Reviewer #1 (Public review):

Summary:

This study provides new insight into the non-canonicial voltage-gating mechanism of BK channels through prolonged (10 us) MD simulations of the Slo1 transmembrane domain conformation and K+ conduction in response to high imposed voltages (300, 750 mV). The results support previous conclusions based on functional and structural data and MD simulations that the voltage-sensor domain (VSD) of Slo1 undergoes limited conformational changes compared to Kv channels, and predicts gating charge movement comparable in magnitude to experimental results. The gating charge calculations further indicate that R213 and R210 in S4 are the main contributors owing to their large side chain movements and the presence of a locally focused electric field, consistent with recent experimental and MD simulation results by Carrasquel-Ursulaez et al.,2022. Most interestingly, changes in pore conformation and K+ conduction driven by VSD activation are resolved, providing information regarding changes in VSD/pore interaction through S4/S5/S6 segments proposed to underly electromechanical coupling.

Strengths:

Include that the prolonged timescale and high voltage of the simulation allow apparent equilibration in the voltage-sensor domain (VSD) conformational changes and at least partial opening of the pore. The study extends the results of previous MD simulations of VSD activation by providing quantitative estimates of gating charge movement, showing how the electric field distribution across the VSD is altered in resting and activated states, and testing the hypothesis that R213 and R210 are the primary gating charges by steered MD simulations. The ability to estimate gating charge contributions of individual residues in the WT channel is useful as a comparison to experimental studies based on mutagenesis which have yielded conflicting results that could reflect perturbations in structure. Use of dynamic community analysis to identify coupling pathways and information flow for VSD-pore (electromechanical) coupling as well as analysis of state-dependent S4/S5/S6 interactions that could mediate coupling provide useful predictions extending beyond what has been experimentally tested.

Weaknesses:

Weaknesses include that a truncated channel (lacking the C-terminal gating ring) was used for simulations, which is known to have reduced single channel conductance and electromechanical coupling compared to the full-length channel. In addition, as VSD activation in BK channels is much faster than opening, the timescale of simulations was likely insufficient to achieve a fully open state as supported by differences in the degree of pore expansion in replicate simulations, which are also smaller than observed in Ca-bound open structures of the full-length channel. Taken together, these limitations suggest that inferences regarding coupling pathways and interactions in the fully open voltage-activated channel may be only partially supported and therefore incomplete. That said, adequate discussion regarding these limitations are provided together with dynamic community analysis based on the Ca-bound open structure. The latter supports the main conclusions based on simulations, while providing an indication of potential interaction differences between simulated and fully open conformations. Another limitation is that while the simulations convincingly demonstrate voltage-dependent channel opening as evidenced by pore expansion and conduction of K+ and water through the pore, single channel conductance is underestimated by at least an order of magnitude, as in previous studies of other K+ channels. These quantitative discrepancies suggest that MD simulations may not yet be sufficiently advanced to provide insight into mechanisms underlying the extraordinarily large conductance of BK channels.

Comments on revisions:

My previous questions and concerns have been adequately addressed.

Reviewer #1 (Public review):

Summary:

This study provides comprehensive instructions for using the chromatophore tracking software, Chromas, to track and analyse the dynamics of large numbers of cephalopod chromatophores across various spatiotemporal scales. This software addresses a long-standing challenge faced by many researchers who study these soft-bodied creatures, known for their remarkable ability to change colour rapidly. The updated software features a user-friendly interface that can be applied to a wide range of applications, making it an essential tool for biologists focused on animal dynamic signalling. It will also be of interest to professionals in the fields of computer vision and image analysis.

Strengths:

This work provides detailed instructions for this tool kit along with examples for potential users to try. The Gitlab inventory hosts the software package, installation documentation, and tutorials, further helping potential users with a less steep learning curve.

Weaknesses:

The evidence supporting the authors' claims is solid, particularly demonstrated through the use of cuttlefish and squid. However, it may not be applicable to all coleoid cephalopods yet, such as octopuses, which have an incredibly versatile ability to change their body forms.

Comments on revisions:

I am pleased to see the more detailed version of this useful tool along with tutorials designed for diverse users who are interested in animal dynamic colouration. This study provides detailed instructions for using the chromatophore tracking software Chromas to track and analyse the dynamics of large numbers of cephalopod chromatophores across various spatiotemporal scales. The software features a user-friendly interface that is highly compelling and can be applied to a wide range of applications.

Reviewer #2 (Public review):

Summary:

The authors show that a combination of arginine methyltransferase inhibitors synergize with PARP inhibitors to kill ovarian and triple negative cancer cell lines in vitro and in vivo using preclinical mouse models.

Strengths and weaknesses

The experiments are well-performed, convincing and have the appropriate controls (using inhibitors and genetic deletions) and use statistics.

They identify the DNA damage protein ERCC1 to be reduced in expression with PRMT inhibitors. As ERCC1 is known to be synthetic lethal with PARPi, this provides a mechanism for the synergy. They use cell lines only for their study in 2D as well as xenograph models.

Joint Public Review:

Summary:

How mechanical forces transmitted by blood flow contribute to cardiac development remains incompletely understood. Using the unique advantages of the zebrafish model, Chen et al make the fundamental discovery that endocardial expression of the transcriptional repressor, Id2b, is maintained in endocardial cells by blood flow. Id1b zebrafish mutants fail to form the valve in the atrioventricular canal (AVC) and show reduced myocardial contractility that they suggest is due to impaired calcium transients. Id2b mutants are largely viable during the first 6 months of life until ~20% display cardiomyopathy characterized by visible edema, structural abnormalities, retrograde blood flow, and reduced systolic function and calcium handling. Mechanistically, the authors suggest that flow-mediated expression of Id2b leads to neuregulin 1 (nrg1) upregulation by physically interacting with and sequestering the Tcf3b transcriptional repressor from conserved tcf3b binding sites upstream of nrg1. Overall, this study advances our understanding of flow-mediated endocardial-myocardial crosstalk during heart development.

Strengths:

The strengths of the study are the significance of the biological question being addressed, use of the zebrafish model, data quality, and use of genetic tools. The text is generally well-written and easy to understand.

Weaknesses:

The main weakness that remains is the lack of rigor surrounding the molecular mechanism where the authors suggest that blood flow induces endocardial expression of Id2b, which binds to Tcf3b and sequesters it from binding the Nrg1 promoter to repress transcription. Although good faith efforts were made to bolster their model, the physical interaction between Id2b and Tcf3b is limited to overexpression of tagged proteins in HEK293 cells. Moreover, no mutagenesis was performed on the tcf3b binding sites identified in the nrg1 promoter to learn their importance in vivo.

Reviewer #1 (Public review):

Summary:

This study uses a cell-based computational model to simulate and study T cell development in the thymus. They initially applied this model to assess the effect of the thymic epithelial cells (TECs) network on thymocyte proliferation and demonstrated that increasing TEC size, density, or protrusions increased the number of thymocytes. They postulated and confirmed that this was due to changes in IL7 signalling and then expanded this work to encompass various environmental and cell-based parameters, including Notch signalling, cell cycle duration, and cell motility. Critical outcomes from the computational model were tested in vivo using medaka fish, such as the role of IL-7 signalling and minimal effect of Notch signalling.

Strengths:

The strength of the paper is the use of computational modelling to obtain unique insights into the niche parameters that control T cell development, such as the role of TEC architecture, while anchoring those findings with in vivo experiments. I can't comment on the model itself, as I am not an expert in modelling, however, the conclusions of the paper seem to be well-supported by the model.

Reviewer #1 (Public review):

Petrovic et al. investigate CCR5 endocytosis via arrestin2, with a particular focus on clathrin and AP2 contributions. The study is thorough and methodologically diverse. The NMR titration data are particularly compelling, clearly demonstrating chemical shift changes at the canonical clathrin-binding site (LIELD), present in both the 2S and 2L arrestin splice variants.

To assess the effect of arrestin activation on clathrin binding, the authors compare: truncated arrestin (1-393), full-length arrestin, and 1-393 incubated with CCR5 phosphopeptides. All three bind clathrin comparably, whereas controls show no binding. These findings are consistent with prior crystal structures showing peptide-like binding of the LIELD motif, with disordered flanking regions. The manuscript also evaluates a non-canonical clathrin binding site specific to the 2L splice variant. Though this region has been shown to enhance beta2-adrenergic receptor binding, it appears not to affect CCR5 internalization.

Similar analyses applied to AP2 show a different result. AP2 binding is activation-dependent and influenced by the presence and level of phosphorylation of CCR5-derived phosphopeptides. These findings are reinforced by cellular internalization assays.

