships between the individual defects remain unclear.

Comments on latest version:

The authors have attempted to address my initial concerns with additional experiments and refutations. Unfortunately, my concerns, especially my specific comments 1-3, remain unaddressed. The present manuscript is descriptive and fails to describe the molecular mechanism by which Sakura exerts its function in the germline. Nevertheless, this reviewer acknowledges that the observed defects in sakura mutant ovaries and the possible physiological significance of the Sakura-Out interaction are worth sharing with the research community, as they may lay the groundwork for future research in functional analysis.

Reviewer #2 (Public review):

Summary:

The authors aimed to determine whether a cryptic pocket in the VP35 protein of Zaire ebolavirus has a functional role in RNA binding and, by extension, in immune evasion. They sought to address whether this pocket could be an effective therapeutic target resistant to evolutionary evasion by studying its role in dsRNA binding among different filovirus VP35 homologs. Through simulations and experiments, they demonstrated that cryptic pocket dynamics modulate the RNA binding modes, directly influencing how VP35 variants block RIG-I and MDA5-mediated immune responses.<br /> The authors successfully achieved their aim, showing that the cryptic pocket is not a random structural feature but rather an allosteric regulator of dsRNA binding. Their results not only explain functional differences in VP35 homologs despite their structural similarity but also suggest that targeting this cryptic pocket may offer a viable strategy for drug development with reduced risk of resistance.

This work represents a significant advance in the field of viral immunoevasion and therapeutic targeting of traditionally "undruggable" protein features. By demonstrating the functional relevance of cryptic pockets, the study challenges long-standing assumptions and provides a compelling basis for exploring new drug discovery strategies targeting these previously overlooked regions.

Strengths:

The combination of molecular simulations and experimental approaches is a major strength, enabling the authors to connect structural dynamics with functional outcomes. The use of homologous VP35 proteins from different filoviruses strengthens the study's generality, and the incorporation of point mutations adds mechanistic depth. Furthermore, the ability to reconcile functional differences that could not be explained by crystal structures alone highlights the utility of dynamic studies in uncovering hidden allosteric features.

Weaknesses:

While the methodology is robust, certain limitations should be acknowledged. For example, the study would benefit from a more detailed quantitative analysis of how specific mutations impact RNA binding and cryptic pocket dynamics, as this could provide greater mechanistic insight. This study would also benefit from providing a clear rationale for the selection of the amber03 force field and considering the inclusion of volume-based approaches for pocket analysis. Such revisions will strengthen the robustness and impact of the study.

Comments on revisions:

The authors addressed the concerns raised.

Reviewer #2 (Public review):

Summary:<br /> This manuscript describes the use of quantitative imaging approaches, that have been a key element of the labs work over the past years, to address one of the major unresolved discussions in trafficking: intra-Golgi transport. The approach used has been clearly described in the labs previous papers, and is thus clearly described. The authors clearly address the weaknesses in this manuscript, and do not overstate the conclusions drawn from the data. The only weakness not addressed is the concept of blocking COPI transport with BFA, which is a strong inhibitor and causes general disruption of the system. This is an interesting element of the paper, which I think could be improved upon by using more specific COPI inhibitors instead, although I understand that this is not necessarily straightforward.

I commend the authors on their clear and precise presentation of this body of work, incorporating mathematical modelling with a fundamental question in cell biology. In all, I think that this is a very robust body of work, that provides a sound conclusion in support of the stable compartment model for the Golgi.

General points:<br /> The manuscript contains a lot of background in its results sections, and the authors may wish to consider rebalancing the text: The section beginning at Line 175 is about 90% background and 10% data. Could some data currently in supplementary be included here to redress this balance, or this part combined with another?

Minor points:<br /> Equation 2: A should be in front of the ln2. It's already resolved in equation 3, so likely only needs changing in the text

Line 152: Why is there a lack of experimental data? High ER background and low golgi signal make it difficult to select ministacks: would be good to see examples of these images. Is 0 a relevant timepoint as cargo is still at the ER? Instead would a timepoint <5' be better demonstrate initial arrival in fast cargo, and 0' discarded?

Table 1 Line 474: 1-3 independent replicates: is there a better way of incorporating this into the table to make it more streamlined? It would be useful to see each cargo as a mean with error. Is there a more demonstrative way to present the table, for example (but does not have to be) fastest cargo first (Tintra) as in Table 2?

Line 264 / Fig 3B: It's unclear to me why the VHH-anti-GFP-mCherry internalisation approach was used, when the cells were expressing GFP, that could be used for imaging. Also, this introduces a question over trafficking of the VHH itself, to access the same compartments as the GFP-proteins are localised. It would be useful to describe the choice of this approach briefly in the text.

446 Typo "internalization"

Post-Revision

I thank the authors for their work revising the paper in light of our comments. I am satisfied with their response, and I have no other comments.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors investigated the interactions between IRE and unfolded peptides using all-atom molecular dynamics simulations. The interactions between a couple of unfolded peptides and IRE might shed light on the activation of the UPR.

Strengths:

(1) Well-written manuscript tailored for a biology audience.

(2) State-of-the-art structural predictions and all-atom simulations.

(3) Validation with existing experimental data

(4) Clear schematic diagram summarizing the mechanisms learned from simulations.

(5) Shared simulation data and code in a public repository.

Weaknesses:

(1) Improving presentation to include more computational details.

(2) More quantitative analysis in addition to visual structures.

Reviewer #2 (Public review):

Summary:

In this work, the authors take a holistic view of Drosophila immunity by selecting four major components of fly immunity often studied separately (Toll signaling, Imd signaling, phagocytosis, and melanization), and studying their combinatory effects on the efficiency of the immune response. They achieve this by using fly lines mutant for one of these components, or modules, as well as for a combination of them, and testing the survival of these flies upon infection with a plethora of pathogens (bacterial, viral, and fungal).

Strengths:

It is clear that this manuscript has required a large amount of hands-on work, considering the number of pathogens, mutations, and timepoints tested. In my opinion, this work is a very welcome addition to the literature on fly immune responses, which obviously do not occur in one type of response at a time, but in parallel, subsequently, and/or are interconnected. I find that the major strength of this work is the overall concept, which is made possible by the mutations designed to target the specific immune function of each module (at least seemingly) without major effects on other functions. I believe that the combinatory mutants will be of use for the fly community and enable further studies of the interplay of these components of immune response in various settings.

To control for the effects arising from the genetic variation other than the intended mutations, the mutants have been backcrossed into a widely used, isogenized Drosophila strain called w1118. Therefore, the differences accounted for by the genotype are controlled.

I also appreciate that the authors have investigated the two possible ways of dealing with an infection: tolerance and resistance, and how the modules play into those.

Weaknesses:

While controlling for the background effects is vital, the w1118 background is problematic (an issue not limited to this manuscript) because of the wide effects of the white mutation on several phenotypes (also other than eye color/eyesight). It is a possibility that the mutation influences the functionality of the immune response components, for example, via effects of the faulty tryptophan handling on the metabolism of the animal.

I acknowledge that it is not reasonable to ask for data in different backgrounds better representing a "wild type" fly (however, that is defined is another question), but I think this matter should be brought up and discussed.

The whole study has been conducted on male flies. Immune responses show quite extensive sex-specific variation across a variety of species studied, also in the fly. But the reasons for this variation are not fully understood. Therefore, I suggest that the authors conduct a subset of experiments on female flies to see if the findings apply to both sexes, especially the infection-specificity of the module combinations.

Reviewer #2 (Public review):

Summary:

The manuscript is important due to the significance of the findings. The strength of evidence is convincing.

Strengths:

(1) Using a Novel SHERLOCK4AAT toolkit for diagnosis.

(2) Identification of various sub-species of Trypanosomes.

(3) Differentiating the animal subspecies from the human one.

Weaknesses:

(1) The title is too long, and the use of definite articles should be reduced in the title.

(2) The route of blood sample collection in the animals should be well defined and explained.

Reviewer #2 (Public review):

Summary:

Tibial nerve (electrical) stimulation (TNS) has emerged over the past 15 years as a non-invasive method to treat bladder overactivity, but interestingly, new animal work has suggested that TNS could actually be used to excite the bladder when appropriately tuning the stimulation frequency, effectively inverting its effect, perhaps opening the door to treat different conditions (e.g., UAB). The present study tests how healthy people respond to low and high frequency TNS, with the authors showing that they can substantially delay people's first sensation of bladder fullness with high frequencies (20Hz, shown many times before) but also that they can slightly hasten people's first sensation with low frequencies (1Hz, new result in humans). Moreover, the authors develop a computational model of interconnected conductance-based simulated neurons arranged in a physiologically plausible circuit that reproduces some aspects of the frequency-dependent effects of TNS. Their simulations suggest that we might expect low-frequency TNS to also increase the duration of bladder contractions in humans. The study highlights a potential new research direction, optimizing TNS stimulation parameters to increase basal bladder excitability.

Strengths:

The main strength of the work is to call attention to a new possibility of inverting the effect of TNS in humans by manipulating stimulation frequency, opening new indications for the therapy. This is highly relevant because of the recent popularity of TNS and its non-invasiveness, which lends itself to rapid testing and evaluation for new conditions and a high willingness to adopt. The authors convincingly demonstrate a modest excitatory effect on bladder sensation with low-frequency TNS, which clearly warrants further investigation.

The high-level design of the hypotheses, concepts, and experiments is clearly articulated in both the methods and in particularly clear diagrams, letting the reader focus their attention on the most important findings.

It is rare to develop a new computational model of the lower urinary tract at a systems level, and even more so for it to incorporate circuits in the spinal cord and brainstem centers, and this work undoubtedly advances the field's ability to engineer such systems. Further, because the model is comprised of linked conductance-based point-neurons, it is an excellent tool to investigate how an arguably plausible wiring diagram for neural control of the LUT could result in stimulation frequency-dependent effects on pelvic efferents. It is a proof of concept demonstrating how their mechanistic hypothesis of TNS could be implemented neurophysiologically by the nervous system.

Weaknesses:

The main drawback of the work is the frequent overinterpretation of the results. The human study and computational model are both proof-of-principle studies because the experimental effect size and sample size are modest, and the computational model is poorly validated and does not generate physiologically typical cystometric responses in simulations that are designed to recapitulate nominal LUT behavior.

Despite the stated caveats about the small effect in the human study, it should be emphasized throughout that this result is most reasonably interpreted as showing the possibility that TNS can have a low-frequency excitatory effect that merits follow-up, rather than a conclusive demonstration. The effect size is small (as the authors note) and should be placed in context with some minimally clinically important difference, if possible. The result is statistically significant, but even this may be subject to revision due to the small sample and the effect of post-hoc outlier removal and data analysis choices.

Given the apparent mismatch between the model and the cystometric behavior at the systems level in the "normal" case (e.g., low capacity, low voiding efficiency, omitted pressure profiles, frequency, etc.) and the absence of quantitative model validation (e.g., it was not compared directly with any experimental data from human urodynamics or rodent cystometry, beyond the initial fit to the neural data, no sensitivity analyses were performed, no goodness of fit computed, etc.) the discussion should be much more circumspect about interpreting the results at a systems level and should probably contain a paragraph explicitly detailing the limitations of the model. The subsequent interpretation should focus narrowly on the neural circuitry, rather than things like contraction duration, where the model is at its strongest. As written, the authors over-interpret what the in silico study can reasonably be used to infer about LUT function.

More justification is needed for why the contraction duration of the model is the central focus of analysis, when it connects only tentatively to the human study results, which focus on urgency. While not necessarily incorrect, a clearer link or motivation should be offered for how this informs our understanding of frequency-dependent TNS afferent or efferent inhibition during filling (which was the focus of the human studies and the abstract). In other words, why doesn't the model reproduce the 1Hz excitation effect of expediting void onset (or urgency in the human study), and why is it justified to look at contraction duration as a surrogate measure?

The authors claim that "voiding behavior occurred earlier [at 1Hz stim in the model]", pointing to Figure 6A as evidence, but this panel appears to show a single example model run where 1Hz voiding occurs only ~1s earlier (display makes this very hard to estimate). This is insufficient evidence to support the claim. Later, it is stated that "TNS did not ... void much earlier". The claims should be made compatible, and all such claims should have reasonable supporting evidence.

There are a number of reporting concerns that can be easily addressed:

(1) Human Study:

(a) To interpret the human study analysis, a fuller description of the "optional 10m inute extension" is necessary. How were participants presented with this option, how was blinding preserved, what fraction of participants accepted, and did phase 1 results influence their decisions to continue?

(b) For reproducibility, details about the TNS parameters should be articulated, such as the method of determining "motor thresholds" (unless this is synonymous with "urge to urinate"), the shape of the stimulation pulses (e.g., biphasic, charge balanced), typical applied current, etc.

(2) The Computational Model

(a) The code availability statement for this type of work is inadequate. The model used for simulations in this work, as well as the code used to initialize (and randomize synaptic connections), needs to be hosted publicly because i) a model this intricate is extremely hard to reproduce/verify without code, ii) simulations are an essential piece of the argument, iii) hosting code requires very little overhead. Although there is an appropriate level of detail in the model description, it would not be possible to reproduce the model in any reasonable amount of time (or at all) because of the implementation-level details that are, understandably, omitted from the methods (e.g., what is a "unit", what 'exactly' do the connections in the PMC and PAG diagrams relate to, what were the final parameters used for all conductances, which parameters were "matched" to the original papers and which were not, etc.).

b) Critical cystometric/urodynamic values that are typically analyzed to assess healthy LUT function are detrusor pressure (timeseries) and/or post-void residual or voiding efficiency (scalars). These should be included to verify that the model is representative of the "normal" case. This is especially important because the model's "normal" behavior appears to have extremely low voiding efficiency (Figure 6A).

Reviewer #3 (Public review):

Summary

In this work, Casu et al. have reported the characterization of a previously uncharacterized membrane protein CisA encoded in a non-canonical contractile injection system of Streptomyces coelicolor, CISSc, which is a cytosolic CISs significantly distinct from both intracellular membrane-anchored T6SSs and extracellular CISs. The authors have presented the first high-resolution structure of the extended CISSc structure. It revealed important structural insights of the extended state of this non-canonical CIS.

To further explore how CISSc interacted with cytoplasmic membrane, they further set out to investigate a membrane protein CisA encoded in the CISSc cluster and previously hypothesized to be the membrane adaptor for CISSc; however, the structure revealed that it was not associated with CISSc. Using a fluorescence microscope and cell fractionation assay, the authors verified that CisA is indeed a membrane-associated protein. They further determined experimentally that CisA had a cytosolic N-terminal domain and a periplasmic C-terminus. The functional analysis of cisA mutant revealed that it is not required for CISSc assembly but is essential for the contraction, as a result, the deletion significantly affects CISSc-mediated cell death upon stress, timely differentiation, as well as secondary metabolite production. Although the work did not resolve the mechanistic detail how CisA interacts with CISSc structure, they used in-silico prediction of protein-protein interactions between monomeric CisA and CISSc components using Alphafold2-Multimer, which identified baseplate protein Cis11 as a potential interaction partner. Such prediction sets out a strong basis for future investigations to explore the molecular mechanistic details how CisA mediates the contraction via interactions with the CIS structural components such as Cis11. Using AlphaFold3, the authors also estimated the oligomerization state of CisA, which can be present as a pentamer. Authors further suggested that such oligomerization is mediated by the interaction of C-terminal solute-binding like domain.

In general, the work provides solid data and a strong foundation for future investigation toward understanding the mechanism of CISSc contraction, and potentially, the relation between the membrane association of CISSc, the sheath contraction and the cell death.

Major Strength:

The paper is well-structured, and the conclusion of the study is supported by solid data and careful data interpretation were presented. The authors provided strong evidence on (1) the high-resolution structure of extended CISSc determined by cryo-EM, and the subsequent comparison with known eCIS structures, which sheds light on both its similarity and different features from other subtypes of eCISs in detail; (2) the topological features of CisA using fluorescence microscopic analysis, cell fractionation and PhoA-LacZα reporter assays, (3) functions of CisA in CISSc-mediated cell death and secondary metabolite production, likely via the regulation of sheath contraction, (4) structural prediction of the oligomerization state of CisA and potential interaction partners of CIS structure.

Weakness:

Due to technical limitations, authors are not able to experimentally demonstrate the direct interaction between CisA with baseplate complex of CISSc, since they could not express cisA in E. coli due to its potential toxicity. Therefore, there is a lack of biochemical analysis of direct interaction between CisA and baseplate wedge. However, they have provided solid AlphaFold2-multimer prediction data and identified baseplate protein Cis11 as a potential interaction partner. Such predictions will guide future work towards biochemical analysis to verify such interaction.