In sum, the results highlight splice-variant-dependent effects and phosphorylation-sensitive arrestin-partner interactions. The data argue against a (rapidly disappearing) one-size-fits-all model for GPCR-arrestin signaling and instead support a nuanced, receptor-specific view, with one example summarized effectively in the mechanistic figure.

Reviewer #1 (Public review):

Summary:

The study shows, perhaps surprisingly, that human fecal homogenates enhance the invasiveness of Salmonella typhimurium into cells of a swine colonic explant. This effect is only seen with chemotactic cells that express the chemoreceptor Tsr. However, two molecules sensed by Tsr that are present at significant concentrations in the fecal homogenates, the repellent indole and the attractant serine, do not, either by themselves or together at the concentrations in which they are present in the fecal homogenates, show this same effect. The authors then go on to study the conflicting repellent response to indole and attractant response to serine in a number of different in vitro assays.

Strengths:

The demonstration that homogenates of human feces enhance the invasiveness of chemotactic Salmonella Typhimurium in a colonic explant is unexpected and interesting. The authors then go on to document the conflicting responses to the repellent indole and the attractant serine, both sensed by the Tsr chemoreceptor, as a function of their relative concentration and the spatial distribution of gradients.

Weaknesses:

The authors do not identify what is the critical compound or combination of compounds in the fecal homogenate that gives the reported response of increased invasiveness. They show it is not indole alone, serine alone, or both in combination that have this effect, although both are sensed by Tsr and both are present in the fecal homogenates. Some of the responses to conflicting stimuli by indole and serine in the in vitro experiments yield interesting results, but they do little to explain the initial interesting observation that fecal homogenates enhance invasiveness.

Reviewer #1 (Public review):

Summary:

The authors developed a tool for simulating multiplexed single-cell RNA-seq and ATAC-seq data with various adjustable settings like ambient RNA/DNA rate and sequencing depth. They used the simulated data with different settings to evaluate the performance of many demultiplexing methods. They also proposed a new metric at single-cell level that correlates with the RNA/DNA contamination level.

Strengths:

The simulation tool has a straightforward design and provides adjustability in multiple parameters that have practical relevance, such as sequencing depth and ambient contamination rate. With the growing use of multiplexing in single-cell RNAseq and ATACseq experiments, the tools and results in this paper can guide the experimental design and tool selection for many researchers. The simulation tool also provides a platform for benchmarking newly developed demultiplexing tools.

Weaknesses:

The usefulness of the proposed new metric of "variant consistency" and how it can guide users in selecting demultiplexing methods seems a little unclear. It correlates with the level of ambient RNA/DNA contamination, which makes it look like a metric on data quality. However, it does depend on the exact demultiplexing method, yet it's not clear how it directly connects to the "accuracy" of each demultiplexing method, which is the most important property that users of these methods care about. Since the simulated data has ground truth of donor identities available, I would suggest using the simulated data to show whether "variant consistency" directly indicates the accuracy of each method, especially the accuracy within those "C2" reads.

I also think the tool and analyses presented in this paper need some further clarification and documentation on the details, such as how the cell-type gene and peak probabilities are determined in the simulation, and how doublets from different cell types are handled in the simulation and analysis. A few analyses and figures also need a more detailed description of the exact methods used.

Reviewer #1 (Public review):

Summary:

The authors had previously found that a brief social isolation could increase the activity of these neurons, and that manipulation of these neurons could alter social behavior in a social rank dependent fashion. This manuscript explored which of the outputs were responsible for this, identifying the central nucleus of the amygdala as the key output region. The authors identified some discrete behavior changes associated with these outputs, and found that during photostimulation of these outputs, neuronal activity appeared altered in 'social response' neurons. In the revised manuscript, the authors address the comments in a rigorous fashion.

Strengths:

Rigorous analysis of the anatomy. Careful examination of the hetergenous effects on cell activity due to stimulation, linking the physiology with the behavior via photostimulation during recording in vivo.

Weaknesses:

The authors have responded to all of my comments.

Reviewer #1 (Public review):

Summary:

This paper reports an intracranial SEEG study of speech coordination, where participants synchronize their speech output with a virtual partner that is designed to vary its synchronization behavior. This allows the authors to identify electrodes throughout the left hemisphere of the brain that have activity (both power and phase) that correlates with the degree of synchronization behavior. They find that high-frequency activity in secondary auditory cortex (superior temporal gyrus) is correlated to synchronization, in contrast to primary auditory regions. Furthermore, activity in inferior frontal gyrus shows a significant phase-amplitude coupling relationship that is interpreted as compensation for deviation from synchronized behavior with the virtual partner.

Strengths:

(1) The development of a virtual partner model trained for each individual participant, which can dynamically vary its synchronization to the participant's behavior in real time, is novel and exciting.

(2) Understanding real-time temporal coordination for behaviors like speech is a critical and understudied area.

(3) The use of SEEG provides the spatial and temporal resolution necessary to address the complex dynamics associated with the behavior.

(4) The paper provides some results that suggest a role for regions like IFG and STG in the dynamic temporal coordination of behavior both within an individual speaker and across speakers performing a coordination task.

Reviewer #1 (Public review):

Summary:

The authors developed a novel theoretical/computational procedure to count bacterial populations without introducing artificial randomness effects due to dilution. Surprisingly, this very important aspect of studies of bacterial systems has been overlooked. The proposed method provides a simple and transparent approach to eliminate the randomness of bacterial accounting procedures, allowing now to fully concentrate on the intrinsic effects of the studied systems.

Strengths:

A very simple and clear procedure is introduced and explained in full detail. This elegant approach finds an excellent compromise between mathematical rigor and computational efficiency, which is important for practical applications. The provided examples are convincing beyond a doubt, clearly indicating the potential strong impact of the proposed framework. Various complications and possible issues are also discussed and analyzed. This seems to be a very powerful novel method that should significantly advance the analysis of complex biological systems.

Weaknesses:

The only minor weakness that I found is the assumption of independence of bacterial species, which is expressed as the well-stirred approximation. One could imagine that bacterial species might cooperate, leading to non-uniform distributions that are real. How to distinguish such situations?

I believe that this method can be extended to determine if this is the case or not before the application. For example, if the bacteria species are independent of each other and one can use the binomial distributions, then the Fano factor would be proportional to the overall relative fraction of bacterial species. Maybe a simple test can be added to test it before the application of REPOP. However, I believe that this is a minor issue.

Reviewer #1 (Public review):

The authors conducted an fMRI study to investigate the neural effects of sustaining attention to areas of different sizes. Participants were instructed to attend to alphanumeric characters arranged in a circular array. The size of attention field was manipulated in four levels, ranging from small (18 deg) to large (162 deg). They used a model-based method to visualize attentional modulation in early visual cortex V1 to V3, and found spatially congruent modulations of the BOLD response, i.e., as the attended area increased in size, the neural modulation also increased in size in the visual cortex. They suggest that this result is a neural manifestation of the zoom-lens model of attention and that the model-based method can effectively reconstruct the neural modulation in the cortical space.

The study is well-designed with sophisticated and comprehensive data analysis. The results are robust and show strong support for a well-known model of spatial attention, the zoom-lens model. Overall, I find the results interesting and useful for the field of visual attention research.

Comments on revisions:

The authors have addressed my previous comments satisfactorily. I would encourage the authors to make data and code publicly available, which appears to be the custom in this era.

Reviewer #1 (Public review):

Henshall et al. delete the highly abundant merozoite surface protein PfMSP2 from two Plasmodium falciparum laboratory lines (3D7 and Dd2) using CRISPR-Cas9. Parasites lacking MSP2 replicate and invade red cells normally, opposing the experimental history that suggests MSP2 is essential. Unexpectedly, the knock-outs become more susceptible to several inhibitory antibodies - most strikingly those that target the apical antigen AMA1-while antibodies to other surface or secreted proteins are largely unaffected. Recombinant MSP2 added in vitro can dampen AMA1-antibody binding, supporting a "conformational masking" model. The reported data suggest that MSP2 helps shield key invasion ligands from host antibodies and may itself be a double-edged vaccine target.

Reviewer #3 (Public review):

The central issue for evaluating the overfilling hypothesis is the identity of the mechanism that causes the very potent (>80% when inter pulse is 20 ms), but very quickly reverting (< 50 ms) paired pulse depression (Fig 1G, I). To summarize: the logic for overfilling at local cortical L2/3 synapses depends critically on the premise that probability of release (pv) for docked and fully primed vesicles is already close to 100%. If so, the reasoning goes, the only way to account for the potent short-term enhancement seen when stimulation is extended beyond 2 pulses would be by concluding that the readily releasable pool overfills. However, the conclusion that pv is close to 100% depends on the premise that the quickly reverting depression is caused by exocytosis dependent depletion of release sites, and the evidence for this is not strong in my opinion. Caution is especially reasonable given that similarly quickly reverting depression at Schaffer collateral synapses, which are morphologically similar, was previously shown to NOT depend on exocytosis (Dobrunz and Stevens 1997). Note that the authors of the 1997 study speculated that Ca2+-channel inactivation might be the cause, but did not rule out a wide variety of other types of mechanisms that have been discovered since, including the transient vesicle undocking/re-docking (and subsequent re-priming) reported by Kusick et al (2020), which seems to have the correct timing.

In an earlier round of review, I suggested raising extracellular Ca2+, to see if this would increase synaptic strength. This is a strong test of the authors' model because there is essentially no room for an increase in synaptic strength. The authors have now done experiments along these lines, but the result is not clear cut. On one hand, the new results suggest an increase in synaptic strength that is not compatible with the authors' model; technically the increase does not reach statistical significance, but, likely, this is only because the data set is small and the variation between experiments is large. Moreover, a more granular analysis of the individual experiments seems to raise more serious problems, even supporting the depletion-independent counter hypothesis to some extent. On the other hand, the increase in synaptic strength that is seen in the newly added experiments does seem to be less at local L2/3 cortical synapses compared to other types of synapses, measured by other groups, which goes in the general direction of supporting the critical premise that pv is unusually high at L2/3 cortical synapses. Overall, I am left wishing that the new data set were larger, and that reversal experiments had been included as explained in the specific points below.

Specific Points:

(1) One of the standard methods for distinguishing between depletion-dependent and depletion-independent depression mechanisms is by analyzing failures during paired pulses of minimal stimulation. The current study includes experiments along these lines showing that pv would have to be extremely close to 1 when Ca2+ is 1.25 mM to preserve the authors' model (Section "High double failure rate ..."). Lower values for pv are not compatible with their model because the k1 parameter already had to be pushed a bit beyond boundaries established by other types of experiments. The authors now report a mean increase in synaptic strength of 23% after raising Ca to 2.5 mM. The mean increase is not quite statistically significant, but this is likely because of the small sample size. I extracted a 95% confidence interval of [-4%, +60%] from their numbers, with a 92% probability that the mean value of the increase in the full population is > 5%. I used the 5% value as the greatest increase that the model could bear because 5% implies pv < 0.9 using the equation from Dodge and Rahamimoff referenced in the rebuttal. My conclusion from this is that the mean result, rather than supporting the model, actually undermines it to some extent. It would have likely taken 1 or 2 more experiments to get above the 95% confidence threshold for statistical significance, but this is ultimately an arbitrary cut off.

(2) The variation between experiments seems to be even more problematic, at least as currently reported. The plot in Figure 3-figure supplement 3 (left) suggests that the variation reflects true variation between synapses, not measurement error. And yet, synaptic strength increased almost 2-fold in 2 of the 8 experiments, which back extrapolates to pv < 0.2. If all of the depression is caused by depletion as assumed, these individuals would exhibit paired pulse facilitation, not depression. And yet, from what I can tell, the individuals depressed, possibly as much as the synapses with low sensitivity to Ca2+, arguing against the critical premise that depression equals depletion, and even arguing - to some extent - for the counter hypothesis that a component of the depression is caused by a mechanism that is independent of depletion. I would strongly recommend adding an additional plot that documents the relationship between the amount of increase in synaptic strength after increasing extracellular Ca2+ and the paired pulse ratio as this seems central.

(3) Decrease in PPR. The authors recognize that the decrease in the paired-pulse ratio after increasing Ca2+ seems problematic for the overfilling hypothesis by stating: "Although a reduction in PPR is often interpreted as an increase in pv, under conditions where pv is already high, it more likely reflects a slight increase in pocc or in the number of TS vesicles, consistent with the previous estimates (Lin et al., 2025)." I looked quickly, but did not immediately find an explanation in Lin et al 2025 involving an increase in pocc or number of TS vesicles, much less a reason to prefer this over the standard explanation that reduced PPR indicates an increase in pv. The authors should explain why the most straightforward interpretation is not the correct one in this particular case to avoid the appearance of cherry picking explanations to fit the hypothesis.

(4) The authors concede in the rebuttal that mean pv must be < 0.7, but I couldn't find any mention of this within the manuscript itself, nor any explanation for how the new estimate could be compatible with the value of > 0.99 in the section about failures.

(5) Although not the main point, comparisons to synapses in other brain regions reported in other studies might not be accurate without directly matching experiments. As it is, 2 of 8 synapses got weaker instead of stronger, hinting at possible rundown, but this cannot be assessed because reversibility was not evaluated. In addition, comparing axons with and without channel rhodopsins might be problematic because the channel rhodopsins might widen action potentials.

(6) Perhaps authors could double check with Schotten et al about whether PDBu does/does not decrease the latency between osmotic shock and transmitter release. This might be an interesting discrepancy, but my understanding is that Schotten et al didn't acquire information about latency because of how the experiments were designed.

(7) The authors state: "These data are difficult to reconcile with a model in which facilitation is mediated by Ca2+-dependent increases in pv." However, I believe that discarding the premise that depression is always caused by depletion would open up wide range of viable possibilities.

Reviewer #1 (Public review):

Summary:

Authors benchmarked five IBD detection methods (hmmIBD, isoRelate, hap-IBD, phasedIBD, and Refined IBD) in Plasmodium falciparum using simulated and empirical data. Plasmodium falciparum has a mutation rate similar to that of humans but a much higher recombination rate and lower SNP density. Thus, the authors evaluated how recombination rate and marker density affect IBD segment detection. Next, they performed parameter optimization for Plasmodium falciparum and benchmarked the robustness of downstream analyses (selection detection and Ne inference) using IBD segments detected by each method. They also tracked the computational efficiency of these methods. The authors' work is valuable for the tested species, and the analyses presented support their claim that users should be cautious when calling IBD in contexts of low SNP density and high recombination rate.

Strengths:

The study design is convincing and well-structured. The authors chose to use P. falciparum, which presents an interesting case due to its high recombination rate and a mutation rate similar to that of humans. The authors note that despite the widespread use of IBD for genomic surveillance, comprehensive evaluation of these methods in high-recombination, low-marker-density contexts has been lacking. Furthermore, they also examined the performance of IBD detection methods developed specifically for P. falciparum, and evaluated it with phased data which broadened the applicability of the work.

Weaknesses:

The authors thoughtfully addressed our prior concerns by 1) expanding the simulations; 2) updating figures and methods for clarity; and 3) more clearly framing the broader utility of their benchmarking effort. These updates strengthen the manuscript and make the relevance of their findings beyond Plasmodium falciparum more apparent.

More specifically:

The authors added three full replicates per simulation scenario and updated figures to reflect uncertainty at relevant levels, which addresses earlier concerns about reproducibility. The limited number of replicates is due to computational intensity. In the future, broader generalizability and deeper exploration of variance in benchmarking accuracy across parameter space would further strengthen the conclusions/generalizability. The author's also emphasized that, while the study is centered on Plasmodium falciparum, the benchmarking framework, not the parameters, are broadly applicable to other sexually recombining species. Lastly, they extensively updated multiple figures to include simulation models, results from simulation replicates, and improved the figures from the previous version of the manuscript.

Reviewer #1 (Public review):

Summary:

Gekko, Nomura et al., show that Drp1 elimination in zygotes using the Trim-Away technique leads to mitochondrial clustering and uneven mitochondrial partitioning during the first embryonic cleavage, resulting in embryonic arrest. They monitor organellar localization and partitioning using specific targeted fluorophores. They also describe the effects of mitochondrial clustering in spindle formation and the detrimental effect of uneven mitochondrial partitioning to daughter cells.

Strengths:

The authors have gathered solid evidence for the uneven segregation of mitochondria upon Drp1 depletion through different means: mitochondrial labelling, ATP labelling and mtDNA copy number assessment in each daughter cell. Authors have also characterised the defects in cleavage mitotic spindles upon Drp1 loss

Weaknesses:

This study convincingly describes the phenotype seen upon Drp1 loss. Further studies should be conducted to elucidate the mechanism by which Drp1 ensures even mitochondrial partitioning during the first embryonic cleavage.

Reviewer #1 (Public review):

Summary:

In this manuscript, Chua, Daugherty, and Smith analyze a new set of archaeal 20S proteasomes obtained by cryo-EM that illustrate how the occupancy of the HbYX binding pocket induces gate opening. They do so primarily through a V24Y mutation in the α-subunit. These results are supported by a limited set of mutations in K66 in the α subunit, bringing new emphasis to this unit.