While there is no direct evidence showing that CisA is responsible for tethering CISSc to the membrane upon stress, and the spatial and temporal relation between membrane association and contraction remains unclear, I recognize that this is above the scope of the current work, so I would expect further investigation to address these questions in future.

Conclusion

Overall, the work provides a valuable contribution to our understanding on the structure of a much less understood subtype of CISs, which is unique compared to both membrane-anchored T6SSs and host-membrane targeting eCISs. Authors have successfully demonstrated the role of CisA in the contraction of CISSc, along with solid and detailed analysis of the contraction state of the particles with or without CisA using cryo-ET. Using structural modeling, authors also identified the potential oligomerization state and possible interaction partner within the CIS particle.

Importantly, the work serves as a strong foundation to further investigate how the sheath contraction works here. The work contributes to expanding our understanding of the diverse CIS superfamilies, with significant novelty.

Reviewer #2 (Public review):

Summary:

Stephens et al. present a comprehensive study of TMEM16-members via coarse-grained MD simulations (CGMD). They particularly focus on the scramblase ability of these proteins and aim to characterize the "energetics of scrambling". Through their simulations, the authors interestingly relate protein conformational states to membrane's thickness and link those to the scrambling ability of TMEM members, measured as the trespassing tendency of lipids across leaflets. They validate their simulation with a direct qualitative comparison with Cryo-EM maps.

Strengths:

The study demonstrates an efficient use of CGMD simulations to explore lipid scrambling across various TMEM16 family members. By leveraging this approach, the authors are able to bypass some of the sampling limitations inherent in all-atom simulations, providing a more comprehensive and high-throughput analysis of lipid scrambling. Their comparison of different protein conformations, including open and closed groove states, presents a detailed exploration of how structural features influence scrambling activity, adding significant value to the field. A key contribution of this study is the finding that groove dilation plays a central role in lipid scrambling. The authors observe that for scrambling-competent TMEM16 structures, there is substantial membrane thinning and groove widening. The open Ca2+-bound nhTMEM16 structure (PDB ID 4WIS) was identified as the fastest scrambler in their simulations, with scrambling rates as high as 24.4 {plus minus} 5.2 events per μs. This structure also shows significant membrane thinning (up to 18 Å), which supports the hypothesis that groove dilation lowers the energetic barrier for lipid translocation, facilitating scrambling.

The study also establishes a correlation between structural features and scrambling competence, though analyses often lack statistical robustness and quantitative comparisons. The simulations differentiate between open and closed conformations of TMEM16 structures, with open-groove structures exhibiting increased scrambling activity, while closed-groove structures do not. This finding aligns with previous research suggesting that the structural dynamics of the groove are critical for scrambling. Furthermore, the authors explore how the physical dimensions of the groove qualitatively correlate with observed scrambling rates. For example, TMEM16K induces increased membrane thinning in its open form, suggesting that membrane properties, along with structural features, play a role in modulating scrambling activity.

Another significant finding is the concept of "out-of-the-groove" scrambling, where lipid translocation occurs outside the protein's groove. This observation introduces the possibility of alternate scrambling mechanisms that do not follow the traditional "credit-card model" of groove-mediated lipid scrambling. In their simulations, the authors note that these out-of-the-groove events predominantly occur at the dimer interface between TM3 and TM10, especially in mammalian TMEM16 structures. While these events were not observed in fungal TMEM16s, they may provide insight into Ca2+-independent scrambling mechanisms, as they do not require groove opening.

Weaknesses:

A significant challenge of the study is the discrepancy between the scrambling rates observed in CGMD simulations and those reported experimentally. Despite the authors' claim that the rates are in line experimentally, the observed differences can mean large energetic discrepancies in describing scrambling (larger than 1kT barrier in reality). For instance, the authors report scrambling rates of 10.7 events per μs for TMEM16F and 24.4 events per μs for nhTMEM16, which are several orders of magnitude faster than experimental rates. While the authors suggest that this discrepancy could be due to the Martini 3 force field's faster diffusion dynamics, this explanation does not fully account for the large difference in rates. A more thorough discussion on how the choice of force field and simulation parameters influence the results, and how these discrepancies can be reconciled with experimental data, would strengthen the conclusions. Likewise, rate calculations in the study are based on 10 μs simulations, while experimental scrambling rates occur over seconds. This timescale discrepancy limits the study's accuracy, as the simulations may not capture rare or slow scrambling events that are observed experimentally and therefore might underestimate the kinetics of scrambling. It's however, important to recognize that it's hard (borderline unachievable) to pinpoint reasonable kinetics for systems like this using the currently available computational power and force field accuracy. The faster diffusion in simulations may lead to overestimated scrambling rates, making the simulation results less comparable to real-world observations. Thus, I would therefore read the findings qualitatively rather than quantitatively. An interesting observation is the asymmetry observed in the scrambling rates of the two monomers. Since MARTINI is known to be limited in correctly sampling protein dynamics, the authors, in order to preserve the fold, have applied a strong (500 kJ mol-1 nm-2) elastic network. However, I am wondering how the ENM applies across the dimer and if any asymmetry can be noticed in the application of restraints for each monomer and at the dimer interface. How can this have potentially biased the asymmetry in the scrambling rates observed between the monomers? Is this artificially obtained from restraining the initial structure, or is the asymmetry somehow gatekeeping the scrambling mechanism to occur majorly across a single monomer? Answering this question would have far-reaching implications to better describe the mechanism of scrambling.

Notably, the manuscript does not explore the impact of membrane composition on scrambling rates. While the authors use a specific lipid composition (DOPC) in their simulations, they acknowledge that membrane composition can influence scrambling activity. However, the study does not explore how different lipids or membrane environments or varying membrane curvature and tension, could alter scrambling behaviour. I appreciate that this might have been beyond the scope of this particular paper and the authors plan to further chase these questions, as this work sets a strong protocol for this study. Contextualizing scrambling in the context of membrane composition is particularly relevant since the authors note that TMEM16K's scrambling rate increases tenfold in thinner membranes, suggesting that lipid-specific or membrane-thickness-dependent effects could play a role.

Comments on revisions:

I have carefully reviewed the replies of the author, which address the points I raised and improved the manuscript by making the changes outlined in their response. Particularly, I am pleased to see that the authors report ensemble averages in Figure 1-supplement 1 and add relevant information in a newly created table. I welcome the refinement of the discussion towards a cautionary approach in describing quantitatively the findings of experiments and computations for what concerns scrambling rates. I still feel that proper statistical analysis to compare the distributions in Figure 3-figure supplement 6 would have made the points claimed even stronger, but - at the same time - I do see the points of the authors in commenting the differences between these distributions more qualitatively. Overall, I support the publication of this manuscript, it has been a pleasure to read it.

Reviewer #1 (Public review):

Summary:

Meteorin proteins were initially described as secreted neurotrophic factors. In this manuscript, Eggeler et al. demonstrate a novel role for Meteorins in establish left-right axis formation in the zebrafish embryo. The authors generated null mutations in each of the three zebrafish meteorin genes - metrn, metrnla, and metrnlab. Triple mutant embryos displayed phenotypes strongly associated with left-right defects such as heart looping and visceral organ placement, and disrupted expression of Nodal-responsive genes, as did single mutants for metrn and metrnla. The authors then go on to demonstrate that these defects in left-right asymmetry are likely to due to defects in Kupffer's Vesicle and the progenitor dorseal forerunner cells including impaired lumen formation and reduced fluid flow, reduced clustering among DFCs, impaired DFC migration, mislocalization of apical proteins ZO-1 and aPKC, and detachment of DFCs from the EVL. Notably, the authors found that expression of marker genes sox32 and sox17 were not affected, suggesting Meteorins are required for DFC/KV morphogenesis but not necessarily fate specification. Finally, the authors show genetic interaction between Meteorins and integrin receptors, which were previously implicated in left-right patterning. In a supplemental figure, the manuscript also presents data showing expression of meteorin genes around the chick Hensen's node, suggesting that the left-right patterning functions may be conserved among vertebrates.

Strengths:

Strengths of this study include the generation of a triple mutant line that targets all known zebrafish meteorin family members. The experiments presented in this study were rigorous especially with respect to quantification and statistical analysis.

Weaknesses:

Although the authors convincingly demonstrate a role for Meteorins in zebrafish left-right patterning, data supporting a conserved role in other vertebrates is compelling but limited to one supplemental figure. This aspect would be interesting to follow up in future studies.

Comments on revisions:

I thank the authors for their thoughtful responses to the reviewers. They have adequately addressed all of my concerns.

Reviewer #2 (Public review):

Summary:

Gylemo et al. present a manuscript focused on identifying the X-inactivation or X-inactivation escape status for 380 genes across 30 normal human tissues. X-inactivation status of X-linked genes across tissues is important for understanding sex-specific differences in X-linked gene expression and therefore traits, and the likely effect of X-linked pathogenic variants in females. These new data are significant as they double the number of genes that have been classified in the human, and double the number of tissues studied previously.

Strengths:

The strengths of this work are that they analyse 3 individuals from the GTex dataset (2 newly identified, 1 previously identified and published) that have highly/ completely skewed X inactivation, which allows the study of escape from X inactivation in bulk RNA-sequencing. The number of individuals and breadth of tissues analysed adds significantly to both the number of genes that have been classified and the weight of evidence for their claims. The additional 198 genes that have been classified and the reclassification of genes that previously had only limited support for their status is useful for the field.

In analysing the data they find that tissue-specific escape from X inactivation appears relatively rare. Rather, if genes escape, even variably, it tends to occur across tissues. Similarly if a gene is inactivated, it is stable across tissues.

Comments on revised version:

The authors have answered all of my queries. While they have not been able to pinpoint the genetic cause of the highly skewed XCI cases in their cohort, I agree this is beyond the scope of this study. I have no further requests.

Reviewer #3 (Public review):

Summary:<br /> Membrane-bound pyrophosphatases (mPPases) are homodimeric proteins that hydrolyze pyrophosphate and pump H+/Na+ across membranes. They are an attractive drug target against protist pathogens. Non-hydrolysable PPi analogue bisphosphonates such as risedronate (RSD) and pamidronate (PMD) serve as primary drugs currently used. Bisphosphonates have a P-C-P bond, with their central carbon can accommodate up to two substituents, allowing a large compound variability. Here authors solved two TmPPase structures in complex with the bisphosphonates etidronate (ETD) and zoledronate (ZLD) and monitored their conformational ensemble using DEER spectroscopy in solution. These results reveal the inhibition mechanism by these compounds, which is crucial for developing future small-molecule inhibitors.

Strengths:<br /> Authors show that seven different bisphosphonates can inhibit TmPPase with IC50 values in the micromolar range. Branched aliphatic and aromatic modifications showed weaker inhibition. High-resolution structures for TmPPase with ETD (3.2 Å) and ZLD (3.3 Å) are determined. These structures reveal the binding mode and shed light on the inhibition mechanism. The nature of modification on the bisphosphonate alters the conformation of the binding pocket. The conformational heterogeneity is further investigated using EPR/DEER spectroscopy under several conditions. Altogether, this provides convincing evidence for a distinct conformational equilibrium of TmPPase in solution and further supports the notion of asymmetric inhibitor binding at the active site, while maintaining a symmetric conformation at the periplasmic interface.

Reviewer #2 (Public review):

Tran and colleagues report evidence supporting the expected yet undemonstrated interaction between the Pkd1 and Pkd2 gene products Pc1 and Pc2 and the Bicc1 protein in vitro, in mice, and collaterally, in Xenopus and HEK293T cells. The authors go on to convincingly identify two large and non-overlapping regions of the Bicc1 protein important for each interaction and to perform gene dosage experiments in mice that suggest that Bicc1 loss of function may compound with Pkd1 and Pkd2 decreased function, resulting in PKD-like renal phenotypes of different severity. These results led to examining a cohort of very early onset PKD patients to find three instances of co-existing mutations in PKD1 (or PKD2) and BICC1. Finally, preliminary transcriptomics of edited lines gave variable and subtle differences that align with the theme that Bicc1 may contribute to the PKD defects, yet are mechanistically inconclusive.

These results are potentially interesting, despite the limitation, also recognized by the authors, that BICC1 mutations seem exceedingly rare in PKD patients and may not "significantly contribute to the mutational load in ADPKD or ARPKD". The manuscript has several intrinsic limitations that must be addressed.

The manuscript contains factual errors, imprecisions, and language ambiguities. This has the effect of making this reviewer wonder how thorough the research reported and analyses have been.

Reviewer #2 (Public review):

Summary:

In the study presented by Itani and colleagues, it is shown that some strains of Aspergillus oryzae - especially those used industrially for the production of sake and soy sauce - develop hyphae with a significantly increased number of nuclei and cell volume over time. These thick hyphae are formed by branching from normal hyphae and grow faster and therefore dominate the colonies. The number of nuclei positively correlates with the thicker hyphae and also the amount of secreted enzymes. The addition of nutrients such as yeast extract or certain amino acids enhanced this effect. Genome and transcriptome analyses identified genes, including rseA, that are associated with the increased number of nuclei and enzyme production. The authors conclude from their data involvement of glycosyltransferases, calcium channels, and the tor regulatory cascade in the regulation of cell volume and number of nuclei. Thicker hyphae and an increased number of nuclei were also observed in high-production strains of other industrially used fungi such as Trichoderma reesei and Penicillium chrysogenum, leading to the hypothesis that the mentioned phenotypes are characteristic of production strains, which is of significant interest for fungal biotechnology.

Strengths:

The study is very comprehensive and involves the application of diverse state-of-the-art cell biological, biochemical, and genetic methods. Overall, the data are properly controlled and analyzed, figures and movies are of excellent quality.<br /> The results are particularly interesting with regard to the elucidation of molecular mechanisms that regulate the size of fungal hyphae and their number of nuclei. For this, the authors have discovered a very good model: (regular) strains with a low number of nuclei and strains with a high number of nuclei. Also, the results can be expected to be of interest for the further optimization of industrially relevant filamentous fungi.

Weaknesses:

There are only a few open questions concerning the activity of the many nuclei in production strains (active versus inactive), their number of chromosomes (haploid/diploid), and whether hyper-branching always leads to propagation of nuclei.

Reviewer #2 (Public review):

Yang et al. describes CCDC32 as a new clathrin mediated endocytosis (CME) accessory protein. The authors show that CCDC32 binds directly to AP2 via a small alpha helical region and cells depleted for this protein show defective CME. Finally, the authors show that the CCDC32 nonsense mutations found in patients with cardio-facial-neuro-developmental syndrome (CFNDS) disrupt the interaction of this protein to the AP2 complex. The results presented suggest that CCDC32 may act as both a chaperone (as recently published) and a structural component of the AP2 complex.

Strengths:<br /> The conclusions presented are generally well supported by experimental data and the authors carefully point out the differences between their results and the results by Wan et al. (PNAS 2024).

Weaknesses:<br /> The experiments regarding the role of CCDC32 in CFNDS still require some clarifications to make them clearer to scientists working on this disease. The authors fail to describe that the CCDC32 isoform they use in their studies is different from the one used when CFNDS patient mutations were described. This may create some confusion. Also, the authors did not discuss that the frame-shift mutations in patients may be leading to nonsense mediated decay.

Reviewer #2 (Public review):

Summary:<br /> This study investigates the impact of mother-child neural synchronization and the quality of parent-child relationships on the development of Theory of Mind (ToM) and social cognition. Utilizing a naturalistic fMRI movie-viewing paradigm, the authors analyzed inter-subject neural synchronization in mother-child dyads and explored the connections between neural maturity, parental caregiving, and social cognitive outcomes. The findings indicate age-related maturation in ToM and social pain networks, emphasizing the importance of dyadic interactions in shaping ToM performance and social skills, thereby enhancing our understanding of the environmental and intrinsic influences on social cognition.

Strengths:<br /> This research addresses a significant question in developmental neuroscience, by linking social brain development with children's behaviors and parenting. It also uses a robust methodology by incorporating neural synchrony measures, naturalistic stimuli, and a substantial sample of mother-child dyads to enhance its ecological validity. Furthermore, the SEM approach provides a nuanced understanding of the developmental pathways associated with Theory of Mind (ToM). The manuscript also addressed many concerns raised in the initial review. The adoption of the neuroconstructivist framework effectively frames neural and cognitive development as reciprocal, addressing prior concerns about causality. The justification for methodological choices, such as omitting resting-state baselines due to scanning challenges in children and using unit-weighted scoring for ToM tasks, further strengthens the study's credibility.