Strengths:

The new structure's analysis is comprehensive, occupying the entire manuscript. As such, the scope of this manuscript is very narrow, but the strength of the data are solid, and they offer an interesting and important new piece to the gate-opening literature.

Weaknesses:

Extrapolating from the core HbYX activating motif shared by Archae and Eukaryotes to the specific operations of gate opening, which is more elaborate in eukaryotes, may prove challenging.

Reviewer #1 (Public review):

N'Guessan et al have improved the report of their study of expression QTL (eQTL) mapping in yeast using single cells. The authors make use of advances in single cell RNAseq (scRNAseq) in yeast to increase the efficiency with which this type of analysis can be undertaken. Building on prior research led by the senior author that entailed genotyping and fitness profiling of almost 100,000 cells derived from a cross between two yeast strains (BY and RM) they performed scRNAseq on a subset of ~5% (n = 4,489) individual cells. To address the sparsity of genotype data in the expression profiling they used a Hidden Markov Model (HMM) to infer genotypes and then identify the most likely known lineage genotype from the original dataset. To address the relationship between variance in fitness and gene expression the authors partition the variance to investigate the sources of variation. They then perform eQTL mapping and study the relationship between eQTL and fitness QTL identified in the earlier study.

This paper seeks to address the question of how quantitative trait variation and expression variation are related. scRNAseq represents an appealing approach to eQTL mapping as it is possible to simultaneously genotype individual cells and measure expression in the same cell. As eQTL mapping requires large sample sizes to identify statistical relationships, the use of scRNAseq is likely to dramatically increase the statistical power of such studies. However, there are several technical challenges associated with scRNAseq and the authors' study is focused on addressing those challenges. The authors have successfully demonstrated their stated goal of developing, and illustrating the benefit of, a one-pot scRNA-seq experiment and analysis for eQTL mapping.

Reviewer #1 (Public review):

Summary:

This is an interesting follow-up to a paper published in Human Molecular Genetics reporting novel roles in corticogenesis of the Kif7 motor protein that can regulate the activator as well as the repressor functions of the Gli transcription factors in Shh signalling. This new work investigates how a null mutation in the Kif7 gene affects the formation of corticofugal and thalamocortical axon tracts and the migration of cortical interneurons. It demonstrates that Kif7 null mutant embryos present with ventriculomegaly and heterotopias as observed in patients carrying KIF7 mutations. The Kif7 mutation also disrupts the connectivity between cortex and thalamus and leads to an abnormal projection of thalamocortical axons. Moreover, cortical interneurons show migratory defects that are mirrored in cortical slices treated with the Shh inhibitor cyclopamine suggesting that the Kif7 mutation results in a down-regulation of Shh signalling. Interestingly, these defects are much less severe at later stages of corticogenesis.

Strengths/weaknesses:

The findings of this manuscript are clearly presented and are based on detailed analyses. Using a compelling set of experiments, especially the live imaging to monitor interneuron migration, the authors convincingly investigate Kif7's roles and their results support their major claims. The migratory defects in interneurons and the potential role of Shh signalling present novel findings and provide some mechanistic insights but rescue experiments would further support Kif7's role in interneuron migration. Similarly, the mechanism underlying the misprojection which has previously been reported in other cilia mutants remains unexplored. Taken together, this manuscript makes novel contributions to our understanding of the role of primary cilia in forebrain development and to the aetiology of the neural symptons in ciliopathy patients.

Comments on revisions:

The authors addressed most of the points I raised in my original review. However, I am not convinced by the figures the authors present on Shh protein expression. The "bright tiny dots" of Shh protein in the cortex are not visible on the images in Figure 7. I wonder whether the authors could present higher magnification and/or black and white images with increased contrast.

Reviewer #1 (Public review):

Summary:

In this study by Li et al., the authors re-investigated the role of cDC1 for atherosclerosis progression using the ApoE model. First, the authors confirmed the accumulation of cDC1 in atherosclerotic lesions in mice and humans. Then, in order to examine the functional relevance of this cell type, the authors developed a new mouse model to selectively target cDC1. Specifically, they inserted the Cre recombinase directly after the start codon of the endogenous XCR1 gene, thereby avoiding off-target activity. Following validation of this model, the authors crossed it with ApoE-deficient mice and found a striking reduction of aortic lesions (numbers and size) following a high-fat diet. The authors further characterized the impact of cDC1 depletion on lesional T cells and their activation state. Also, they provide in-depth transcriptomic analyses of lesional in comparison to splenic and nodal cDC1. These results imply cellular interactions between lesion T cells and cDC1. Finally, the authors show that the chemokine XCL1, which is produced by activated CD8 T cells (and NK cells), plays a key role in the interaction with XCR1-expressing cDC1 and particularly in the atherosclerotic disease progression.

Strengths:

The surprising results on XCL1 represent a very important gain in knowledge. The role of cDC1 is clarified with a new genetic mouse model.

Weaknesses:

My criticism is limited to the analysis of the scRNAseq data of the cDC1. I think it would be important to match these data with published data sets on cDC1. In particular, the data set by Sophie Janssen's group on splenic cDC1 might be helpful here (PMID: 37172103; https://www.single-cell.be/spleen_cDC_homeostatic_maturation/datasets/cdc1). It would be good to assign a cluster based on the categories used there (early/late, immature/mature, at least for splenic DC).

Reviewer #1 (Public review):

Summary:

This study presents a compelling strategy for ultrasound-mediated immunomodulation in HCC, supported by robust scRNA-Seq data. While the mechanistic depth and translational validation require further refinement, the work significantly advances the field of noninvasive cancer immunotherapy. Addressing the major concerns, particularly regarding calcium signaling specificity and STNDs@Ca²⁺ safety, will strengthen the manuscript's impact.

Strengths:

(1) Innovative Approach:<br /> The integration of noninvasive ultrasound with calcium-targeted nanotechnology (STNDs@Ca²⁺) represents a significant advancement in cancer immunotherapy. The dual mechanism - direct immunomodulation via FUS and calcium delivery via nanoparticles - is both novel and promising.

(2) Comprehensive Mechanistic Insights:<br /> The use of scRNA-seq and flow cytometry provides a detailed map of immune cell dynamics, highlighting key pathways (TNF, NFκB, MAPK) and cellular transitions (e.g., MDSC suppression, CD8⁺/NK cell activation).

(3) Robust Preclinical Validation:<br /> The study validates findings in two distinct HCC models (H22 and Hepal-6), demonstrating consistent tumor suppression (>70-90%) and prolonged survival, which strengthens translational relevance.

Weaknesses:

Major Issues:

(1) Mechanistic Specificity of Calcium Influx:<br /> While the study attributes immunomodulation to ultrasound-induced calcium influx, the exact mechanism (e.g., involvement of mechanosensitive channels like Piezo1 or TRP families) remains underdiscussed. The qRT-PCR data shows no changes in TRP channels, but the upregulation of Piezo1 warrants deeper exploration.

Suggestion: The authors should include experiments to inhibit Piezo1 or other calcium channels to confirm their role in FUS-mediated effects.

(2) STNDs@Ca²⁺ Biodistribution and Safety:<br /> Although biodistribution data show splenic accumulation, potential off-target effects (e.g., liver/lung uptake) and long-term toxicity are not fully addressed. The serum biochemical analysis (Table 2) lacks critical markers like inflammatory cytokines or immune cell counts.

Suggestion: The authors should provide longitudinal toxicity data (e.g., histopathology beyond 3 hours) and assess systemic immune activation/inflammation.

(3) Statistical and Technical Clarifications:<br /> The statistical methods for multi-group comparisons (e.g., ANOVA vs. t-test) are inconsistently described. For instance, Figure 1 labels significance without specifying correction for multiple comparisons.

Suggestion: the authors should clarify statistical methods in figure legends and the Methods section; apply Bonferroni or FDR correction where appropriate.

(4) Interpretation of scRNA-seq Data:<br /> The clustering of MDSCs using surface markers (Itgam/Ly6c2/Ly6g) overlaps with conventional myeloid populations (Supplementary Figure 16), raising questions about subset specificity.

Suggestion: The authors should validate MDSC identity using functional assays (e.g., T cell suppression) or additional markers (e.g., Arg1, iNOS).

Reviewer #1 (Public review):

Summary:

In this fMRI study, the authors wished to assess neural mechanisms supporting flexible "temporal construals". For this, human participants learned a story consisting of fifteen events. During fMRI, events were shown to them and they were instructed to consider the event from "an internal" or from "an external" perspective. The authors found opposite patterns of brain activity in the posterior parietal cortex and the anterior hippocampus for the internal and the external viewpoint. They conclude that allocentric sequences are stored in the hippocampus, whereas egocentric sequences are used in the parietal cortex. The claims align with previous fMRI work addressing this question.

Strengths:

The research topic is fascinating, and very few labs in the world are asking the question of how time is represented in the human brain. Working hypotheses have been recently formulated, and this work seems to want to tackle some of them.

Weaknesses:

The current writing is fuzzy both conceptually and experimentally. I cannot provide a sufficiently well-informed assessment of the quality of the experimental work because there is a paucity of details provided in the report. Any future revisions will likely improve transparency.

(1) Improving writing and presentation:

The abstract and the introduction make use of loaded terms such as "construals", "mental timeline", "panoramic views" in very metaphoric and unexplained ways. The authors do not provide a comprehensive and scholarly overview of these terms, which results in verbiage and keywords/name-dropping without a clear general framework being presented. Some of these terms are not metaphors. They do refer to computational concepts that the authors should didactically explain to their readership. This is all the more important that some statements in the Introduction are misattributed or factually incorrect; some statements lack attributions (uncited published work).

Once the theory, the question, and the working hypothesis are clarified, the authors should carefully explain the task.

(2) The experimental approach lacks sufficient details to be comprehensible to a general audience. In my opinion, the results are thus currently uninterpretable. I highlight only a couple of specific points (out of many). I recommend revision and clarification.

a) No explanation of the narrative is being provided. The authors report a distribution of durations with no clear description of the actual sequence of events. The authors should provide the text that was used, how they controlled for low-level and high-level linguistic confounds.

b) The authors state, "we randomly assigned 15 phrases to the events twice". It is impossible to comprehend what this means. Were these considered stimuli? Controls? IT is also not clear which event or stimulus is part of the "learning set" and whether these were indicated to be such to participants.

c) The left/right counterbalancing is not being clearly explained. The authors state that there is counterbalancing, but do not sufficiently explain what it means concretely in the experiment. If a weak correlation exists between sequential position and distance, it also means that the position and the distance have not been equated within. How do the authors control for these?

d) The authors used two tasks. In the "external perspective" one, the authors asked participants to report whether events were part of the same or a different part of the day. In the "internal perspective one", the authors asked participants to project themselves to the reference event and to determine whether the target event occurred before or after the projected viewpoint. The first task is a same/different recognition task. The second task is a temporal order task (e.g., Arzy et al. 2009). These two asks are radically different and do not require the same operationalization. The authors should minimally provide a comprehensive comparison of task requirements, their operationalization, and, more importantly, assess the behavioral biases inherent to each of these tasks that may confound brain activity observed with fMRI.

e) The authors systematically report interpreted results, not factual data. For instance, while not showing the results on behavioral outcomes, the authors directly interpret them as symbolic distance effects.

Crucially, the authors do not comment on the obvious differences in task difficulty in these two tasks, which demonstrates a substantial lack of control in the experimental design. The same/different task (task 1 called "external perspective") comes with known biases in psychophysics that are not present in the temporal order task (task 2 called " internal perspective"). The authors also did not discuss or try to match the performance level in these two tasks. Accordingly, the authors claim that participants had greater accuracy in the external (same/different) task than in the internal task, although no data are shown and provided to support this report. Further, the behavioral effect is trivialized by the report of a performance accuracy trade-off that further illustrates that there is a difference in the task requirements, preventing accurate comparison of the two tasks.

All fMRI contrasts are also confounded by this experimental shortcoming, seeing as they are all reported at the interaction level across a task. For instance, in Figure 4, the authors report a significant beta difference between internal and external tasks. It is impossible to disentangle whether this effect is simply due to task difference or to an actual processing of the duration that differs across tasks, or to the nature of the representation (the most difficult to tackle, and the one chosen by the authors).

Conclusion:

In conclusion, the current experimental work is confounded and lacks controls. Any behavioral or fMRI contrasts between the two proposed tasks can be parsimoniously accounted for by difficulty or attentional differences, not the claim of representational differences being argued for here.

Reviewer #1 (Public review):

Summary:

This is a rigorous data-driven modeling study, extending the authors' previous model of spinal locomotor central pattern generator (CPG) circuits developed for the mouse spinal cord and adapted here to the rat to explore potential circuit-level changes underlying altered speed-dependent gaits, due to asymmetric (lateral) thoracic spinal hemisection and symmetric midline contusion. The model reproduces key features of the rat speed-dependent gait-related experimental data before injury and after recovery from these two different thoracic spinal cord injuries and suggests injury-specific mechanisms of circuit reorganization underlying functional recovery. There is much interest in the mechanisms of locomotor behavior recovery after spinal cord injury, and data-driven behaviorally relevant circuit modeling is an important approach. This study represents an important advance in the authors' previous experimental and modeling work on locomotor circuitry and in the motor control field.

Strengths:

(1) The authors use an advanced computational model of spinal locomotor circuitry to investigate potential reorganization of neural connectivity underlying locomotor control following recovery from symmetrical midline thoracic contusion and asymmetrical (lateral) hemisection injuries, based on an extensive dataset for the rat model of spinal cord injury.

(2) The rat dataset used is from an in vivo experimental paradigm involving challenging animals to perform overground locomotion across the full range of speeds before and after the two distinct spinal cord injury models, enabling the authors to more completely reveal injury-specific deficits in speed-dependent interlimb coordination and locomotor gaits.

(3) The model reproduces the rat gait-related experimental data before injury and after recovery from these two different thoracic spinal cord injuries, which exhibit roughly comparable functional recovery, and suggests injury-specific, compensatory mechanisms of circuit reorganization underlying recovery.

(4) The model simulations suggest that recovery after lateral hemisection mechanistically involves partial functional restoration of descending drive and long propriospinal pathways. In contrast, recovery following midline contusion relies on reorganization of sublesional lumbar circuitry combined with altered descending control of cervical networks.

(5) These observations suggest that symmetrical (contusion) and asymmetrical (lateral hemisection) injuries induce distinct types of plasticity in different spinal cord regions, suggesting that injury symmetry partly dictates the location and type of neural plasticity supporting recovery.

(6) The authors suggest that therapeutic strategies may be more effective by targeting specific circuits according to injury symmetry.

Weaknesses:

The recovery mechanisms implemented in the model involve circuit connectivity/connection weights adjustment based on assumptions about the structures involved and compensatory responses to the injury. As the authors acknowledge, other factors affecting locomotor patterns and compensation, such as somatosensory afferent feedback, neurochemical modulator influences, and limb/body biomechanics, are not considered in the model.

Reviewer #1 (Public review):

In this manuscript, Saeb et al reported the mechanistic roles of the flexible stalk domain in sTREM2 function using molecular dynamics simulations. They have reported some interesting molecular bases explaining why sTREM2 shows protective effects during AD, such as partial extracellular stalk domain promoting binding preference and stabilities of sTREM2 with its ligand even in the presence of known AD-risk mutation, R47H. Furthermore, they found that the stalk domain itself acts as the site for ligand binding by providing an "expanded surface", known as 'Expanded Surface 2' together with the Ig-like domain. Also, they observed no difference in the binding free energy of phosphatidyl-serine with wild TREM2-Ig and mutant TREM2-Ig, which is a bit inconsistent with the previous report with experiment studies by Journal of Biological Chemistry 293, (2018), Alzheimer's and Dementia 17, 475-488 (2021), Cell 160, 1061-1071 (2015).

Reviewer #1 (Public review):

Summary:

Odor- and taste-sensing are mediated by two different systems, the olfactory and gustatory systems, and have different behavioral roles. In this study, Wei et al. challenge this dichotomy by showing that odors can activate gustatory receptor neurons (GRNs) in Drosophila to promote feeding responses, including the proboscis extension response (PER) that was previously thought to be driven only by taste. While previous studies suggested that odors can promote PER to appetitive tastants, Wei et al. go further to show that odors alone cause PER, this effect is mediated through sweet-sensing GRNs, and sugar receptors are required. The study also shows that odor detection by bitter-sensing GRNs suppresses PER. The authors' conclusions are supported by behavioral assays, calcium imaging, electrophysiological recordings, and genetic manipulations. The observation that both attractive and aversive odors promote PER leaves an open question as to why this effect is adaptive. Overall, the study sheds new light on chemosensation and multimodal integration by showing that odor and taste detection converge at the level of sensory neurons, a finding that is interesting and surprising while also being supported by another recent study (Dweck & Carlson, Sci Advances 2023).

Strengths:

(1) The main finding that odors alone can promote PER by activating sweet-sensing GRNs is interesting and novel.

(2) The study uses video tracking of the proboscis to quantify PER rather than manual scoring, which is typically used in the field. The tracking method is less subjective and provides a higher-resolution readout of the behavior.

(3) The study uses calcium imaging and electrophysiology to show that odors activate GRNs. These represent complementary techniques that measure activity at different parts of the GRN (axons versus dendrites, respectively) and strengthen the evidence for this conclusion.