Weaknesses:<br /> (1) The revised introduction has improved, particularly in framing the first goal-developmental changes in ToM and SPM networks-as a "developmental anchor" for goals 2 and 3. However, given prior research on age-related changes in these networks (e.g., Richardson et al., 2018), the authors should clarify whether this goal seeks to replicate prior findings or to extend them under new contexts. Specifying how this part differs from existing work and articulating specific hypotheses would enhance the focus.<br /> (2) I still have some reservations about retaining the slightly causal term "shape" in the title. While the manuscript now carefully avoids causal claims, the title may still be interpreted as implying directionality, especially by non-specialist audiences.<br /> (3) One more question about Figure 2A and 2B: adults and children showed highly similar response curves for video frames, yet some peaks (e.g., T02, T05, T06) are identified as ToM or SPM events only in adults. Whether statistical methods account for the differences? Or whether the corresponding video frames contain subtle social cues that only adults can process?

Reviewer #2 (Public review):

Summary:

The authors present a clear expansion of biophysical (thermodynamic) theory regarding the binding of proteins to membrane-bound receptors, accounting for higher local concentration effects of the protein. To partially test the expanded theory, the authors perform in vitro experiments on the binding of ZO1 proteins to Claudin2 C-terminal receptors anchored to a supported lipid bilayer, and capture the effects that surface phase separation of ZO1 has on its adsorption to the membrane.

Strengths:

(1) The derived theoretical framework is consistent and largely well-explained.

(2) The experimental and numerical methodologies are transparent.

(3) The comparison between the best parameterized non-dilute theory is in reasonable agreement with experiments.

Weaknesses:

(1) In the theoretical section, what has previously been known, compared to which equations are new, should be made more clear.

(2) Some assumptions in the model are made purely for convenience and without sufficient accompanying physical justification. E.g., the authors should justify, on physical grounds, why binding rate effects are/could be larger than the other fluxes.

(3) I feel that further mechanistic explanation as to why bulk phase separation widens the regime of surface phase separation is warranted.

(4) The major advantage of the non-dilute theory as compared with a best parameterized dilute (or homogenous) theory requires further clarification/evidence with respect to capturing the experimental data.

(5) Discrete (particle-based) molecular modelling could help to delineate the quantitative improvements that the non-dilute theory has over the previous state-of-the-art. Also, this could help test theoretical statements regarding the roles of bulk-phase separation, which were not explored experimentally.

(6) Discussion of the caveats and limitations of the theory and modelling is missing from the text.

Reviewer #2 (Public review):

In the present manuscript, Golf et al. investigate the consequences of astrocyte-specific deletion of Neuroligin (Nlgn) family cell adhesion proteins on synapse structure and function in the brain. Decades of prior research had shown that Neuroligins mediate their effects at synapses through their role in the postsynaptic compartment of neurons and their transsynaptic interaction with presynaptic Neurexins. More recently, it was proposed for the first time that Neuroligins expressed by astrocytes can also bind to presynaptic Neurexins to regulate synaptogenesis (Stogsdill et al. 2017, Nature). However, several aspects of the model proposed by Stogsdill et al. on astrocytic Neuroligin function conflict with prior evidence on the role of Neuroligins at synapse, prompting Golf et al. to further investigate astrocytic Neuroligin function in the current study. Using postnatal conditional deletion of Nlgn1-3 specifically from astrocytes in mice, Golf et al. show that virtually no changes in the expression of synaptic proteins or in the properties of synaptic transmission at either excitatory or inhibitory synapses are observed. Moreover, no alterations in the morphology of astrocytes themselves were found. To further extend this finding, the authors additionally analyzed human neurons co-cultured with mouse glia lacking expression of Nlgn1-4. No difference in excitatory synaptic transmission was observed between neurons cultured in the presence of wildtype vs. Nlgn1-4 conditional knockout glia. The authors conclude that while Neuroligins are indeed expressed in astrocytes and are hence likely to play some role there, this role does not include any direct consequences on synaptic structure and function, in direct contrast to the model proposed by Stogsdill et al.

Overall, this is a strong study that addresses a fundamental and highly relevant question in the field of synaptic neuroscience. Neuroligins are not only key regulators of synaptic function, they have also been linked to numerous psychiatric and neurodevelopmental disorders, highlighting the need to precisely define their mechanisms of action. The authors take a wide range of approaches to convincingly demonstrate that under their experimental conditions, Nlgn1-3 are efficiently deleted from astrocytes in vivo, and that this deletion does not lead to major alterations in the levels of synaptic proteins or in synaptic transmission at excitatory or inhibitory synapses, or in the morphology of astrocytes. The authors have conducted an elegant and compelling analysis demonstrating efficient deletion of astrocytic Nlgn1-3, with deletion rates of 83-96% for Nlgn2 and Nlgn3, and 65-72% for Nlgn1. While the co-culture experiments provide additional support, they are not essential as the in vivo data on astrocytic Nlgn1-3 deletion are compelling on their own. Together, the data from this study provide compelling and important evidence that, whatever the role of astrocytic Neuroligins may be, they do not contribute substantially to synapse formation or function under the conditions investigated.

Comments on revisions:

All of my concerns have been satisfactorily addressed.<br /> The authors have fully addressed my concerns, and have in particular conducted a very elegant and compelling analysis of the degree of deletion of astrocytic Nlgn1-3/4 in their models. This greatly strengthens the main claims of their study and the fundamental nature of their conclusions for the field of synapse biology.<br /> Regarding the co-culture experiments, while I was initially concerned about the lack of controls demonstrating that glia affect synapse formation in human neurons, the authors have appropriately addressed this by clarifying the missing references and explaining that their culture system has been extensively validated in previous studies. Since the data on astrocytic Nlgn1-3 deletion in vivo are compelling on their own, the co-culture experiment provides useful additional support for the main conclusions.<br /> The authors have also added the mouse strain background information to the methods section as requested, which is important for interpreting potential differences with other studies.

Reviewer #2 (public review):

The manuscript by Christopher N. Rudzitis et al. describes the role of TGFβ2 in the transcription and functional expression of mechanosensitive channel isoforms, alongside studies on TM contractility in biomimetic hydrogels and intraocular pressure. Overall, it is a very interesting study, nicely designed, and will contribute to the available literature on TRPV4 sensitivity to mechanical forces.

Reviewer #2 (Public review):

Summary:

Garcia-Mora et al. presented a two-step bioinformatics pipeline using H3K27ac ChIP-seq and RNA-seq data from 11 human embryonic tissues published by the same groups of senior authors. "First Search" identifies motifs for TFs that are both tissue-restricted in expression and enriched in tissue-specific enhancers. "Second Search" then looks for additional motifs that co-occur near each "First Search" motif. The authors here went further than previous motif co-occurrence/co-enrichment analyses by identifying TEAD motifs as (1) representing a ubiquitously expressed family and (2) showing high co-occurrence with tissue-specific motifs at tissue-specific enhancers. They then elaborate on this finding and speculate that "TEAD, in concert with cardiac-restricted transcriptional regulators, may contribute to the recruitment of CHD4 and may play a role in attenuating the activity of enhancers involved in cardiomyocyte differentiation." They also discussed validation experiments using the luciferase assay.

Strengths:

The manuscript is well-written and easy to follow for the most part.

Weaknesses:

My main concerns and criticisms are about the sensitivity of the method and the validation of experiment designs and conclusions. Some examples where validation could be improved are as follows:

(1) The authors propose a mechanism of a TF trio (TEAD - CHD4 - tissue-specific TFs). However, only one validation experiment checked CHD4. CHD4 binding was not mentioned at all in the other cases.

(2) The authors integrated E12.5 TEAD binding with E11.5 acetylation data, and it would be important to show that this experimental approach is valid or otherwise qualify its limitations.

(3) Motif co-occurrence analysis was extended to claiming TF interactions without further validation.

Reviewer #2 (Public review):

Summary:

The authors repeatedly measured the behavior of individual flies across several environmental situations in custom-made behavioral phenotyping rigs.

Strengths:

The study uses several different behavioral phenotyping devices to quantify individual behavior in a number of different situations and over time. It seems to be a very impressive amount of data. The authors also make all their behavioral phenotyping rig design and tracking software available, which I think is great, and I'm sure other folks will be interested in using and adapting to their own needs.

Weaknesses/Limitations:

I think an important limitation is that while the authors measured the flies under different environmental scenarios (i.e. with different lighting, temperature) they didn't really alter the "context" of the environment. At least within behavioral ecology, context would refer to the potential functionality of the expressed behaviors so for example, an anti-predator context, or a mating context, or foraging. Here, the authors seem to really just be measuring aspects of locomotion under benign (relatively low risk perception) contexts. This is not a flaw of the study, but rather a limitation to how strongly the authors can really say that this demonstrates that individuality is generalized across many different contexts. It's quite possible that rank-order of locomotor (or other) behaviors may shift when the flies are in a mating or risky context.

I think the authors are missing an opportunity to use much more robust statistical methods It appears as though the authors used pearson correlations across time/situations to estimate individual variation; however far more sophisticated and elegant methods exist. The problem is that pearson correlation coefficients can be anti-conservative and additionally, the authors have thus had to perform many many tests to correlate behaviors across the different trials/scenarios. I don't see any evidence that the authors are controlling for multiple testing which I think would also help. Alternatively, though, the paper would be a lot stronger, and my guess is, much more streamlined if the authors employ hierarchical mixed models to analyse these data, which are the standard analytical tools in the study of individual behavioral variation. In this way, the authors could partition the behavioral variance into its among- and within-individual components and quantify repeatability of different behaviors across trials/scenarios simultaneously. This would remove the need to estimate 3 different correlations for day 1 & day 2, day 1 & 3, day 2 & 3 (or stripe 0 & stripe 1, etc) and instead just report a single repeatability for e.g. the time spent walking among the different strip patterns (eg. figure 3). Additionally, the authors could then use multivariate models where the response variables are all the behaviors combined and the authors could estimate the among-individual covariance in these behaviors. I see that the authors state they include generalized linear mixed models in their updated MS, but I struggled a bit to understand exactly how these models were fit? What exactly was the response? what exactly were the predictors (I just don't understand what Line404 means "a GLM was trained using the environmental parameters as predictors (0 when the parameter was not changed, 1 if it was) and the resulting individual rank differences as the response"). So were different models run for each scenario? for different behaviors? Across scenarios? What exactly? I just harp on this because I'm actually really interested in these data and think that updating these methods can really help clarify the results and make the main messages much clearer!

I appreciate that the authors now included their sample sizes in the main body of text (as opposed to the supplement) but I think that it would still help if the authors included a brief overview of their design at the start of the methods. It is still unclear to me how many rigs each individual fly was run through? Were the same individuals measured in multiple different rigs/scenarios? Or just one?

I really think a variance partitioning modeling framework could certainly improve their statistical inference and likely highlight some other cool patterns as these methods could better estimate stability and covariance in individual intercepts (and potentially slopes) across time and situation. I also genuinely think that this will improve the impact and reach of this paper as they'll be using methods that are standard in the study of individual behavioral variation

Reviewer #2 (Public review):

Summary:

In this study, Hill and colleagues use a novel reinforcement-based motor learning task ("RML"), asking how aspects of RML change over the course of development from toddler years through adolescence. Multiple versions of the RML task were used in different samples, which varied on two dimensions: whether the reward probability of a given hand movement direction was deterministic or probabilistic, and whether the solution space had continuous reach targets or discrete reach targets. Using analyses of both raw behavioral data and model fits, the authors report four main results: First, developmental improvements reflected 3 clear changes, including increases in exploration, an increase in the RL learning rate, and a reduction of intrinsic motor noise. Second, changes to the task that made it discrete and/or deterministic both rescued performance in the youngest age groups, suggesting that observed deficits could be linked to continuous/probabilistic learning settings. Overall, the results shed light on how RML changes throughout human development, and the modeling characterizes the specific learning deficits seen in the youngest ages.

Strengths:

(1) This impressive work addresses an understudied subfield of motor control/psychology - the developmental trajectory of motor learning. It is thus timely and will interest many researchers.

(2) The task, analysis, and modeling methods are very strong. The empirical findings are rather clear and compelling, and the analysis approaches are convincing. Thus, at the empirical level, this study has very few weaknesses.

(3) The large sample sizes and in-lab replications further reflect the laudable rigor of the study.

(4) The main and supplemental figures are clear and concise.

Reviewer #2 (Public review):

This manuscript examines network mechanisms that allow networks of neurons to perform context-dependent decision-making.<br /> In a recent study, Pagan and colleagues identified two distinct mechanisms by which recurrent neural networks can perform such computations. They termed these two mechanisms input-modulation and selection-vector modulation. Pagan and colleagues demonstrated that recurrent neural networks can be trained to implement combinations of these two mechanisms, and related this range of computational strategies with inter-individual variability in rats performing the same task. What type of structure in the recurrent connectivity favors one or the other mechanism however remained an open question.

The present manuscript addresses this specific question by using a class of mechanistically interpretable recurrent neural networks, low-rank RNNs.<br /> The manuscript starts by demonstrating that unit-rank RNNs can only implement the input-modulation mechanism, but not the selection-vector modulation. The authors then build rank three networks which implement selection-vector modulation, and show how the two mechanisms can be combined. Finally, they relate the amount of selection-vector modulation with the effective rank, ie the dimensionality of activity, of a trained full-rank RNN.

Strength:

- The manuscript is written in an obvious manner<br /> - The analytic approach adopted in the manuscript is impressive<br /> - Very clear identification of the mechanisms leading to the two types of context-dependent modulation<br /> - Altogether, this manuscript reports remarkable insights on a very timely question

Reviewer #2 (Public review):

Summary/Significance of the findings:

The authors have done a great job by extensively carrying out transcriptomic and epigenomic analyses in the primary human/mouse monocytes/macrophages to investigate TNF-PGE2 (TP) crosstalk and their regulation by IFN-γ in the Rheumatoid arthritis (RA) synovial macrophages. They proposed that TP induces inflammatory genes via a novel regulatory axis whereby IFN-γ and PGE2 oppose each other to determine the balance between two distinct TNF-induced inflammatory gene expression programs relevant to RA and ICI-arthritis.

Strengths:

The authors have done a great job on RT-qPCR analysis of gene expression in primary human monocytes stimulated with TNF and showing the selective agonists of PGE2 receptors EP2 and EP4 22 that signal predominantly via cAMP. They have beautifully shown IFN-γ opposes the effects of PGE2 on TNF-induced gene expression. They found that TP signature genes are activated by cooperation of PGE2-induced AP-1, CEBP, and NR4A with TNF-induced NF-κB activity. On the other hand, they found that IFN-γ suppressed induction of AP-1, CEBP, and NR4A activity to ablate induction of IL-1, Notch, and neutrophil chemokine genes but promoted expression of distinct inflammatory genes such as TNF and T cell chemokines like CXCL10 indicating that TP induces inflammatory genes via IFN-γ in the RA and ICI-arthritis.

Comments on latest version:

The authors have answered my questions and i recommend this manuscript for publication.

Reviewer #2 (Public review):

Before providing my review of the revised version of this study by Berger et al., which explores potential deliberate burials of Homo naledi within the Rising Star Cave System, I would like to briefly summarize the key points from my previous review of the earlier version (in 2023). Summarizing my previous review will provide context for assessing how effectively the revised study addresses the concerns I raised previously (in 2023).

In my earlier comments, I highlighted significant methodological and analytical shortcomings that, in my view, undermined the authors' claim of intentional burials by Homo naledi. While the study presented detailed geological and fossil data, I found the evidence for intentional burials unconvincing due to insufficient application of archaeothanatological principles and other methodological gaps.

My key concerns included:

(1) The absence of a comprehensive archaeothanatological analysis, particularly with respect to taphonomic changes, bone articulations, and displacement patterns such as the collapse of sediments and bone remains into voids created by decomposition.

(2) Missing or unclear illustrations of bone arrangements, which are critical for interpreting burial positions and processes.

(3) A lack of detailed discussion on the sequence of decomposition, joint disarticulation, sediment infill, and secondary bone displacement.

To convincingly support claims of deliberate burial, I argued that the study must reconstruct the timeline and processes surrounding death and deposition while clearly distinguishing natural taphonomic changes from intentional human actions. I emphasized the importance of integrating established archaeothanatological frameworks, such as those outlined by Duday et al. or Boulestin et al., to provide the necessary analytical rigor.

I will now explain how the revised version of this study has successfully addressed all the concerns raised in my previous review and why I now think that the authors provide sufficient evidence for the presence of "repeated and patterned" deliberate burials (referred to as "cultural burials" by the authors) by Homo naledi within the Rising Star Cave System.

In their revised manuscript, the authors have implemented substantial improvements in methodology, analytical depth, and overall presentation, which have effectively resolved the critical issues I previously highlighted. These revisions greatly strengthen their argument for intentional funerary practices. Importantly, the authors remain cautious in their interpretation of the evidence, explicitly refraining from inferring "symbolic" behavior or complex cognitive motivations behind these burials. Instead, they focus on presenting clear evidence for deliberate, patterned practices while leaving the broader implications for Homo naledi's cultural and cognitive capacities open for further investigation. This cautious approach adds to the credibility of their conclusions and avoids overextending the interpretation of the data.

The authors' enhanced application of archaeothanatological principles now offers a more comprehensive and convincing interpretation of the burial features. Key gaps in the earlier version, such as the absence of detailed reconstructions of taphonomic processes, bone articulations, and displacement patterns, have been addressed with thorough analyses and clearer illustrations. The study also now includes a well-structured timeline of events surrounding death and deposition, demonstrating an improved ability to differentiate between natural processes and deliberate human actions. These additions lend greater clarity and rigor to the evidence, making the argument for intentional burials both robust and persuasive.

Furthermore, the revised study presents detailed data on skeletal arrangements, decomposition sequences, and spatial patterns. This information is now relatively well illustrated and contextualized, enabling readers to better understand the complex processes involved in these burial practices. Importantly, the authors provide a stronger theoretical framework, integrating established archaeothanatological methodologies and taphonomic studies that situate their findings within broader archaeological and anthropological discussions of funerary behavior.

That being said, there remain relatively minor issues that could be refined further. Addressing these would help ensure the study is as clear and accessible as possible to the reader. Such adjustments would enhance the overall readability and reinforce the study's impact within the scientific community.

A - Suggested changes:

While the revised version of this study marks a significant improvement, successfully addresses my previous major concerns and provides a convincing argument for deliberate burials by Homo naledi, I believe that including both one summary table + one summary figure for each of the three main locations and the-Hill Antechamber, and Dinaledi Chamber (Feature 1 and Puzzle Box)-would further enhance the clarity and accessibility of the findings. Such tables and figures would serve as a valuable reference, allowing readers to more easily follow how the detailed patterns observed at each site fit the criteria for distinguishing intentional from natural processes.

The summary tables should consolidate key information for each location, such as:

(1) Bone articulations: A comprehensive list of articulated skeletal elements, categorized by their anatomical relationships (e.g., labile vs. stable articulations).

(2) Displacement patterns: Documentation of any spatial shifts in bone positions, noting directions and extents of disarticulation.

(3) Sequence of decomposition: Observations regarding the sequence of decomposition, joint disarticulation and associated changes in bone arrangements.

(4) Sediment interaction: Notes on sediment infill and its timing relative to decomposition, including evidence of secondary voids or delayed sediment deposition.

(5) Distinguishing criteria: Clear indications of how each observed pattern supports intentional burial (e.g., structured placement, lack of natural transport mechanisms) versus natural processes (e.g., random dispersal, sediment-driven bone displacement).<br /> Including such tables would not only summarize the complex taphonomic and archaeothanatological data but also allow readers to quickly assess how the evidence supports the authors' conclusions. This approach would bridge the gap between the detailed narrative descriptions and the criteria necessary to differentiate deliberate funerary practices from natural occurrences.

To streamline the main text further, many of the detailed descriptions of individual bones, specific displacement measurements, and other intricate observations could be moved to the supplementary data. This reorganization would maintain the richness of the data for those who wish to explore it in depth, while the summary tables would present the key findings concisely in the main text. This balance between accessibility and detail would ensure that the study appeals to both specialists requiring comprehensive data and readers looking for an overarching understanding of the findings.

In addition to these structural changes, it is crucial to ensure that evidence is consistently illustrated throughout the text.

Importantly the skeletal part representation is provided for Dinaledi Feature 1 in Figure 14, but similar data is not presented for the other burial features, such as those in the Hill Antechamber or Puzzle Box. This inconsistency could make it more challenging for readers to compare the features and fully appreciate the patterns of burial behavior across the different locations. Ensuring that similar types of evidence and analyses are presented uniformly for all features would strengthen the study and make its conclusions more cohesive and compelling.

Adding supplementary figures to represent the skeletal part distribution (as in Figure 14) within each excavated area (i.e., not only for Dinaledi Feature 1 but also for Hill Antechamber and Puzzle Box) would significantly enhance the study's clarity and accessibility. These figures could provide a visual summary of skeletal part representation, allowing readers to easily understand the nature of human remains within each burial context.

Specifically, such figures could:

(1) Illustrate Skeletal Part Representation: By visually mapping the presence and location of various skeletal elements, the figures would make it easier for readers to assess the completeness and arrangement of remains in each feature. This is particularly important for interpreting patterns of bone articulation and disarticulation.<br /> For example, it is quite challenging to determine the exact number and characteristics of the human skeletal remains identified within the Puzzle Box and those recovered through the "subsurface collection" in its surrounding area. The authors state that "at least six individuals" were identified in this area (during "subsurface collection") but provide no further clarification. They simply mention that "most elements" were described previously, without specifying which elements or where this prior description can be found.

(2) Highlight Articulations and Displacements: Figures could indicate which bones are articulated and their relative positions, as well as the spatial distribution of disarticulated elements. This would provide a clear visual context to support interpretations of taphonomic processes.

(3) Facilitate Comparisons Across Locations: By presenting skeletal part representation consistently for each location, the figures would enable readers to directly compare features, reinforcing the argument for "repeated and patterned" behavior.

(4) Simplify Complex Data: Instead of relying solely on textual descriptions, the visual format would allow readers to quickly grasp the key findings, making the study more accessible to a broader audience

By including such figures alongside the proposed summary tables in the main text, the study would achieve a balance between detailed narrative descriptions and concise, visual representation of the data. This approach would strengthen the overall presentation and support the authors' conclusions effectively.

Again, by presenting the data in a structured and comparative format, the new tables + figures could also highlight the differences and similarities between the three locations. This would reinforce the argument for "repeated and patterned" behavior, as the tables would make it easier to observe consistent burial practices across different contexts within the Rising Star Cave System.

Adding these summary tables + figures, ensuring consistent presentation of evidence, and reallocating detailed descriptions to supplementary materials would not require significant new analysis. However, these organizational adjustments would greatly enhance the study's clarity, readability, and overall impact.

B - A few additional changes are needed:

Figure 8: This figure is critical but lacks clarity. Specifically:

Panels 8a-c suffer from low contrast, making details difficult to discern.<br /> Panel 8d (sediment profile) is too small and lacks annotations that would aid interpretation.<br /> Figure S7: While this figure has significantly better contrast than Figures 8a-c, I am unable to identify the "articulated foot ... at right of frame," as mentioned in the caption. Please clarify this by adding annotations directly to the figure.

Page 4, 2nd paragraph: In the sentence "Researchers thus have diverse opinions about how to test whether ...," the word "opinions" should be replaced with a more precise term, such as "approaches."

C - In conclusion, I am impressed by the significant effort and meticulous work that has gone into this revised version of the study. The quality of the new evidence presented is commendable, and the findings now convincingly demonstrate not only clear evidence of intentional burial practices by Homo naledi but also compelling indications of post-depositional reworking. These advancements reflect a major improvement in the study's analytical rigor and the robustness of its conclusions, making it a valuable contribution to the understanding of early hominin funerary behavior.

Reviewer #2 (Public review):

Summary:

The manuscript describes synaptic connectivity in Songbird cortex four main classes of sensory neurons afferents onto three known classes of projection neurons of the pre-motor cortical region HVC. HVC is a region associated with the generation of learned bird song. Investigators here use all male zebra finches to examine the functional anatomy of this region using patch clamp methods combined with optogenetic activation of select neuronal groups.

Strengths:

The quality of the recordings is extremely high and the quantity of data is on a very significant scale, this will certainly aid the field.

Weaknesses:

Could make the figures a little easier to navigate by having some atlas drawings.

Comments on revisions:

The authors have addressed the minor concerns and suggestions

Reviewer #2 (Public review):

In this study, Wang and colleagues aimed to explore brain-wide activation patterns associated with NREM sleep oscillations, including slow oscillations (SOs), spindles, and SO-spindle coupling events. Their findings reveal that SO-spindle events corresponded with increased activation in both the thalamus and hippocampus. Additionally, they observed that SO-spindle coupling was linked to heightened functional connectivity from the hippocampus to the thalamus, and from the thalamus to the medial prefrontal cortex-three key regions involved in memory consolidation and episodic memory processes.

This study's findings are timely and highly relevant to the field. The authors' extensive data collection, involving 107 participants sleeping in an fMRI while undergoing simultaneous EEG recording, deserves special recognition. If shared, this unique dataset could lead to further valuable insights.

Comments on revisions:

The authors' efforts in revising the manuscript and addressing the reviewers' comments are certainly commendable. However, I remain concerned about potential issues in detecting sleep-related oscillations (SOs, spindles, and consequently coupled SO-spindle events), which may arise due to suboptimal parameter selection or inaccurate sleep staging, potentially impacting all subsequent analyses.

A review of Supplementary Tables 1-4 reveals an unusually high number of detected SOs and spindles during sleep stage N1 and REM sleep. While the authors correctly note that a percentile-based detection approach will always identify a certain number of events across sleep stages, the particularly high counts in N1 and REM are concerning. To mitigate the limitations of this method, the authors could have performed event detection independently of sleep stages (i.e., across the entire dataset for each participant) and subsequently assigned the detected events to the corresponding sleep stages. If the event counts in N1 and REM remained disproportionately high, this would indicate a fundamental issue with the detection procedure.

Reviewer #2 (Public review):

Summary:

In this work the authors trained RNN to perform a reversal task also performed by animals while PFC activity is recorded. The authors devised a new method to train RNN on this type of reversal task, which in principle ensures that the behavior of the RNN matches the behavior of the animal. They then performed some analysis of neural activity, both RNN and PFC recording, focusing on the neural representation of the reversal probability and its evolution across trials. Given the analysis presented, it has been difficult for me to asses at which point RNN can reasonably be compared to PFC recordings.

Strengths:

Focusing on a reversal task, the authors address a challenge in RNN training, as they do not use a standard supervised learning procedure where the desired output is available for each trial. They propose a new way of doing that.

They attempt to confront RNN and neural recordings in behaving animals.

Weaknesses:

It would be nice to better articulate the analysis results of the two training set-ups (with and without 0 response during fixation). The dynamical system analysis is confusing, the notions of stationary and non-stationary dynamics and its relationship with attractors are puzzling. Is there a line attractor in one case (with inputs orthogonal to the integration direction being called back to the attractor, and reward input aligned with the stable direction)? In the other case, do we have a cylindrical attracting manifold on which activity circles around and is pushed along the axis of the cylinder by reward inputs? Which case is closest to the PFC recordings?

Reviewer #2 (Public review):

Summary:

In this study, the authors build a statistical model that stochastically samples from a time-interval distribution of reorientation rates. The form of the distribution is extracted from a large array of behavioral data, is then used to describe not only the dynamics of individual worms (including the inter-individual variability in behavior), but also the aggregate population behavior. The authors note that the model does not require an assumption about behavioral state transitions, or evidence accumulation, as has been done previously, but rather that the stochastic nature of behavior is "simply the product of stochastic sampling from an exponential function".

Strengths:

This model provides a strong juxtaposition to other foraging models in the worm. Rather than evoking a behavioral transition function (that might arise from a change in internal state or the activity of a cell type in the network), or evidence accumulation (which again maps onto a cell type, or the activity of a network) - this model explains behavior via the stochastic sampling of a function of an exponential decay. The underlying model and the dynamics being simulated, as well as the process of stochastic sampling are well described and the model fits the exponential function (equation 1) to data on a large array of worms exhibiting diverse behaviors (1600+ worms from Lopez-Cruz et al). The work of this study is able to explain or describe the inter-individual diversity of worm behavior across a large population. The model is also able to capture two aspects of the reorientations, including the dynamics (to switch or not to switch) and the kinetics (slow vs fast reorientations). The authors also work to compare their model to a few others including the Levy walk (whose construction arises from a Markov process) to a simple exponential distribution, all of which have been used to study foraging and search behaviors.

Weaknesses:

This manuscript has two weaknesses that dampen the enthusiasm for the results. First, in all of the examples the authors cite where a Gillespie algorithm is used to sample from a distribution, be it the kinetics associated with chemical dynamics, or a Lotka-Volterra Competition Model, there are underlying processes that govern the evolution of the dynamics, and thus the sampling from distributions. In one of their references for instance, the stochasticity arises from the birth and death rates, thereby influencing the genetic drift in the model. In these examples, the process governing the dynamics (and thus generating the distributions from which one samples) are distinct from the behavior being studied. In this manuscript, the distribution being sampled from is the exponential decay function of the reorientation rate (lines 100-102). This appears to be tautological - a decay function fitted to the reorientation data is then sampled to generate the distributions of the reorientation data. That the model performs well, and matches the data is commendable, but it is unclear how that could not be the case if the underlying function generating the distribution was fit to the data.

The second weakness is somewhat related to the first, in that absent an underlying mechanism or framework, one is left wondering what insight the model provides. Stochastic sampling a function generated by fitting the data to produce stochastic behavior is where one ends up in this framework, and the authors indeed point this out: "simple stochastic models should be sufficient to explain observably stochastic behaviors." (Line 233-234). But if that is the case, what do we learn about how the foraging is happening. The authors suggest that the decay parameter M can be considered a memory timescale; which offers some suggestion, but then go on to say that the "physical basis of M can come from multiple sources". Here is where one is left for want: The mechanisms suggested, including loss of sensory stimuli, alternations in motor integration, ionotropic glutamate signaling, dopamine, and neuropeptides are all suggested: this is basically all of the possible biological sources that can govern behavior, and one is left not knowing what insight the model provides. The array of biological processes listed are so variable in dynamics and meaning, that their explanation of what govern M is at best unsatisfying. Molecular dynamics models that generate distributions can point to certain properties of the model, such as the binding kinetics (on and off rates, etc.) as explanations for the mechanisms generating the distributions, and therefore point to how a change in the biology affects the stochasticity of the process. It is unclear how this model provides such a connection, especially taken in aggregate with the previous weakness.

Providing a roadmap of how to think about the processes generating M, the meaning of those processes in search, and potential frameworks that are more constrained and with more precise biological underpinning (beyond the array of possibilities described) would go a long way to assuaging the weaknesses.

Comments on revised version:

The authors have addressed the main concerns of the manuscript.

Reviewer #2 (Public review):

The manuscript by Lacy et al. is well written, with a clear and compelling introduction that effectively conveys the significance of the study. The methods are appropriate and well-executed, and the results, both in the main text and supplementary materials, are presented in a clear and detailed manner. The authors interpret their findings with appropriate caution.

This work makes a valuable contribution to our understanding of the evolution of complementary sex determination (CSD) in ants. In particular, it provides important evidence for the ancient origin of a non-coding locus implicated in sex determination, and shows that, remarkably, this sex locus is conserved even in an ant species with a non-canonical reproductive system that typically does not produce males. I found this to be an excellent and well-rounded study, carefully analyzed and well contextualized.

That said, I do have a few minor comments, primarily concerning the discussion of the potential 'ghost' CSD locus. While the authors acknowledge (line 367) that they currently have no data to distinguish among the alternative hypotheses, I found the evidence for an additional CSD locus presented in the results (lines 261-302) somewhat limited and at times a bit difficult to follow. I wonder whether further clarification or supporting evidence could already be extracted from the existing data. Specifically:

(1) Line 268: I doubt the relevance of comparing the proportion of diploid males among all males between lines A and B to infer the presence of additional CSD loci. Since the mechanisms producing these two types of males differ, it might be more appropriate to compare the proportion of diploid males among all diploid offspring. This ratio has been used in previous studies on CSD in Hymenoptera to estimate the number of sex loci (see, for example, Cook 1993, de Boer et al. 2008, 2012, Ma et al. 2013, and Chen et al., 2021). The exact method might not be applicable to clonal raider ants, but I think comparing the percentage of diploid males among the total number of (diploid) offspring produced between the two lineages might be a better argument for a difference in CSD loci number.

(2) If line B indeed carries an additional CSD locus, one would expect that some females could be homozygous at the ANTSR locus but still viable, being heterozygous only at the other locus. Do the authors detect any females in line B that are homozygous at the ANTSR locus? If so, this would support the existence of an additional, functionally independent CSD locus.

(3) Line 281: The description of the two tra-containing CSD loci as "conserved" between Vollenhovia and the honey bee may be misleading. It suggests shared ancestry, whereas the honey bee csd gene is known to have arisen via a relatively recent gene duplication from fem/tra (10.1038/nature07052). It would be more accurate to refer to this similarity as a case of convergent evolution rather than conservation.

(4) Finally, since the authors successfully identified multiple alleles of the first CSD locus using previously sequenced haploid males, I wonder whether they also observed comparable allelic diversity at the candidate second CSD locus. This would provide useful supporting evidence for its functional relevance.