(4) Genetic manipulations show that odor-evoked PER is primarily driven by sugar GRNs and sugar receptors rather than olfactory neurons. This is a major finding that distinguishes this work from previous studies of odor effects on PER and feeding (e.g., Reisenman & Scott, 2019; Shiraiwa, 2008) that assumed or demonstrated that odors were acting through olfactory neurons.

Weaknesses/Limitations:

(1) Many of the odor effects on behavior or neuronal responses were only observed at very high concentrations. Most effects seemed to require concentrations of at least 10^-2 (0.01 v/v), which is at the high end of the concentration range used in olfactory studies (e.g., Hallem et al., 2004), and most experiments in the paper used a far higher concentration of 0.5 v/v. It is unclear whether these are concentrations that would be naturally encountered by flies. In addition, it is difficult to compare the concentrations used for electrophysiology and behavior given that they are presented in solution versus volatile form.

(2) The timecourse of GRN activation by odors seems quite prolonged (and possibly delayed, depending on the exact timing of odor onset to the fly), and this timecourse is not directly compared with activation by tastes to determine whether it is a property of the calcium sensor or a real difference.

(3) While the overall effect of different conditions is tested using appropriate statistical methods, post-hoc tests are not always used to determine which specific groups are different from each other (e.g., which odors and concentrations elicit significant PER compared to air or mineral oil controls in Fig. 1; which odors show impaired responses without olfactory organs in Fig. 2A).

Discrepancies with previous studies:

These discrepancies are important to note but should not necessarily be considered "weaknesses" of the present study.

(1) It is not entirely clear why PER to odors alone has not been previously reported, especially as this study shows that it is a broad effect evoked by many different odors. Previous studies (Oh et al., 2021; Reisenman & Scott, 2019; Shiraiwa, 2008) tested the effect of odors on PER and only observed enhancement of PER to sugar rather than odor-evoked PER; some of these studies explicitly show no effect of odor alone or odor with low sugar concentration. In the Response to Reviewers, the authors propose that genetic background may explain discrepancies, but this is not discussed much in the paper itself. Differences in behavioral quantification (automated vs. manual scoring, quantification of PER duration versus probability) may also contribute.

(2) The calcium imaging data showing that sugar GRNs respond to a broad set of odors contrasts with results from Dweck & Carlson (Sci Adv, 2023) who recorded sugar neurons with electrophysiology and observed responses to organic acids, but not other odors. This discrepancy is mentioned in the Discussion but the underlying reason is not clear.

Reviewer #1 (Public review):

Summary:

The authors attempted to dissect the function of a long non-coding RNA, lnc-FANCI-2, in cervical cancer. They profiled lnc-FANCI-2 in different cell lines and tissues, generated knockout cell lines, and characterized the gene using multiple assays.

Strengths:

A large body of experimental data has been presented and can serve as a useful resource for the scientific community, including transcriptomics and proteomics datasets. The reported results also span different parts of the regulatory network and open up multiple avenues for future research.

Weaknesses:

The write-up is somewhat unfocused and lacks deep mechanistic insights in some places.

Comments on revisions:

The manuscript is much improved. I am satisfied with the authors' responses.

Reviewer #1 (Public review):

Summary

Detecting unexpected epistatic interactions between multiple mutations requires a robust null expectation-or neutral function-that predicts the combined effects of multiple mutations on phenotype based on the individual effects of single mutations. This study evaluated the relevance of the product neutrality function, where double-mutant fitness is represented as a multiplicative combination of single-mutant fitness in the absence of epistatic interactions. The authors used a recent large dataset on fitness, specifically yeast colony size, to analyze epistatic interactions.

The study confirmed that the product function outperformed other neutral functions in predicting double-mutant fitness, showing no bias between negative and positive epistatic interactions. Additionally, in the theoretical portion of the study, the authors employed a previously established theoretical model of bacterial cell growth to simulate growth rates of both single- and double-mutants under multiple parameters. The simulations similarly demonstrated that the product function was superior to other functions in predicting the fitness of hypothetical double-mutants. Based on these findings, the authors concluded that the product function is a robust tool for analyzing epistatic interactions in growth fitness and effectively reflects how growth rates depend on the combination of multiple biochemical pathways.

Strength

By leveraging a previously published large dataset of yeast colony sizes for single- and double-knockout mutants, this study validated the relevance of the product function, which has frequently been used in genetics to analyze epistatic interactions. The confirmation that the product function provides a more reliable prediction of double-mutant fitness compared to other neutral functions is valuable for researchers analyzing epistatic interactions, particularly those working with the same dataset.<br /> Notably, this dataset has been previously used in studies exploring epistatic interactions with the product neutrality function. This study's findings affirm the validity of using the product function, which could enhance confidence in the conclusions drawn by those earlier studies. Consequently, both researchers utilizing this dataset and readers of prior research will benefit from the confirmation provided by this study.

Weakness

This study contains several serious problems, primarily stemming from the following issues: ignoring the substantial differences in the mechanisms regulating cell growth between prokaryotes and eukaryotes and adopting an overly specific and unrealistic set of assumptions in the mutation model. Below, the details are discussed.

(1) Misapplication of prokaryotic growth models

The mechanistic origin of the multiplicative model observed in yeast colony fitness is explained using a bacterial cell growth model. However, there is no valid justification for linking these two systems. The bacterial growth model, the Scott-Hwa model, heavily rely on specific molecular mechanisms, such as ppGpp-mediated regulation, which adjusts ribosome expression and activity during translation. In particular, this mechanism is critical to ensure growth-dependency of the fraction of ribosome in proteome in the Scott-Hwa model [https://doi.org/10.1111/j.1462-2920.2010.02357.x; https://doi.org/10.1073/pnas.2201585119]. Yeast cells lack this regulatory mechanism, making it inappropriate to directly apply bacterial growth models to yeast.<br /> The Weiße model is based on a larger set of underlying equations and involves more parameters than the Scott-Hwa model. In the original paper by Weiße et al. (PNAS, 2015), however, the model parameters were fitted solely to experimental data from E. coli, and the model's applicability to yeast was never assessed. In summary, for neither the Scott-Hwa model nor the Weiße model has it been demonstrated that the entire model quantitatively fits experimental data from yeast. A positive correlation between growth rate and RNA/protein ratio, often observed in yeast, supports only a limited portion of either model, and does not constitute validation of the models as a whole.

(2) Overly specific assumptions in the theoretical model

The theoretical model assumes that two mutations affect only independent parameters of specific biochemical processes. However, this overly restrictive assumption weakens the model's validity in explaining the general occurrence of the multiplicative model in mutations. Furthermore, experimental evidence suggests limitations of this approach. For example, in most viable yeast deletion mutants with reduced growth rates, the expression of ribosomal proteins remained largely unchanged, contrary to the predictions of the Scott-Hwa model [https://doi.org/10.7554/eLife.28034]. This discrepancy highlights that the Scott-Hwa model and its derivatives cannot reliably explain mutants' growth rates based on current experimental evidence.

(3) Limited reliability of the mechanistic origin of the multiplicative model

The authors seem to regard growth-optimizing feedback as the mechanistic origin of the multiplicative model. However, the importance of growth-optimizing feedback in explaining product neutrality heavily depends on the very specific framework of the Scott-Hwa model. As I pointed out above, the Scott-Hwa model is a bacterial growth model that considers only a narrowly defined set of biochemical reactions. Using such a narrow model to explore the mechanistic origin of product neutrality observed on a genome-wide scale appears to be inappropriate. Arguments based on either the Scott-Hwa model or the Weiße model fail to account for the generality of product neutrality across diverse genetic perturbations. These models, in their current form, do not explain the broader patterns of product neutrality observed experimentally.

Reviewer #1 (Public review):

Summary:

This work provides a new potential tool to manipulate Tregs function for therapeutic use. It focuses on the role of PGAM in Tregs differentiation and function. The authors, interrogating publicly available transcriptomic and proteomic data of human regulatory T cells and CD4 T cells, state that Tregs express higher levels of PGAM (at both message and protein levels) compared to CD4 T cells. They then inhibit PGAM by using a known inhibitor ECGC and show that this inhibition affects Tregs differentiation. This result was also observed when they used antisense oligonucleotides (ASOs) to knockdown PGAM1.

PGAM1 catalyzes the conversion of 3PG to 2PG in the glycolysis cascade. However, the authors focused their attention on the additional role of 3PG: acting as starting material for the de novo synthesis of serine.