Overall, these are relatively minor points in the context of a strong manuscript, but I believe addressing them would improve the clarity and robustness of the authors' conclusions.

Reviewer #2 (Public review):

Summary:

The authors apply the recently developed VARX model, which explicitly models intrinsic dynamics and the effect of extrinsic inputs, to simulated data and intracranial EEG recordings. This method provides a directed method of 'intrinsic connectivity'. They argue this model is better suited to the analysis of task neuroimaging data because it separates the intrinsic and extrinsic activity. They show: that intrinsic connectivity is largely unaltered during a movie-watching task compared to eyes open rest; intrinsic noise is reduced in the task; and there is intrinsic directed connectivity from sensory to higher-order brain areas.

Strengths:

(1) The paper tackles an important issue with an appropriate method.

(2) The authors validated their method on data simulated with a neural mass model.

(3) They use intracranial EEG, which provides a direct measure of neuronal activity.

(4) Code is made publicly available and the paper is written well.

Comments on revisions:'

The authors have addressed my comments.

Reviewer #3 (Public review):

Summary:

Protein-DNA interactions and sequence readout represent a challenging and rapidly evolving field of study. Recognizing the complexity of this task, the authors have developed a compact and elegant model. They applied well-established approaches to address a difficult problem, effectively enhancing the information extracted from sparse contact maps by integrating an artificial decoy sequence set and available experimental data. This has resulted in a practical tool that can be adapted for use with other proteins.

Strengths:

The authors integrate sparse information with available experimental data to construct a model whose utility extends beyond the limited set of structures used for training.

A comprehensive methods section is included, ensuring reproducibility.

The authors provide a well-represented performance comparison between their model and other existing models.

Additionally, the authors have shared their model as a GitHub project, reflecting their commitment to research transparency.

Weaknesses:

The coarse-graining procedure is quite convoluted, but the authors provide reasoning for the proposed scheme. The authors acknowledge discrepancies between data-driven and simulation models.

Reviewer #2 (Public review):

Summary:

The authors used deep full-length single-cell sequencing to study the human photoreceptor development, with a particular emphasis on the characteristics of photoreceptors that may contribute to retinoblastoma.

Strengths:

This single-cell study captures gene regulation in photoreceptors across different developmental stages, defining post-mitotic cone and rod populations by highlighting their unique gene expression profiles through analyses such as RNA velocity and SCENIC. By leveraging full-length sequencing data, the study identifies differentially expressed isoforms of NRL and THRB in L/M cone and rod precursors, illustrating the dynamic gene regulation involved in photoreceptor fate commitment. Additionally, the authors performed high-resolution clustering to explore markers defining developing photoreceptors across the fovea and peripheral retina, particularly characterizing SYK's role in the proliferative response of cones in the RB loss background. The study provides an in-depth analysis of developing human photoreceptors, with the authors conducting thorough analyses using full-length single-cell RNA sequencing. The strength of the study lies in its design, which integrates single-cell full-length RNA-seq, long-read RNA-seq, and follow-up histological and functional experiments to provide compelling evidence supporting their conclusions. The model of cell type-dependent splicing for NRL and THRB is particularly intriguing. Moreover, the potential involvement of the SYK and MYC pathways with RB in cone progenitor cells aligns with previous literature, offering additional insights into RB development.

Weaknesses:

The manuscript feels somewhat unfocused, with a lack of a strong connection between the analysis of developing photoreceptors, which constitutes the bulk of the manuscript, and the discussion on retinoblastoma. Additionally, given the recent publication of several single-cell studies on developing human retina, it is important for the authors to cross-validate their findings and adjust their statements where appropriate.

Comments on revisions:

The authors have done quite thorough work addressing concerns raised by myself and other reviewers. The identification of unresolved developing state of rod/cone precursor cell is interesting and intriguing. I do not have much more to add.

Reviewer #2 (Public review):

In this study, the authors used scanning electron microscopy (SEM) to image and analyze eleven Utah multielectrode arrays (including eight chronically implanted in four macaques). Four of the eight arrays had previously been used to deliver electrolytic lesions. Each intact electrode was scored in five damage categories. They found that damage disproportionately occurred to the outer edges of arrays. Importantly, the authors conclude that their electrolytic Lesioning protocol does not significantly increase material degradation compared to normal chronic use without lesion. Additionally, the authors have released a substantial public dataset of single-electrode SEM images of explanted Utah arrays.

The paper is well-written and addresses an important stability issue for long-term chronically implanted array recordings and electrolytic lesioning, which is relevant to both basic science and translational research. By comparing lesioning and non-lesioning electrodes on the same array and within the same animal, the study effectively controls for confounds related to the animal and surgical procedures. The shared dataset, accessible via interactive plots, enhances transparency and serves as a valuable reference for future investigations. Below, we outline some major and minor concerns that could help improve the work.

Major concerns:

(1) Electrode impedance is a critical measurement to evaluate the performance of recording electrodes. It would be helpful if the authors could provide pre-explant and post-explant impedance values for each electrode alongside the five SEM damage scores. This would allow the readers to assess how well the morphological scores align with functional degradation.

(2) The lesion parameters differ across experiments and electrodes. It would be helpful if the authors could evaluate whether damage scores (and/or impedance changes) correlate with total charge, current amplitude, duration, or frequency.

Reviewer #2 (Public review):

Summary:

This is the first study to show how a L-R bias in the relationship between numerical magnitude and space depends on brain lateralisation, and moreover, how is modulated by in ovo conditions.

Strengths:

Novel methodology for investigating the innateness and neural basis of an L-R bias in the relationship between number and space.

Weaknesses:

I would query the way the experiment was contextualised. They ask whether culture or innate pre-wiring determines the 'left-to-right orientation of the MNL [mental number line]'.

The term, 'Mental Number Line' is an inference from experimental tasks. One of the first experimental demonstrations of a preference or bias for small numbers in the left of space and larger numbers in the right of space, was more carefully described as the spatial-numerical association of response codes - the SNARC effect (Dehaene, S., Bossini, S., & Giraux, P. (1993). The mental representation of parity and numerical magnitude. Journal of Experimental Psychology: General, 122, 371-396).

This has meant that the background to the study is confusing. First, the authors note, correctly, that many other creatures, including insects, can show this bias, though in none of these has neural lateralisation been shown to be a cause. Second, their clever experiment shows that an experimental manipulation creates the bias. If it were innate and common to other species, the experimental manipulation shouldn't matter. There would always be an L-R bias. Third, they seem to be asserting that humans have a left-to-right (L-R) MNL. This is highly contentious, and in some studies, reading direction affects it, as the original study by Dehaene et al showed; and in others, task affects direction (e.g. Bachtold, D., Baumüller, M., & Brugger, P. (1998). Stimulus-response compatibility in representational space. Neuropsychologia, 36, 731-735, not cited). Moreover, a very careful study of adult humans, found no L-R bias (Karolis, V., Iuculano, T., & Butterworth, B. (2011), not cited, Mapping numerical magnitudes along the right lines: Differentiating between scale and bias. Journal of Experimental Psychology: General, 140(4), 693-706). Indeed, Rugani et al claim, incorrectly, that the L-R bias was first reported by Galton in 1880. There are two errors here: first, Galton was reporting what he called 'visualised numerals', which are typically referred to now as 'number forms' - spontaneous and habitual conscious visual representations - not an inference from a number line task. Second, Galton reported right-to-left, circular, and vertical visualised numerals, and no simple left-to-right examples (Galton, F. (1880). Visualised numerals. Nature, 21, 252-256.). So in fact did Bertillon, J. (1880). De la vision des nombres. La Nature, 378, 196-198, and more recently Seron, X., Pesenti, M., Noël, M.-P., Deloche, G., & Cornet, J.-A. (1992). Images of numbers, or "When 98 is upper left and 6 sky blue". Cognition, 44, 159-196, and Tang, J., Ward, J., & Butterworth, B. (2008). Number forms in the brain. Journal of Cognitive Neuroscience, 20(9), 1547-1556.

If the authors are committed to chicks' MN Line they should test a series of numbers showing that the bias to the left is greater for 2 and 3 than for 4, etc.

What does all this mean? I think that the paper should be shorn of its misleading contextualisation, including the term 'Mental Number Line'. The authors also speculate, usefully, on why chicks and other species might have a L-R bias. I don't think the speculations are convincing, but at least if there is an evolutionary basis for the bias, it should at least be discussed.

This paper is very interesting with its focus on why the L-R bias exists, and where and why it does not.

Reviewer #2 (Public review):

Summary:

In this study, Ye et al. have developed a theoretical model of osmotic pressure adaptation by osmolyte production and wall synthesis.

Strengths:

They validate their model predictions of a rapid increase in growth rate on osmotic shock experimentally using fission yeast. The study has several interesting insights which are of interest to the wider community of cell size and mechanics.

Comments on revisions:

The authors have in the revised manuscript addressed the aspects of the writing that were unclear. , that are listed previously as major and minor comments. We believe the issues raised by this reviewer have been adequately addressed in the manuscript.

Reviewer #4 (Public review):

Thank you for the opportunity to provide a peer-review of this manuscript, which I first reviewed in 2023 under the title of '241,000 to 335,000 Years Old Rock Engravings Made by Homo naledi in the Rising Star Cave system, South Africa'. My review is brief as the authors state they have made "relatively minimal changes", so most of the comments I made in 2023 still stand. Some of the language is a little more temperate but the main issues of this potentially landmark study remain and undermine scientific acceptance of the findings claim. The fact that this is an initial report does not excuse it from the normal conventions of building arguments supported by empirical data. Again, the absence of a rock art expert on the authorial team causes recurring weaknesses still to be evident (would one ask a rock art expert to analyse a new fossil hominin skull for example?). Specifically, there are two major issues that need to be resolved before there is necessary and sufficient cause to assign the term 'rock engravings' to the marks in the Dinaledi chamber. These are authorship and dating.

 Authorship: The assertion that the 'rock engravings' are anthropogenic remains unsupported by empirical evidence, with a number of possible natural factors that could just as likely have caused the marks. Not to use image enhancements - which is standard in most rock art research and has been for some time - is a critical omission. The concerns stated about AI and data standards are not developed and the authors are directed to the literature in this field, for example this 2025 overview - https://www.sciencedirect.com/science/article/pii/S1296207424002516. Again, having a rock art expert would show the AI concern to be valid but easily addressed using Data Standards. In the almost 2 years since the first pre-print was released, there has been ample time for high resolution photographs and scans of the purported 'rock engravings'; analysis of which by relevant experts could properly physically characterise the marks and thus establish more or less likely agents for their production. European-based researchers in particular has utilised this approach on material such as the Blombos ochre and marked bone from Europe and Africa. None of these methods is invasive or destructive.

To then go on and link Homo naledi to these markings is premature, especially when this landscape has been home to multiple hominins. Most rock art sites do not contain the physical bodily remains of their makers so we assign authorship based on dating (such as for Neanderthal era art in Europe for example); the second critical issue in this report:

 Dating: There is no direct or closely associated chronometric dating of the 'rock engravings' or their immediate context, so the age range claimed is unsupported. Rock art dating is notoriously difficult - and why researchers closely scrutinise dates produced. In this case, however, the chronological context is physically so far removed from these rock markings, as to be misleading at best and need to be discounted until a proper programme of dating has commenced. The sources cited for rock art dating tend to be out of date and it would be standard practice to have a geochronologist assess the rock-marked areas and then establish dating protocols.

Authorship and dating are cornerstone of archaeological/paleoanthropological work and need to established in the first instance. Until that has been done commensurate with current standards in global rock art research this potentially landmark finding cannot be taken as probable, only as possible. This is a pity as the last decade or so has revolutionised our understanding of the socially complex world multiple hominin species lived in, and marked in utilitarian and symbolic ways. The conditions for acceptance of ancient rock art has thus never been better, but the Dinaledi example needs to revisit research first principles around authorship and dating to be included as a credible part of this larger context. It would have been good to see a commitment to a coherent research programme to this end for this case study.

I hope these observations are useful. As above I keep them short as there has been minimal change to the 2023 ms, and my detailed comments on that remain with the first version of the work.

Reviewer #2 (Public review):

Summary:

The authors present an interesting paper where they test the antagonistic pleiotropy theory. Based on this theory they hypothesize that genetic variants associated with later onset of age at menarche and age at first birth have a positive causal effect on a multitude of health outcomes later in life, such as epigenetic aging and prevalence of chronic diseases. Using a mendelian randomization and colocalization approach, the authors show that SNPs associated with later age at menarche are associated with delayed aging measurements, such as slower epigenetic aging and reduced facial aging and a lower risk of chronic diseases, such as type 2 diabetes and hypertension. Moreover, they identify 128 fertility-related SNPs that associate with age-related outcomes and they identified BMI as a mediating factor for disease risk, discussing this finding in the context of evolutionary theory.

Strengths:

The major strength of this manuscript is that it addresses the antagonistic pleiotropy theory in aging. Aging theories are not frequently empirically tested although this is highly necessary. The work is therefore relevant for the aging field as well as beyond this field, as the antagonistic pleiotropy theory addresses the link between fitness (early life health and reproduction) and aging.

Weaknesses:

The authors report evidence in support of the antagonistic pleiotropy theory in aging and discuss the discuss the disposable soma theory. Although both theories describe distinct mechanisms, separating them in empirical research is complicated and needs further studies in future research.

Reviewer #2 (Public review):

Summary:

The investigation provides a computational as well as biochemical insights into the (un)binding mechanisms of a pair of psychoactive substances into cannabinoid receptors. A combination of molecular dynamics simulation and a set of state-of-the art statistical post-processing techniques were employed to exploit GPCR-ligand dynamics.

Strengths:

The strength of the manuscript lies in usage and comparison of TRAM as well as Markov state modelling (MSM) for investigating ligand binding kinetics and thermodynamics. Usually MSMs have been more commonly used for this purpose. But as the authors have pointed out, implicit in the usage of MSMs lie the assumption of detailed balance, which would not hold true for many cases especially those with skewed binding affinities. In this regard, the author's usage of TRAM which harnesses both biased and unbiased simulations for extracting the same, provides a more appropriate way-out.

Weaknesses:

(1) While the authors have used TRAM (by citing MSM to be inadequate in these cases), the thermodynamic comparisons of both techniques provide similar values. In this case, one would wonder what advantage TRAM would hold in this particular case.

(2) The initiation of unbiased simulations from previously run biased metadynamics simulations would almost surely introduce hysteresis in the analysis. The authors need to address these issues.

(3) The choice of ligands in the current work seems very forced and none of the results compare directly with any experimental data. An ideal case would have been to use the seminal D.E. Shaw research paper on GPCR/ligand binding as a benchmark and then show how TRAM, using much lesser biased simulation times, would fare against the experimental kinetics or even unbiased simulated kinetics of the previous report

(4) The method section of the manuscript seems to suggest all the simulations were started from a docked structure. This casts doubt on the reliability of the kinetics derived from these simulations that were spawned from docked structure, instead of any crystallographic pose. Ideally, the authors should have been more careful in choosing the ligands in this work based on the availability of the crystallographic structures.

(5) The last part of using a machine learning-based approach to analyse allosteric interaction seems to be very much forced, as there are numerous distance-based more traditional precedent analyses that do a fair job of identifying an allosteric job.

(6) While getting busy with the methodological details of TRAM vs MSM, the manuscript fails to share with sufficient clairty what the distinctive features of two ligand binding mechanisms are.

Comments on revisions:

The authors have addressed most of the queries of the reviewer in an adequate manner. However, The current code availability section just provides the link to Python files to generate the plots. It is not very useful in its current form. The code availability section should provide a proper GitHub page that shows the usage of TRAM for the readers to execute. While Pyemma has been cited for TRAM, a python note book to reproduce the TRAM would be very instructive.

Reviewer #2 (Public review):

Summary:

The authors use a genetic screen in C. elegans to investigate the physiological roles of polyunsaturated fatty acids (PUFAs). They screen for mutations that rescue fat-2 mutants, which have strong reductions in PUFAs. As a result, either mutations in fat-2 itself, or mutations in genes involved in the HIF-1 pathway, were found to rescue fat-2 mutants. Mutants in the HIF-1 pathway rescue fat-2 mutants by boosting its catalytic activity (via upregulated Fe2+). Thus, the authors show that in the context of fat-2 mutation, the sole genetic means to rescue PUFA insufficiency is to restore PUFA levels.

Strengths:

As C. elegans can produce PUFAs de novo as essential lipids, the genetic model is well suited to study the fundamental roles of PUFAs. The genetic screen finds mutations in convergent pathways, suggesting that it has reached near-saturation. The authors extensively validate the results of the screening and provide sufficient mechanistic insights to show how PUFA levels are restored in HIF-1 pathway mutants. As many of the mutations found to rescue fat-2 mutants are of gain-of-function, it is unlikely that similar discoveries could have been made with other approaches like genome-wide CRISPR screenings, making the current study distinctive. Consequently, the study provides important messages. First, it shows that PUFAs are essential for life. The inability to genetically rescue PUFA deficiency, except for mutations that restore PUFA levels, suggests that they have pleiotropic essential functions. In addition, the results suggest that the most essential functions of PUFAs are not in fluidity regulation, which is consistent with recent reviews proposing that the importance of unsaturation goes beyond fluidity (doi: 10.1016/j.tibs.2023.08.004 and doi: 10.1101/cshperspect.a041409). Thus, the study provides fundamental insights about how membrane lipid composition can be linked to biological functions.

Weaknesses:

The authors did a lot of efforts to answer the questions that arose through peer review, and now all the claims seem to be supported by experimental data. Thus, I do not see obvious weaknesses. Of course, it remains still unclear what PUFAs do beyond fluidity regulation, but this is something that cannot be answered from a single study. I just have one final proposition to make.

I still do not agree with the answer to my previous comment 6 regarding Figure S2E. The authors claim that hif-1(et69) suppresses fat-2(wa17) in a ftn-2 null background (in Figure S2 legend for example). To claim so, they would need to compare the triple mutant with fat-2(wa17);ftn-2(ok404) and show some rescue. However, we see in Figure 5H that ftn-2(ok404) alone rescues fat-2(wa17). Thus, by comparing both figures, I see no additional effect of hif-1(et69) in an ftn-2(ok404) background. I actually think that this makes more sense, since the authors claim that hif-1(et69) is a gain-of-function mutation that acts through suppression of ftn-2 expression. Thus, I would expect that without ftn-2 from the beginning, hif-1(et69) does not have an additional effect, and this seems to be what we see from the data. Thus, I would suggest that the authors reformulate their claims regarding the effect of hif-1(et69) in the ftn-2(ok404) background, which seems to be absent (consistently with what one would expect).

Reviewer #2 (Public review):

This work is a nice contribution to the literature in articulating a specific, testable theory of how psychedelics act to generate hallucinations and plasticity. The connection to replay, however - including in the title, abstract, and framing throughout the paper - is not well fleshed out.

In particular, the paper's framing seems to conflate replay, dreams, and top-down processing, but these are not one and the same. Picard-Delano et al. TICS 2023 provides a useful review of the differences between replay and dreams. One key point is that most replay has been observed during NREM sleep, but our canonically bizarre / vivid dreams occur during REM. Top-down connections have also been proposed to be used for many processes aside from replay. The paper would benefit from much more precision and nuance on these points.

I believe the paper is missing demonstrations or speculation about how plasticity under various doses of psychedelics relates to changes in performance, which would be an important link to the replay-dependent learning literature.

Are there renderings available for 'ripple' effects of psychedelics that could be included, to allow readers to compare the model's hallucinations to humans'? Short of this, it would be useful to have a more detailed description of what rippling is. (For those readers without firsthand knowledge!) It is currently difficult to assess how close the match is.

Reviewer #2 (Public review):

Summary:

This methods paper proposes two changes to classic RSA, a popular method to probe neural representation in neuroimaging experiments: computing RSA at row/column level of RDM, and using mixed linear modeling to compute second-level statistics, using the individual row/columns to estimate a random effect of stimulus. The benefit of the new method is demonstrated using simulations and a re-analysis of a prior fMRI dataset on object perception and memory encoding.

Strengths:

(1) The paper is clearly written and features clear illustrations of the proposed method.

(2) The combination of simulation and real data works well, with the same factors being examined in both simulations and real data, resulting in a convincing demonstration of the benefits of tRSA in realistic experimental scenarios.

(3) I find the author's claim that tRSA is a promising approach to perform more complete modeling of cogneuro data, but also to conceptualize representation at the single trial/event level (cf Discussion section on P42), quite appealing.

Weaknesses:

(1) While I generally welcome the contribution (see above), I take some issue with the accusatory tone of the manuscript in the Introduction. The text there (using words such as 'ignored variances', 'errouneous inferences', 'one must', 'not well-suited', 'misleading') appears aimed at turning cRSA in a 'straw man' with many limitations that other researchers have not recognized but that the new proposed method supposedly resolves. This can be written in a more nuanced, constructive manner without accusing the numerous users of this popular method of ignorance.

(2) The described limitations are also not entirely correct, in my view: for example, statistical inference in cRSA is not always done using classic parametric statistics such as t-tests (cf Figure 1): the rsatoolbox paper by Nili et al. (2014) outlines non-parametric alternatives based on permutation tests, bootstrapping and sign tests, which are commonly used in the field. Nor has RSA ever been conducted at the row/column level (here referred to by the authors as 'trial level'; cf King et al., 2018).

(3) One of the advantages of cRSA is its simplicity. Adding linear mixed effects modeling to RSA introduces a host of additional 'analysis parameters' pertaining to the choice of the model setup (random effects, fixed effects, interactions, what error terms to use) - how should future users of tRSA navigate this?

(4) Here, only a single real fMRI dataset is used with a quite complicated experimental design for the memory part; it's not clear if there is any benefit of using tRSA on a simpler real dataset. What's the benefit of tRSA in classic RSA datasets (e.g., Kriegeskorte et al., 2008), with fixed stimulus conditions and no behavior?

(5) The cells of an RDM/RSM reflect pairwise comparisons between response patterns (typically a brain but can be any system; cf Sucholutsky et al., 2023). Because the response patterns are repeatedly compared, the cells of this matrix are not independent of one another. Does this raise issues with the validity of the linear mixed effects model? Does it assume the observations are linearly independent?

(6) The manuscript assumes the reader is familiar with technical statistical terms such as Type I/II error, sensitivity, specificity, homoscedasticity assumptions, as well as linear mixed models (fixed effects, random effects, etc). I am concerned that this jargon makes the paper difficult to understand for a broad readership or even researchers currently using cRSA that might be interested in trying tRSA.

(7) I could not find any statement on data availability or code availability. Given that the manuscript reuses prior data and proposes a new method, making data and code/tutorials openly available would greatly enhance the potential impact and utility for the community.

References

King, M. L., Groen, I. I., Steel, A., Kravitz, D. J., & Baker, C. I. (2019). Similarity judgments and cortical visual responses reflect different properties of object and scene categories in naturalistic images. NeuroImage, 197, 368-382.

Kriegeskorte, N., Mur, M., Ruff, D. A., Kiani, R., Bodurka, J., Esteky, H., ... & Bandettini, P. A. (2008). Matching categorical object representations in inferior temporal cortex of man and monkey. Neuron, 60(6), 1126-1141.

Nili, H., Wingfield, C., Walther, A., Su, L., Marslen-Wilson, W., & Kriegeskorte, N. (2014). A toolbox for representational similarity analysis. PLoS computational biology, 10(4), e1003553.

Sucholutsky, I., Muttenthaler, L., Weller, A., Peng, A., Bobu, A., Kim, B., ... & Griffiths, T. L. (2023). Getting aligned on representational alignment. arXiv preprint arXiv:2310.13018.

Reviewer #2 (Public review):

Summary:

The authors aimed to show that connectivity patterns within spinal circuits composed of specific excitatory and inhibitory connectivity and with varying degrees of modularity could achieve tail beats at various frequencies as well as proper left-right coordination and rostrocaudal propagation speeds.

Strengths:

The model is simple, and the connectivity patterns explored are well supported by the literature.

The conclusions are intuitive and support many experimental studies on zebrafish spinal circuits for swimming. The simulations provide strong support for the sufficiency of connectivity patterns to produce and control many hallmark features of swimming in zebrafish.

Weaknesses:

I only have two minor suggestions:

(1) Figure 1A, if I interpret Figure 1B correctly, should there not be long descending projections as well that don't seem to be illustrated?

(2) Page 5, It would be good to define what is meant by slow and fast here, as this definition changes with age in zebrafish (what developmental age)?

Reviewer #2 (Public review):

Summary:

This comparative study of macaque species and the type of social interaction is both ambitious and inevitably comes with a lot of caveats. The overall conclusion is that more intolerant species have a larger amygdala. There are also opposing development profiles regarding amygdala volume depending on whether it is a tolerant or intolerant species.

To achieve any sort of power, they have combined data from 4 centres, which have all used different scanning methods, and there are some resolution differences. The authors have also had to group species into 4 classifications - again to assist with any generalisations and power. They have focussed on the volumes of two structures, the amygdala and the hippocampus, which seems appropriate. Neither structure is homogeneous and so it may well be that a targeted focus on specific nuclei or subfields would help (the authors may well do this next) - but as the variables would only increase further along with the number of potential comparisons, alongside small group numbers, it seems only prudent to treat these findings are preliminary. That said, it is highly unlikely that large numbers of macaque brains will become available in the near future.

This introduction is by way of saying that the study achieves what it sets out to do, but there are many reasons to see this study as preliminary. The main message seems to be twofold: (1) that more intolerant species have relatively larger amygdalae, and (2) that with development, there is an opposite pattern of volume change (increasing with age in intolerant species and decreasing with age in tolerant species). Finding 1 is the opposite of that predicted in Table 1 - this is fine, but it should be made clearer in the Discussion that this is the case, otherwise the reader may feel confused. As I read it, the authors have switched their prediction in the Discussion, which feels uncomfortable.

It is inevitable that the data in a study of this complexity are all too prone to post hoc considerations, to which the authors indulge. In the case of Grade 1 species, the individuals have a lot to learn, especially if they are not top of the hierarchy, but at the same time, there are fewer individuals in the troop, making predictions very tricky. As noted above, I am concerned by the seemingly opposite predictions in Table 1 and those in the Discussion regarding tolerance and amygdala volume. (It may be that the predictions in Table 1 are the opposite of how I read them, in which case the Table and preceding text need to align.)

Reviewer #2 (Public review):

Summary:

The authors produce a new tool, BEHAV3D to analyse tracking data and to integrate these analyses with large and small scale architectural features of the tissue. This is similar to several other published methods to analyse spatio-temporal data, however, the connection to tissue features is a nice addition, as is the lack of requirement for coding. The tool is then used to analyse tracking data of tumour cells in diffuse midline glioma. They suggest 7 clusters exist within these tracks and that they differ spatially. They ultimately suggest that there these behaviours occur in distinct spatial areas as determined by CytoMAP.

Strengths:

- The tool appears relatively user-friendly and is open source. The combination with CytoMAP represents a nice option for researchers.

- The identification of associations between cell track phenotype and spatial features is exciting and the diffuse midline glioma data nicely demonstrates how this could be used.

Weaknesses:

- The revision has dealt with many concerns, however, the statistics generated by the process are still flawed. While the statistics have been clarified within the legends and this is a great improvement in terms of clarity the underlying assumptions of the tests used are violated. The problem is that individual imaging positions or tracks are treated as independent and then analysed by ANOVA. As separate imaging positions within the same mouse are not independent, nor are individual cells within a single mouse, this makes the statistical analyses inappropriate. For a deeper analysis of this that is feasible within a review please see Lord, Samuel J., et al. "SuperPlots: Communicating reproducibility and variability in cell biology." The Journal of cell biology 219.6 (2020): e202001064. Ultimately, while this is a neat piece of software facilitating the analysis of complex data, the fact that it will produce flawed statistical analysis is a major problem. This problem is compounded by the fact that much imaging analysis has been analysed in this inappropriate manner in the past, leading to issues of interpretation and ultimately reproducibility.

Reviewer #2 (Public review):

Gekko et al investigate the impact of perturbing mitochondrial during early embryo development, through modulation of the mitochondrial fission protein Drp1 using Trim-Away technology. They aimed to validate a role for mitochondrial dynamics in modulating chromosomal segregation, mitochondrial inheritance and embryo development and achieve this through the examination of mitochondrial and endoplasmic reticulum distribution, as well as actin filament involvement, using targeted plasmids, molecular probes and TEM in pronuclear stage embryos through the first cleavages divisions. Drp1 deletion perturbed mitochondrial distribution, leading to asymmetric partitioning of mitochondria to the 2-cell stage embryo, prevented appropriate chromosomal segregation and culminated in embryo arrest. Resultant 2-cell embryos displayed altered ATP, mtDNA and calcium levels. Microinjection of Drp1 mRNA partially rescued embryo development. A role for actin filaments in mitochondrial inheritance is described, however the actin-based motor Myo19 does not appear to contribute.

Overall, this study builds upon their previous work and provides further support for a role of mitochondrial dynamics in mediating chromosomal segregation and mitochondrial inheritance. In particular, Drp1 is required for redistribution of mitochondria to support symmetric partitioning and support ongoing development.

Strengths:<br /> The study is well designed, the methods appropriate and the results clearly presented. The findings are nicely summarised in a schematic.

The addition of further quantification, including mitochondrial cluster size, elongation/aspect ratio and ROS, as requested by the reviewers, has provided further evidence for the impact of Drp1 depletion on mitochondrial morphology and function.

Understanding the role of mitochondria in binucleation and mitochondrial inheritance is of clinical relevance for patients undergoing infertility treatment, particularly those undergoing mitochondrial replacement therapy.

Weaknesses (original manuscript):<br /> The authors first describe the redistribution of mitochondria during normal development, followed by alterations induced by Drp1 depletion. It would be useful to indicate time post-hCG for imaging of fertilised zygotes (first paragraph of the results/Figure 1) to compare with subsequent Drp1 depletion experiments.

It is noted that Drp1 protein levels were undetectable 5h post-injection, suggesting earlier times were not examined, yet in Figure 3A it would seem that aggregation has occurred within 2 hours (relative to Figure 1).

Mitochondria appear to be slightly more aggregated in Drp1 fl/fl embryos than in control, though comparison with untreated controls does not appear to have been undertaken. There also appears to be some variability in mitochondrial aggregation patterns following Drp1 depletion (Figure 2-suppl 1 B) which are not discussed.

The authors use western blotting to validate the depletion of Drp1, however do not quantify band intensity. It is also unclear whether pooled embryo samples were used for western blot analysis.

Likewise, intracellular ROS levels are examined however quantification is not provided. It is therefore unclear whether 'highly accumulated levels' are of significance or related to Drp1 depletion.

In previous work, Drp1 was found to have a role as a spindle assembly checkpoint (SAC) protein. It is therefore unclear from the experiments performed whether aggregation of mitochondria separating the pronuclei physically (or other aspects of mitochondrial function) prevents appropriate chromosome segregation or whether Drp1 is acting directly on the SAC.

Weaknesses (revised manuscript):

The only remaining weakness is that the authors have not undertaken additional experiments to clarify any role for mitochondrial transport following Drp1 depletion.

Reviewer #2 (Public review):

Summary:

Primates are a particularly important and oft-applied model for understanding the evolution of, e.g., life history and senescence in humans. Although there is a growing body of work on aging in primates, there are three components of primate senescence research that have been underutilized or understudied: (1) longitudinal datasets, (2) wild populations, and (3) (stone) tool-use behaviors. Therefore, the goal of this study was to (1) use a 17-year longitudinal dataset (2) of wild chimpanzees in the Bossou forest, (3) visiting a site for field experiments on nut-cracking. They sampled and analyzed data from five field seasons for five chimpanzees of old age. From this sample, Howard-Spink and colleagues noted a decline in tool-use and tool-use efficiency in some individuals, but not in others. The authors then conclude that there is a measurable effect of senescence on chimpanzee behavior, but that it varies individually. The study has major intellectual value as a building block for future research, but there are several major caveats.

Strengths:

With this study, Howard-Spink and colleagues make a foray into a neglected topic of research: the impact of the physiological and cognitive changes due to senescence on stone tool use in chimpanzees. Based on novelty alone, this is a valuable study. The authors cleverly make use of a longitudinal record covering 17 years of field data, which provides a window into long-term changes in the behavior of wild chimpanzees, which I agree cannot be understood through cross-sectional comparisons.

The metrics of 'efficiency' (see caveats below) are suitable for measuring changes in technological behavior over time, as specifically tailored to the nut-cracking (e.g., time, number of actions, number of strikes, tool changes). The ethogram and the coding protocol are also suitable for studying the target questions and objectives. I would recommend, however, the inclusion of further variables that will assist in improving the amount of valid data that can be extrapolated (see also below).

With this pilot, Howard-Spink and colleagues have established a foundation upon which future research can be designed, including further investigation with the Bossou dataset and other existing video archives, but especially future targeted data collection, which can be designed to overcome some of the limits and confounds that can be identified in the current study.

Weaknesses:

Although I agree with the reasoning behind conducting this research and understand that, as the authors state, there are logistical considerations that have to be made when planning and executing such a study, there are a number of methodological and theoretical shortcomings that either need to be more explicitly stated by the authors or would require additional data collection and analysis.