They hypothesized that PGAM1 regulates Tregs differentiation by regulating the levels of 3PG that are available for de novo synthesis of serine, which has a negative impact on Tregs differentiation. Indeed, they tested whether the effect on Tregs differentiation observed by reducing PGAM1 levels was reverted by inhibiting the enzyme that catalyzes the synthesis of serine from 3PG.

The authors continued by testing whether both synthesized and exogenous serine affect Tregs differentiation and continued with in vivo experiments to examine the effects of dietary serine restriction on Tregs function.

In order to understand the mechanism by which serine impacts Tregs function, the authors assessed whether this depends on the contribution of serine to one-carbon metabolism and to DNA methylation.

The authors therefore propose that extracellular serine and serine whose synthesis is regulated by PGAM1 induce methylation of genes Tregs associated, downregulating their expression and overall impacting Tregs differentiation and suppressive functions.

Strengths:

The strength of this paper is the number of approaches taken by the authors to verify their hypothesis. Indeed, by using both pharmacological and genetic tools in in vitro and in vivo systems they identified a potential new metabolic regulation of Tregs differentiation and function.

Reviewer #1 (Public review):

Summary:

The authors strived for an inventory of GPCRs and GPCR pathway component genes within the genomes of 23 choanoflagellates and other close relatives of metazoans.

Strengths:

The authors generated a solid phylogenetic overview of the GPCR superfamily in these species. Intriguingly, they discover novel GPCR families, novel assortments of domain combinations, and novel insights into the evolution of those groups within the Opisthokonta clade. A particular focus is laid on adhesion GPCRs, for which the authors discover many hitherto unknown subfamilies based on Hidden Markov Models of the 7TM domain sequences, which were also reflected by combinations of extracellular domains of the homologs. In addition, the authors provide bioinformatic evidence that aGPCRs of choanoflagellates also contain a GAIN domain, which is self-cleavable, thereby reflecting the most remarkable biochemical feat of aGPCRs.

Weaknesses:

The chosen classification scheme for aGPCRs may require reassessment and amendment by the authors in order to prevent confusion with previously issued classification attempts of this family.

Reviewer #1 (Public review):

Summary:

This is an interesting theoretical study examining the viability of Virtual Circular Genome (VCG) model, a recently proposed scenario of prebiotic replication in which a relatively long sequence is stored as a collection of its shorter subsequences (and their compliments). It was previously pointed out that VCG model is prone to so-called sequence scrambling which limits the overall length of such a genome. In the present paper, additional limitations are identified. Specifically, it is shown that VCG is well replicated when the oligomers are elongated by sufficiently short chains from "feedstock" pool. However, ligation of oligomers from VCG itself results in a high error rate. I believe the research is of high quality and well written. However, the presentation could be improved and the key messages could be clarified.

(1) It is not clear from the paper whether the observed error has the same nature as sequence scrambling

(2) The authors introduce two important lengths LS1 and LS2 only in the conclusions and do not explain enough which each of them is important. It would make sense to discuss this early in the manuscript.

(3) It is not entirely clear why specific length distribution for VCG oligomers has to be assumed rather than emerged from simulations.

(4) Furthermore, the problem has another important length, L0 that is never introduced or discussed: a minimal hybridization length with a lifetime longer than the ligation time. From the parameters given, it appears that L0 is sufficiently long (~10 bases). In other words, it appears that the study is done is a somewhat suboptimal regime: most hybridization events do not lead to a ligation. Am I right in this assessment? If that is the case, the authors might want to explore another regime, L0<br /> Strengths:

High-quality theoretical modeling of an important problem is implemented.

Reviewer #1 (Public review):

Summary:

The authors have assembled a cohort of 10 SiNET, 1 SiAdeno, and 1 lung MiNEN samples to explore the biology of neuroendocrine neoplasms. They employ single-cell RNA sequencing to profile 5 samples (siAdeno, SiNETs 1-3, MiNEN) and single-nuclei RNA sequencing to profile seven frozen samples (SiNET 4-10).

They identify two subtypes of siNETs, characterized by either epithelial or neuronal NE cells, through a series of DE analyses. They also report findings of higher proliferation in non-malignant cell types across both subtypes. Additionally, they identify a potential progenitor cell population in a single lung MiNEN samples.

In the revised study, they have addressed my points and I have no further comments.

Reviewer #2 (Public review):

Summary:

In this manuscript by Peto et al., the authors describe the impact of different antimicrobials on gut microbiota in a prospective observational study of 225 participants (healthy volunteers, inpatients and outpatients). Both cross-sectional data (all participants) and longitudinal data (subset of 79 haematopoietic cell transplant patients) were used. Using metagenomic sequencing, they estimated the impact of antibiotic exposure on gut microbiota composition and resistance genes. In their models, the authors aim to correct for potential confounders (e.g. demographics, non-antimicrobial exposures and physiological abnormalities), and for differences in the recency and total duration of antibiotic exposure. I consider these comprehensive models an important strength of this observational study. Yet, the underlying assumptions of such models may have impacted the study findings and residual confounding is likely. Other strengths include the presence of both cross-sectional and longitudinal exposure data and presence of both healthy volunteers and patients. Together, these observational findings expand on previous studies (both observational and RCTs) describing the impact of antimicrobials on gut microbiota.

Weaknesses:

(1) The main weaknesses result from the observational design. This hampers causal interpretation and makes correction for potential confounding necessary. While the authors have used comprehensive models to correct for potential confounders and for differences between participants in duration of antibiotic exposure and time between exposure and sample collection, I believe residual confounding is likely (which is mentioned as a limitation in the discussion).<br /> For their models, the authors found a disruption half-life of 6 days to be the best fit based on Shannon diversity. Yet, the disruption caused by antimicrobials may be longer than represented in this model - as highlighted in the discussion.

(2) Another consequence of the observational design of this study is the relatively small number of participants available for some comparisons (e.g. oral clindamycin was only used by 6 participants). Care should be taken when drawing any conclusions from such small numbers.

Comments on revisions:

The authors have adequately addressed all of my comments.

Reviewer #1 (Public review):

Summary:

The study by Cao et al. provides a compelling investigation into the role of mutational input in the rapid evolution of pesticide resistance, focusing on the two-spotted spider mite's response to the recent introduction of the acaricide cyetpyrafen. This well-documented introduction of the pesticide-and thus a clearly defined history of selection-offers a powerful framework for studying the temporal dynamics of rapid adaptation. The authors combine resistance phenotyping across multiple populations, extensive resequencing to track the frequency of resistance alleles, and genomic analyses of selection in both contemporary and historical samples. These approaches are further complemented by laboratory-based experimental evolution, which serves as a baseline for understanding the genetic architecture of resistance across mite populations in China. Their analyses identify two key resistance-associated genes, sdhB and sdhD, within which they detect 15 mutations in wild-collected samples. Protein modeling reveals that these mutations cluster around the pesticide's binding site, suggesting a direct functional role in resistance. The authors further examine signatures of selective sweeps and their distribution across populations to infer the mechanisms-such as de novo mutation or gene flow-driving the spread of resistance, a crucial consideration for predicting evolutionary responses to extreme selection pressure. Overall, this is a well-rounded, thoughtfully designed and well-written manuscript. It shows significant novelty, as it is relatively rare to integrate broad-scale evolutionary inference from natural populations with experimentally informed bioassays, however, follow up work will be needed to fully resolve haplotype structure and the functional effects of resistance mutations in the system.

Strengths:

One of the most compelling aspects of this study is its integration of genomic time-series data in natural populations with controlled experimental evolution. By coupling genome sequencing of resistant field populations with laboratory selection experiments, the authors tease apart the individual effects of resistance alleles along with regions of the genome where selection is expected to occur, and compare that to the observed frequency in the wild populations over space and time. Their temporal data clearly demonstrates the pace at which evolution can occur in response to extreme selection. This type of approach is a powerful roadmap for the rest of the field of rapid adaptation.

The study effectively links specific genetic changes to resistance phenotypes. The identification of sdhB and sdhD mutations as major drivers of cyetpyrafen resistance is well supported by allele frequency shifts in both field and experimental populations. The scope of their sampling clearly facilitated the remarkable number of observed mutations within these target genes, and the authors provide a careful discussion of the likelihood of these mutations from de novo or standing variation. Furthermore, the discovered cross-resistance that these mutations confer to other mitochondrial complex II inhibitors highlights the potential for broader resistance management and evolution.

Weaknesses:

(1) Pleiotropy without pesticide modes of action (cyflumetofen and cyetpyrafen) may also play a role in the rapid response to the focal pesticide in this study<br /> (2) Other aspects of the environment that might influence selection were not considered in the structure of resistance alleles (i.e. climate, elevation)<br /> (3) Very little data were used for haplotype reconstruction, only 8 SNPs, and this excluded all heterozygous alleles, which could dramatically influence the complexity of these inferred haplotype networks.<br /> (4) Single Mutations and Their Effects:<br /> - Allelic effects were not estimated in isogenic lines, so the effects presented also include heterogeneity from allelic interactions with the genomic background<br /> - The authors see populations that segregate for resistance mutations but that have no survival to pesticides. This suggests either not all of the resistance mutations studied here actually have functional effects or that dominance is playing a role in masking their effects in the heterozygous state.