One of the main limitations of this study is the small sample size. There are only 5 of the old-aged individuals, which is not enough to draw any inferences about aging for chimpanzees more generally. Howard-Spink and colleagues also study data from only five of the 17 years of recorded data at Bossou. The selection of this subset of data requires clarification: why were these intervals chosen, why this number of data points, and how do we know that it provides a representative picture of the age-related changes of the full 17 years?

With measuring and interpreting the 'efficiency' of behaviors, there are in-built assumptions about the goals of the agents and how we can define efficiency. First, it may be that efficiency is not an intentional goal for nut-cracking at all, but rather, e.g., productivity as far as the number of uncrushed kernels (cf. Putt 2015). Second, what is 'efficient' for the human observer might not be efficient for the chimpanzee who is performing the behavior. More instances of tool-switching may be considered inefficient, but it might also be a valid strategy for extracting more from the nuts, etc. Understanding the goals of chimpanzees may be a difficult proposition, but these are uncertainties that must be kept in mind when interpreting and discussing 'decline' or any change in technological behaviors over time.

For the study of the physiological impact of senescence of tool use (i.e., on strength and coordination), the study would benefit from the inclusion of variables like grip type and (approximate) stone size (Neufuss et al., 2016). The size and shape of stones for nut-cracking have been shown to influence the efficacy and 'efficiency' of tool use (i.e., the same metrics of 'efficiency' implemented by Howard-Spink et al. in the current study), meaning raw material properties are a potential confound that the authors have not evaluated.

Similarly, inter- and intraspecific variation in the properties of nuts being processed is another confound (Falótico et al., 2022; Proffitt et al., 2022). If oil palm nuts were varying year-to-year, for example, this would theoretically have an effect on the behavioral forms and strategies employed by the chimpanzees, and thus, any metric of efficiency being collected and analyzed. Further, it is perplexing that the authors analyze only one year where the coula nuts were provided at the test site, but these were provided during multiple field seasons. It would be more useful to compare data from a similar number of field seasons with both species if we are to study age-related changes in nut processing over time (one season of coula nut-cracking certainly does not achieve this).

Both individual personality (especially neophilia versus neophobia; e.g., Forss & Willems, 2022) and motivation factors (Tennie & Call, 2023) are further confounds that can contribute to a more valid interpretation of the patterns found. To draw any conclusions about age-related changes in diet and food preferences, we would need to have data on the overall food intake/preferences of the individuals and the food availability in the home range. The authors refer briefly to this limitation, but the implications for the interpretation of the data are not sufficiently underlined (e.g., for the relevance of age-related decline in stone tool-use ability for individual survival).

Generally speaking, there is a lack of consideration for temporal variation in ecological factors. As a control for these, Howard-Spink and colleagues have examined behavioral data for younger individuals from Bossou in the same years, to ostensibly show that patterns in older adults are different from patterns in younger adults, which is fair given the available data. Nonetheless, they seem to focus mostly on the start and end points and not patterns that occur in between. For example, there is a curious drop in attendance rate for all individuals in the 2008 season, the implications of which are not discussed by the authors.

As far as attendance, Howard-Spink and colleagues also discuss how this might be explained by changes in social standing in later life (i.e., chimpanzees move to the fringes of the social network and become less likely to visit gathering sites). This is not senescence in the sense of physiological and cognitive decline with older age. Instead, the reduced attendance due to changes in social standing seems rather to exacerbate signs of aging rather than be an indicator of it itself. The authors also mention a flu-like epidemic that caused the death of 5 individuals; the subsequent population decline and related changes in demography also warrant more discussion and characterization in the manuscript.

Understandably, some of these issues cannot be evaluated or corrected with the presented dataset. Nonetheless, these undermine how certain and/or deterministic their conclusions can really be considered. Howard-Spink et al. have not strongly 'demonstrated' the validity of relationships between the variables of the study. If anything, their cursory observations provide us with methods to apply and hypotheses to test in future studies. It is likely that with higher-resolution datasets, the individual variability in age-related decline in tool-use abilities will be replicated. For now, this can be considered a starting point, which will hopefully inspire future attempts to research these questions.

Falótico, T., Valença, T., Verderane, M. & Fogaça, M. D. Stone tools differences across three capuchin monkey populations: food's physical properties, ecology, and culture. Sci. Rep. 12, 14365 (2022).<br /> Forss, S. & Willems, E. The curious case of great ape curiosity and how it is shaped by sociality. Ethology 128, 552-563 (2022).<br /> Neufuss, J., Humle, T., Cremaschi, A. & Kivell, T. L. Nut-cracking behaviour in wild-born, rehabilitated bonobos (Pan paniscus): a comprehensive study of hand-preference, hand grips and efficiency. Am. J. Primatol. 79, e22589 (2016).<br /> Proffitt, T., Reeves, J. S., Pacome, S. S. & Luncz, L. V. Identifying functional and regional differences in chimpanzee stone tool technology. R. Soc. Open Sci. 9, 220826 (2022).<br /> Putt, S. S. The origins of stone tool reduction and the transition to knapping: An experimental approach. J. Archaeol. Sci.: Rep. 2, 51-60 (2015).<br /> Tennie, C. & Call, J. Unmotivated subjects cannot provide interpretable data and tasks with sensitive learning periods require appropriately aged subjects: A Commentary on Koops et al. (2022) "Field experiments find no evidence that chimpanzee nut cracking can be independently innovated". ABC 10, 89-94 (2023).

Comments on Revised Version (from BRE):

The authors have revised their methods to clarify why certain field seasons were chosen and have clarified aspects of their analysis relevant to this reviewer's concerns. The coula nut cracking data and results which were of a single season have now been restricted to the Supplementary. The revised discussion now includes a much more detailed limitations section including both ecological factors but also the effects of social aging. Stone tool size, grip and other factors are also acknowledged as being potentially important for measuring efficiency but the authors were unable to include in this study due to the nature of the dataset.

Reviewer #2 (Public review):

Summary:

The Commander complex is a key player in endosomal recycling which recruits cargo proteins and facilitates the formation of tubulo-vesicular carriers. Squiers et al found COMMD3, a subunit of the Commander complex, could interact directly with ARF1 and regulate endosomal recycling.

Strengths:

Overall, this is a nice study that provides some interesting knowledge on the function of the Commander complex.

Comments on revisions:

The authors have addressed all my previous concerns

Reviewer #2 (Public review):

Summary:

Ngo et al. use AlphaFold2 and Rosetta to model closed, open, and inactive states of the human ion channel hERG. Subsequent MD simulations and comparisons with experiment support the plausibility of their models.

Strengths:

Ngo et al. employ various computational methods to enhance AlphaFold2's prediction capabilities for the human voltage-gated potassium channel hERG. They guide AlphaFold2 to explore different protein conformations and states, including its open, closed, and inactivated forms, using targeted templates. Additionally, they applied the Rosetta FastRelax protocol with an implicit membrane to refine the conformation of each residue in the predictions and address steric clashes, along with molecular dynamics (MD) simulations to account for membrane-pore flexibility. The methodology is well-described, and the figures are clear and descriptive.

The authors have addressed some of the concerns raised during the first round of reviews. For instance, to mitigate potential bias in selecting the inactivated conformation, they evaluated conformational variability via backbone dihedral angles at specific residues in the selectivity filter and the drug binding sites. They also evaluated the top representative model from inactivated-state-sampling Cluster 3 (termed "AF ic3"), which was initially excluded. This model is now included in the revised manuscript as Figure S9a, b. MD simulations confirmed that this state could be a potential alternative open-state conformation. The authors also acknowledged the limitation of their study by not incorporating other enhanced sampling methods and AF3.

In the revised manuscript, the authors provided more extensive explanations of their methods. For example, they explained that their approach to template selection was guided by their experience-AlphaFold2 with larger templates often overly constraining predictions to the input structure, reducing its flexibility to explore alternative conformations. In contrast, smaller, targeted fragments increase the likelihood that AlphaFold2 will incorporate the desired structural features while predicting the rest of the protein. They also noted that pLDDT scores are not always reliable for selecting new or alternative conformations, citing proper references. They included a model from cluster 3 of the inactivated-state sampling process, which exhibited lower pLDDT scores to illustrate this further.

Another point raised by the reviewers was the exclusion of the N-terminal PAS domain due to GPU memory limitations and its impact on the study. This omission may overlook the PAS domain's potential roles in gating kinetics and allosteric effects on drug binding. The authors acknowledged these limitations in the main text and highlighted the need for future studies to explore these regions in greater detail. They also alluded to potential future research to address these points. Additionally, they have made some of their analysis scripts and tools available on GitHub as a community resource.

Weakness:

The primary issue with the study is the lack of a general pipeline or strategy that can be universally applied to any system, even if limited to ion channels or membrane proteins. A related paper assessed the conformational variability in voltage-sensing domains (VSDs) by applying both the default MSA depth and a range of reduced MSA depths to enhance conformational diversity (please see https://doi.org/10.1101/2025.03.12.642934). They generated 600 models for 32 members of the voltage-gated cation channel superfamily and demonstrated that AlphaFold2 can predict a range of diverse structures of the VSDs, representing activated, deactivated, and intermediate conformations, with more diversity observed for some VSDs compared to others.

The authors have addressed one of the reviewer's concerns about generalizability by including an example in Figure S14 of the modified text, showing how their approach can be applied to model another ion channel system. However, some outstanding questions remain: Is this method better suited for ion channels or membrane proteins with already solved structures and extensive research available? Can this pipeline be applied to other systems as well? Additionally, how does this method compare to other methods using MSA subsampling and other enhanced AF-based techniques to generate alternative conformations of proteins?

Reviewer #2 (Public review):

Summary:

This is important work that helps to uncover how the process of autophagy is initiated - via structural analyses of the initiating ULK1 complex. High resolution structural details and a mechanistic insight of this complex have been lacking and understanding how it assembles and functions is a major goal of a field that impacts many aspects of cell and disease biology. While we know components of the ULK1 complex are essential for autophagy, how they physically interact is far from clear. The work presented makes use of AlphaFold2 to structurally predict interaction sites between the different subunits of the ULK1 complex (namely ULK1, ATG13 and FIP200). Importantly, the authors go on to experimentally validate that these predicted sites are critical for complex formation by using site-directed mutagenesis and then go on to show that the three-way interaction between these components is necessary to induce autophagy in cells.

Strengths:

The data are very clear. Each binding interface of ATG13 (ATG13 with FIP300/ATG13 with ULK1) is confirmed biochemically with ITC and IP experiments from cells. Likewise, IP experiments with ULK1 and FIP200 also validate interaction domains. A real strength of the work is in the analyses of the consequences of disrupting ATG13's interactions in cells. The authors make CRISPR KI mutations of the binding interface point mutants. This is not a trivial task and is the best approach as everything is monitored under endogenous conditions. Using these cells the authors show that ATG13's ability to interact with both ULK1 and FIP200 is essential for a full autophagy response.

Weaknesses:

I think a main weakness here is the failure to acknowledge and compare results with an earlier preprint that shows essentially the same thing (https://doi.org/10.1101/2023.06.01.543278). Arguably, this earlier work is much stronger from a structural point of view as it relies not only on AlphaFold2 but also actual experimental structural determinations (and takes the mechanisms of autophagy activation further by providing evidence for a super complex between the ULK1 and VPS34 complexes). That is not to say that this work is not important, as in the least it independently helps to build a consensus for ULK1 complex structure. Another weakness is that the downstream "functional" consequences of disrupting the ULK1 complex are only minimally addressed. The authors perform a Halotag-LC3 autophagy assay, which essentially monitors the endpoint of the process. There are a lot of steps in between, knowledge of which could help with mechanistic understanding. Not in the least is the kinase activity of ULK1 - how is this altered by disrupting its interactions with ATG13 and/or FIP200?

Update:

I feel the authors have addressed my concerns in their revised manuscript

Reviewer #2 (Public review):

Summary

Bigge and colleagues use a sophisticated free-flight setup to study visuo-motor responses elicited in different parts of the visual field in the hummingbird hawkmoth. Hawkmoths have been previously shown to rely on translational optic flow information for flight control exclusively in the ventral and lateral parts of their visual field. Dorsally presented patterns, elicit a formerly completely unknown response - instead of using dorsal patterns to maintain straight flight paths, hawkmoths fly, more often, in a direction aligned with the main axis of the pattern presented (Bigge et al, 2021). Here, the authors go further and put ventral/lateral and dorsal visual cues into conflict. They found that the different visuomotor pathways act in parallel, and they identified a 'hierarchy': the avoidance of dorsal patterns had the strongest weight and optic flow-based speed regulation the lowest weight. The authors linked their behavioral results to visual scene statistics in the hawkmoths' natural environment. The partition of ventral and dorsal visuomotor pathways is well in line with differences in visual cue frequencies. The response hierarchy, however, seems to be dominated by dorsal features, that are less frequent, but presumably highly relevant for the animals' flight safety.

Strengths

The data are very interesting and unique. The manuscript provides a thorough analysis of free-flight behavior in a non-model organism that is extremely interesting for comparative reasons (and on its own). These data are both difficult to obtain and very valuable to the field.

Weaknesses

While the present manuscript clearly goes beyond Bigge et al, 2021, the advance could have perhaps been even stronger with a more fine-grained investigation of the visual responses in the dorsal visual field. Do hawkmoths, for example, show optomotor responses to rotational optic flow in the dorsal visual field?

I find the majority of the data, which are also the data supporting the main claims of the paper, compelling. However, the measurements of flight height are less solid than the rest and I think these data should be interpreted more carefully.

Reviewer #2 (Public review):

This manuscript asks an interesting and important question: what part of 'cerebellar' motor dysfunction is an acute control problem vs a compensatory strategy to the acute control issue? The authors use a cerebellar 'blockade' protocol, consisting of high frequency stimuli applied to the cerebellar peduncle which is thought to interfere with outflow signals. This protocol was applied in monkeys performing center out reaching movements and has been published from this laboratory in several preceding studies. I found the take-home-message broadly convincing and clarifying - that cerebellar block reduces muscle activation acutely particularly in movements that involve multiple joints and therefore invoke interaction torques, and that movements progressively slow down to in effect 'compensate' for these acute tone deficits. The manuscript was generally well written, data were clear, convincing and novel. The key strengths are differentiating acute from sub-acute (within session but not immediate) kinematic consequences of cerebellar block.

Reviewer #3 (Public review):

Summary:

The manuscript by Flowers et al. aimed to enhance the accuracy of automated ligand model building by refining the qFit-ligand algorithm. Recognizing that ligands can exhibit conformational flexibility even when bound to receptors, the authors developed a bioinformatic pipeline to model alternate ligand conformations while improving fitting and more energetically favorable conformations.

Strengths:

The authors present a computational pipeline designed to automatically model and fit ligands into electron density maps, identifying potential alternative conformations within the structures.

Weaknesses:

Ligand modeling, particularly in cases of poorly defined electron density, remains a challenging task. The procedure presented in this manuscript exhibits limitations in low-resolution electron density maps (lower than 2.0 Å) and low-occupancy scenarios. Considering that the maps used to establish the operational bounds of qFit-ligand were synthetically generated, it's likely that the resolution cutoff will be even stricter when applied to real-world data.

Reviewer #2 (Public review):

Summary:

The authors aimed to develop a large-scale drug screen to identify B-lp modulators in a vertebrate whole-animal system. Using the zebrafish LipoGlo system that the authors had previously published and validated, the authors screened 2762 drug candidates to generate 49 hits and ultimately validated 19 drugs as genuine ApoB-lowering drugs. Using LipoGlo-Electrophoresis, the authors are able to obtain insights into the ApoB-lipoprotein size/subclass distribution. The authors further validate and study the mechanism of a strong hit, Enoxolone, known as also known as 18β-Glycyrrhetinic acid, which has previously been reported to modulate lipid metabolism. The authors also show that Enoxolone effects are mediated through HNF4⍺, which has been previously shown in the mouse system, but this is the first time it has been shown in the zebrafish.

Strengths:

The study was methodical and robust, using a published and well-validated zebrafish LipoGlo model. The authors validated the hits from the screen independently and considered the possibility that some drugs may have been detected as false positive results due to effects on the enzymatic activity of NanoLuciferase; only one hit, verteporfin, was shown to be a false positive. Using LipoGlo-Electrophoresis, the authors are able to obtain extra insights into the ApoB-lipoprotein size/subclass distribution. They showed that while enoxolone treatment reduces total B-lps, there are no overt changes in B-lp size distribution compared to vehicle-treated animals, other than a slight increase in the zero mobility (ZM) fraction, which contains very large particles and/or tissue aggregates. In contrast, the positive control, lomitapide, does show a change in B-lp size distribution compared to vehicle-treated animals - an increase in frequency of LDLs (low-density lipoprotein), but a decrease in VLDLs (very low-density lipoprotein). This study also assesses the LipoGlo-Electrophoresis profile of HNF4⍺ inhibitors. Work in the zebrafish larvae means that the effect on overall development and an entire vertebrate organism can also be assessed. Finally, the authors applied a thorough statistical measure to define a hit, using the Strictly Standardized Mean Difference (SSMD) method.

Weaknesses:

While the screen was thorough and well-validated, the authors missed a chance to provide a lot of extra significance to a wide range of readership. While the hits were thoroughly validated and displayed, the authors could have also presented the LipoGlo-Electrophoresis for all validated hits or at least a number of them. This would hugely increase the insights into these compounds. Also, the authors chose to validate and follow up a mechanism for Enoxolone, yet this hit was already known to modulate lipid metabolism through HNF4⍺, therefore, hugely limiting the impact of the paper. So what the authors have shown that is novel is only subtly added to this - consistent in vertebrate models, RNA sequencing of pathways, further validation of the HNF4⍺ pathway, and a profile of resulting B-lp size distribution. It seemed an easy way out to pick such a candidate, and they could have followed up by validating more thoroughly a completely novel drug. Also, the authors' prior paper showing the methodology also depicted complementary EM and LipoGlo-microscopy approaches. The microscopy especially, would have been an easy complementary add-on to the screen to really give extra insights into B-lp metabolism in a whole organism for all candidates. This felt like a missed opportunity.

Reviewer #2 (Public review):

Summary:

This study focused on the roles of the nuclear envelope proteins lamin A and C, as well as nesprin-2, encoded by the LMNA and SYNE2 genes, respectively, on gene expression and chromatin mobility. It is motivated by the established role of lamins in tethering heterochromatin to the nuclear periphery in lamina-associated domains (LADs) and modulating chromatin organization. The authors show that depletion of lamin A, lamin A and C, or nesprin-2 results in differential effects of mRNA and lncRNA expression, primarily affecting genes outside established LADs. In addition, the authors used fluorescent dCas9 labeling of telomeric genomic regions combined with live-cell imaging to demonstrate that depletion of either lamin A, lamin A/C, or nesprin-2 increased the mobility of chromatin, suggesting an important role of lamins and nesprin-2 in chromatin dynamics.

Strengths:

The major strength of this study is the detailed characterization of changes in transcript levels and isoforms resulting from depletion of either lamin A, lamin A/C, or nesprin-2 in human osteosarcoma (U2OS) cells. The authors use a variety of advanced tools to demonstrate the effect of protein depletion on specific gene isoforms and to compare the effects on mRNA and lncRNA levels.

The TIRF imaging of dCas9-labeled telomeres allows for high-resolution tracking of multiple telomeres per cell, thus enabling the authors to obtain detailed measurements of the mobility of telomeres within living cells and the effect of lamin A/C or nesprin-2 depletion.

Weaknesses:

Although the findings presented by the authors overall confirm existing knowledge about the ability of lamins A/C and nesprin to broadly affect gene expression, chromatin organization, and chromatin dynamics, the specific interpretation and the conclusions drawn from the data presented in this manuscript are limited by several technical and conceptual challenges.

One major limitation is that the authors only assess the knockdown of their target genes on the mRNA level, where they observe reductions of around 70%. Given that lamins A and C have long half-lives, the effect at the protein level might be even lower. This incomplete and poorly characterized depletion on the protein level makes interpretation of the results difficult. The description for the shRNA targeting the LMNA gene encoding lamins A and C given by the authors is at times difficult to follow and might confuse some readers, as the authors do not clearly indicate which regions of the gene are targeted by the shRNA, and they do not make it obvious that lamin A and C result from alternative splicing of the same LMNA gene. Based on the shRNA sequences provided in the manuscript, one can conclude that the shLaminA shRNA targets the 3' UTR region of the LMNA gene specific to prelamin A (which undergoes posttranslational processing in the cell to yield lamin A). In contrast, the shRNA described by the authors as 'shLMNA' targets a region within the coding sequence of the LMNA gene that is common to both lamin A and C, i.e., the region corresponding to amino acids 122-129 (KKEGDLIA) of lamin A and C. The authors confirm the isoform-specific effect of the shLaminA isoform, although they seem somewhat surprised by it, but do not confirm the effect of the shLMNA construct. Assessing the effect of the knockdown on the protein level would provide more detailed information both on the extent of the actual protein depletion and the effect on specific lamin isoforms. Similarly, given that nesprin-2 has numerous isoforms resulting from alternative splicing and transcription initiation. In the current form of the manuscript, it remains unclear which specific nesprin-2 isoforms were depleted, and to what extent (on the protein level).

Another substantial limitation of the manuscript is that the current analysis, with the exception of the chromatin mobility measurements, is exclusively based on transcriptomic measurements by RNA-seq and qRT-PCR, without any experimental validation of the predicted protein levels or proposed functional consequences. As such, conclusions about the importance of lamin A/C on RNA synthesis and other functions are derived entirely from gene ontology terms and are not sufficiently supported by experimental data. Thus, the true functional consequences of lamin A/C or nesprin depletion remain unclear. Statements included in the manuscript such as "our findings reveal that lamin A is essential for RNA synthesis, ..." (Lines 79-80) are thus either inaccurate or misleading, as the current data do not show that lamin A is ESSENTIAL for RNA synthesis, and lamin A/C and lamin A deficient cells and mice are viable, suggesting that they are capable of RNA synthesis.

Another substantial weakness is that the data and analysis presented in the manuscript raise some concerns about the robustness of the findings. Given that the 'shLMNA' construct is expected to deplete both lamin A and C, i.e., its effect encompasses the depletion of lamin A, which is achieved by the 'shLaminA' construct, one would expect a substantial overlap between the DEGs in the shLMNA and shLaminA conditions, with the shLMNA depletion producing a broader effect as it targets both lamin A and C. However, the Venn Diagram in Figure 4a, the genomic loci distribution in Figure 4b, and the correlation analysis in Supplementary Figure S2 show little overlap between the shLMNA and shLaminA conditions, which is quite surprising. In the mapping of the DEGs shown in Figure 4b, it is also surprising not to see the gene targeted by the shRNA, LMNA, found on chromosome 1, in the results for the shLMNA and shLamin A depletion.

The correlation analysis in Supplementary Figure S2 raises further questions. The authors use doc-inducible shRNA constructs to target lamin A (shLaminA), lamin A/C (shLMNA), or nesprin-2 (shSYNE2). Thus, the no-dox control (Ctr) for each of these constructs would be expected to be very similar to the non-target scrambled controls (Ctrl.shScramble and Dox.shScramble). However, in the correlation matrix, each of the no-dox controls clusters more closely with the corresponding dox-induced shRNA condition than with the Ctrl.shScramble or Dox.shScramble conditions, suggesting either a very leaky dox-inducible system, strong effects from clonal selection, or substantial batch effects in the processing. Either of these scenarios could substantially affect the interpretation of the findings. For example, differences between different clonal cell lines used for the studies, independent of the targeted gene, could explain the limited overlap between the different shRNA constructs and result in apparent differences when comparing these clones to the scrambled controls, which were derived from different clones.

The manuscript also contains several factually inaccurate or incorrect statements or depictions. For example, the depiction of the nuclear envelope in Figure 1 shows a single bilipid layer, instead of the actual double bi-lipid layer of the inner and outer nuclear membranes that span the nuclear lumen. The depiction further lacks SUN domain proteins, which, together with nesprins, form the LINC complex essential to transmit forces across the nuclear envelope. The statement in line 214 that "Linker of nucleoskeleton and cytoskeleton (LINC) complex component nesprin-2 locates in the nuclear envelope to link the actin cytoskeleton and the nuclear lamina" is not quite accurate, as nesprin-2 also links to microtubules via dynein and kinesin.

The statement that "Our data show that Lamin A knockdown specifically reduced the usage of its primary isoform, suggesting a potential role in chromatin architecture regulation, while other LMNA isoforms remained unaffected, highlighting a selective effect" (lines 407-409) is confusing, as the 'shLaminA' shRNA specifically targets the 3' UTR of lamin A that is not present in the other isoforms. Thus, the observed effect is entirely consistent with the shRNA-mediated depletion, independent of any effects on chromatin architecture.

The premise of the authors that lamins would only affect peripheral chromatin and genes at LADs neglects the fact that lamins A and C are also found in the nuclear interior, where they form stable structure and influence chromatin organization, and the fact that lamins A and C and nesprins additionally interact with numerous transcriptional regulators such as Rb, c-Fos, and beta-catenins, which could further modulate gene expression when lamins or nesprins are depleted.

The comparison of the identified DEGs to genes contained in LADs might be confounded by the fact that the authors relied on the identification of LADs from a previous study (ref #28), which used a different human cell type (human skin fibroblasts) instead of the U2OS osteosarcoma cells used in the present study. As LADs are often highly cell-type specific, the use of the fibroblast data set could lead to substantial differences in LADs.

Another limitation of the current manuscript is that, in the current form, some of the figures and results depicted in the figures are difficult to interpret for a reader not deeply familiar with the techniques, based in part on the insufficient labeling and figure legends. This applies, for example, to the isoform use analysis shown in Figure 3d or the GenometriCorr analysis quantifying spatial distance between LADs and DEGs shown in Figure 4c.

Overall appraisal and context:

Despite its limitations, the present study further illustrates the important roles the nuclear envelope proteins lamin A, lamin C, and nesprin-2 have in chromatin organization, dynamics, and gene expression. It thus confirms results from previous studies (not always fully acknowledged in the current manuscript) previously reported for lamin A/C depletion. For example, the effect of lamin A/C depletion on increasing mobility of chromatin had already been demonstrated by several other groups, such as Bronshtein et al. Nature Comm 2015 (PMID: 26299252) and Ranade et al. BMC Mol Cel Biol 2019 (PMID: 31117946). Additionally, the effect of lamin A/C depletion on gene and protein expression has already been extensively studied in a variety of other cell lines and model systems, including detailed proteomic studies (PMIDs 23990565 and 35896617).

The finding that that lamin A/C or nesprin depletion not only affects genes at the nuclear periphery but also the nuclear interior is not particularly surprising giving the previous studies and the fact that lamins A and C are also founding within the nuclear interior, where they affect chromatin organization and dynamics, and that lamins A/C and nesprins directly interact with numerous transcriptional regulators that could further affect gene expression independent from their role in chromatin organization.

The authors provide a detailed analysis of isoform switching in response to lamin A/C or nesprin depletion, but the underlying mechanism remains unclear. Similarly, their analysis of the genomic location of the observed DEGs shows the wide-ranging effects of lamin A/C or nesprin depletion, but lets the reader wonder how these effects are mediated. A more in-depth analysis of predicted regulator factors and their potential interaction with lamins A/C or nesprin would be beneficial in gaining more mechanistic insights.

Reviewer #2 (Public review):

Summary:

The manuscript explores the role of PRMT1 in AMKL, highlighting its overexpression as a driver of metabolic reprogramming. PRMT1 overexpression enhances the glycolytic phenotype and extracellular acidification by increasing lactate production in AMKL cells. Treatment with the PRMT1 inhibitor MS023 significantly reduces AMKL cell viability and improves survival in tumor-bearing mice. Intriguingly, PRMT1 overexpression also increases mitochondrial number and mtDNA content. High PRMT1-expressing cells demonstrate the ability to utilize alternative energy sources dependent on mitochondrial energetics, in contrast to parental cells with lower PRMT1 levels.

Strengths:

This is a conceptually novel and important finding as PRMT1 has never been shown to enhance glycolysis in AMKL, and provides a novel point of therapeutic intervention for AMKL.

Comments on revisions:

The author has responded satisfactorily to the review comments and revised the manuscript accordingly.

Reviewer #2 (Public review):

Summary:<br /> In the presented work by Wu et al. the authors investigate the role of the transcription factor Pu.1 in the survival and maintenance of microglia, the tissue resident macrophage population in the brain. To this end they generated a sophisticated new conditional pu.1 allele in zebrafish using CRISPR mediated genome editing which allows visual detection of expression of the mutant allele through a switch from GFP to dsRed after Cre-mediated recombination. Using EdU pulse-chase labelling, they first estimate the daily turnover rate of microglia in the adult zebrafish brain which was found to be higher than rates previously estimated for mice and humans. After conditional deletion of pu.1 in coro1a positive cells, they do not find a difference in microglia number at 2 and 8 days or 1 month post injection of Tamoxifen. However, at 3 month post injection, a strong decrease in mutant microglia could be detected. While no change in microglia number was detected at 1mpi, an increase in apoptotic cells and decreased proliferation as observed. RNA-seq analysis of WT and mutant microglia revealed an upregulation of tp53, which was shown to play a role in the depletion of pu.1 mutant microglia as deletion in tp53-/- mutants did not lead to a decrease in microglia number at 3mpi. Through analysis of microglia number in pU.1 mutants, the authors further show that the depletion of microglia in the conditional mutants is dependent on the presence of WT microglia. To show that the phenomenon is conserved between species, similar experiments were also performed in mice.

This work expands on previous in vitro studies using primary human microglia. The majority of conclusions are well supported by the data, addition of controls and experimental details would strengthen the conclusions and rigor of the paper.

Strengths:

Generation of an elegantly designed conditional pu.1 allele in zebrafish that allows for the visual detection of expression of the knockout allele.<br /> The combination of analysis of pu.1 function in two model systems, zebrafish and mouse, strengthens the conclusions of the paper.<br /> Confirmation of the functional significance of the observed upregulation of tp53 in mutant microglia through double mutant analysis provides some mechanistic insight.

Weaknesses:

(1) The presented RNA-Seq analysis of mutant microglia is underpowered and details on how the data was analyzed is missing. Only 9-15 cells were analyzed in total (3 pools of 3-5 cells each). Further the variability in relative gene expression of ccl35b.1, which was used as a quality control and inclusion criterion to define pools consisting of microglia, is extremely high (between ~4 and ~1600, Fig. S7A).

(2) The authors conclude that the reduction of microglia observed in the adult brain after cKO of pu.1 in the spi-b mutant background is due to apoptosis (Lines 213-215). However, they only provide evidence of apoptosis in 3-5 dpf embryos, a stage at which loss of pu.1 alone does lead to a complete loss of microglia (Fig.2E). A control of pu.1 KI/d839 mutants treated with 4-OHT should be added to show that this effect is indeed dependent on the loss of spi-b. In addition, experiments should be performed to show apoptosis in the adult brain after cKO of pu.1 in spi-b mutants as there seems to be a difference in requirement of pu.1 in embryonic and adult stages.

Comments on Revised Version (from BRE):

The authors have elaborated on the details of the RNA-Seq procedure and clarified the distinct phenotypes observed with global versus condition pu.1 knockout. In addition, the authors' proposed collaborative relationship between Pu.1 and Spi-b has been expanded in the revised manuscript. The authors have addressed all the minor concerns raised by the reviewer.

Reviewer #2 (Public review):

Summary:<br /> This manuscript by Tubert et al. presents the role of D5 receptors (D5R) in regulating the striatal cholinergic interneuron (CIN) pause response through D5R-cAMP-Kv1 inhibitory signaling. Their findings provide a compelling model explaining the "on/off" switch of the CIN pause, driven by the distinct dopamine affinities and the balance of D2R and D5R. Furthermore, the study bridges their previous finding of CIN hyperexcitability (Paz et al., Movement Disorder 2022) with the loss of the pause response in LID mice and demonstrates the restore of the pause through D1/D5 inverse agonist clozapine.

Strengths:<br /> The study presents solid findings, and the writing is logically structured and easy to follow. The experiments are well-designed, properly combining ex vivo electrophysiology recording, optogenetics, and pharmacological treatment to dissect / rule out most, if not all, alternative mechanisms in their model.

Weaknesses (fixed in this revision):<br /> In this round of revision, the authors have included additional experiments examining the role of D2R, and the possible clozapine effects on serotonin receptors in the LID off -L-DOPA ex vivo slices. Although, to our surprise, D2R agonism using quinpirole and sumanirole failed to restore the CIN pause, this study still provides new insights into the balance between D2R and D5R in modulating CIN pause.

Overall, the authors' response adequately addressed concerns raised in the previous revision.

Reviewer #2 (Public review):

The authors used a clever and powerful approach to explore how Nav1.2 and Nav1.6 channels, which are both present in neocortical pyramidal neurons, differentially control firing properties of the neurons. Overall, the approach worked very well, and the results show very interesting differences when one or the other channel is partially inhibited. The experimental data is solid and the experimental data is very nicely complemented by a computational model incorporating the different localization of the two types of sodium channels.

The revised manuscript has re-organized figures that make the results and interpretation easier to follow.