Reviewer #1 (Public review):

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.

Latest comments:

The reviewer acknowledges the importance of sharing the observed defects in Sakura mutant ovaries and the possible physiological significance of the Sakura-Out interaction with the research community, as this information could lay the groundwork for future functional analysis research.

Reviewer #2 (Public review):

Summary:

The authors report the discovery of a population of gingival fibroblasts displaying the expression of cellular senescence markers P21 and P16 in human periodontitis samples and a murine ligature-induced periodontitis (LIP) model. They support this finding in the murine model through bulk RNA-sequencing and show that differentially expressed genes are significantly enriched in the SenMayo cellular senescence in aging dataset. They then show that Ligature-Induced Periodontitis (LIP) mice treated with the senomorphic drug metformin display overall diminished bone damage, reduced histomorphic alterations, and a reduction in P21 and P16 immunostaining signal. To explore the cell types expressing cellular senescence markers in periodontitis, the authors make use of a combination of bioinformatic analyses on publicly available scRNA-seq data, immunostainings on patient samples and their LIP model; as well as in vitro culture of healthy human gingival fibroblasts treated with LPS. They found that fibroblasts are a cell population expressing P16 in periodontitis which are also enriched for SenMayo genes, suggesting they have a senescent phenotype. They then point to a subgroup of fibroblasts expressing CD81+ with the highest enrichment for a SASP geneset in periodontitis. They also show that treatment of LIP mice and human LPS-treated gingival fibroblasts with metformin leads to a reduction of P21 and P16-positive cells, as well as the senescence-associated beta-galactosidase (SA-beta-gal) marker. Finally, they show evidence suggesting that CD81+ senescent fibroblasts are the source of C3 complement protein, which they stipulate signals through the C3AR1 receptor present in neutrophils in periodontitis. The authors observed that both CD81+ fibroblast and C3AR1+ neutrophil populations are expanded in periodontitis, that both populations appear to be in close contact, and that treatment with metformin reduced both C3 and the neutrophil marker MPO in their mouse LIP model.

After a round of revision, the authors have made significant improvements to their manuscript, such as improving the quality of the data/evidence and also included new data from experiments using a well-known senolytic and the senomorphic metformin, which all together provide a solid support to their main claims.

Strengths:

The study implements several different techniques and tools on human samples, mouse models, fibroblast cultures, and publicly available data to support their conclusions. In summary, they provide solid evidence showing that in the context of periodontitis, there is an expansion of cells expressing senescence markers P21, and P16, as well as members of the SASP, and that this includes CD81+ fibroblasts.

Weaknesses:

The fact that in this study the periodontitis samples belonged to patients with a significantly higher median age (all older than 50 years of age) and the healthy samples belonged to young adults (all younger than 35 years of age), raises the need for caution in interpretation due to a possible effect of aging in the accumulation of CD81+ senescent fibroblasts. However, the recruitment of similar age groups in this case is of course difficult due to the higher prevalence of periodontitis in older adults. In this regard it is important to note that the authors still support their findings using a mouse ligature model. Similar studies comparing healthy and periodontitic patients from similar age groups will be of great importance in the future.

Reviewer #1 (Public review):

Summary:

In this paper, Wang et al show that differentiated peridermal cells of the zebrafish epidermis extend cytoneme-like protrusions toward the less differentiated, intermediate layer below. They present evidence that expression of a dominant-negative cdc42, inhibits cytoneme formation and leads to elevated expression of a marker of undifferentiated keratinocytes, krtt1c19e, in the periderm layer. It is demonstrated that Delta-Notch signaling is involved in keratinocyte differentiation and that loss of cytonemes correlates with a loss of Notch signaling. Finally, changes in expression of the inflammatory cytokine IL-17 and its receptors is shown to affect cytoneme number and periderm structure in a manner similar to Notch and cdc42 perturbations.

Strengths:

Overall, the idea that differentiated cells signal to underlying undifferentiated cells via membrane protrusions in skin keratinocytes is interesting and novel, and it is clear that periderm cells send out thin membrane protrusions that contain a Notch ligand. Further, and perturbations that affect cytoneme number, Notch signaling and IL-17 expression clearly lead to changes in periderm structure and gene expression.

Weaknesses:

The mechanisms by which IL-17 affects cytoneme formation requires further investigation.

Reviewer #1 (Public review):

Summary:

This fundamental study presents a practical modification of the orthogonal hybridization chain reaction (HCR) technique, a promising yet underutilized method with broad potential for future applications across various fields. The authors advance this technique by integrating peptide ligation technology and nanobody-based antibody mimetics-cost-effective and scalable alternatives to conventional antibodies-into a DNA-immunoassay framework that merges oligonucleotide-based detection with immunoassay methodologies. They demonstrate this with compelling evidence that this approach facilitates a modified ELISA platform capable of simultaneously quantifying multiple target protein expression levels within a single protein mixture sample.

Strengths:

The hybridization chain reaction (HCR) technique was initially developed to enable the simultaneous detection of multiple mRNA expression levels within the same tissue. This method has since evolved into immuno-HCR, which extends its application to protein detection by utilizing antibodies. A key requirement of immuno-HCR is the coupling of oligonucleotides to antibodies, a process that can be challenging due to the inherent difficulties in expressing and purifying conventional antibodies.<br /> In this study, the authors present an innovative approach that circumvents these limitations by employing nanobody-based antibody mimetics, which recognize antibodies, instead of directly coupling oligonucleotides to conventional antibodies. This strategy facilitates oligonucleotide conjugation-designed to target the initiator hairpin oligonucleotide of HCR-through peptide ligation and click chemistry.

Weaknesses:

The sandwich-format technique presented in this study, which employs a nanobody that recognizes primary IgG antibodies, may have limited scalability compared to existing methods that directly couple oligonucleotides to primary antibodies. This limitation arises because the C-region types of primary antibodies are relatively restricted, meaning that the use of nanobody-based detection may constrain the number of target proteins that can be analyzed simultaneously. In contrast, the conventional approach of directly conjugating oligonucleotides to primary antibodies allows for a broader range of protein targets to be analyzed in parallel.

Additionally, in the context of HCR-based protein detection, the number of proteins that can be analyzed simultaneously is inherently constrained by fluorescence wavelength overlap in microscopy, which limits its multiplexing capability. By comparison, direct coupling oligonucleotides to primary antibodies can facilitate the simultaneous measurement of a significantly greater number of protein targets than the sandwich-based nanobody approach in the barcode-ELISA/NGS-based technique.

Comments on revisions:

The previous suggestions were well incorporated in the revised manuscript.

Reviewer #2 (Public review):

Summary:

This study explores how a simple sensorimotor circuit in the nematode C. elegans enables it to navigate salt gradients based on past experiences. Using computational simulations and previously described neural connections, the study demonstrates how a single neuron, ASER, can change its signaling behavior in response to different salt conditions, with which the worm is able to "remember" prior environments and adjust its navigation toward "preferred" salinity accordingly.

Strengths:

The key novelty and strength of this paper is the explicit demonstration of computational neurobehavioral modeling and evolutionary algorithms to elucidate the synaptic plasticity in a minimal neural circuit that is sufficient to replicate memory-based chemotaxis. In particular, with changes in ASER's glutamate release and sensitivity of downstream neurons, the ASER neuron adjusts its output to be either excitatory or inhibitory depending on ambient salt concentration, enabling the worm to navigate toward or away from salt gradients based on prior exposure to salt concentration.

Weaknesses:

While the model successfully replicates some behaviors observed in previous experiments, some key assumptions of the work still need to be verified by biological validation of further experiments.

Comments on revisions:

Thank you for the authors' response. The revision and their response have substantially addressed my concerns.

Reviewer #1 (Public review):

Summary:

In the present study, Chen et al. investigate the role of Endophilin A1 in regulating GABAergic synapse formation and function. To this end, the authors use constitutive or conditional knockout of Endophilin A1 (EEN1) to assess the consequences on GABAergic synapse composition and function, as well as the outcome for PTZ-induced seizure susceptibility. The authors show that EEN1 KO mice show a higher susceptibility to PTZ-induced seizures, accompanied by a reduction in the GABAergic synaptic scaffolding protein gephyrin as well as specific GABAAR subunits and eIPSCs. The authors then investigate the underlying mechanisms, demonstrating that Endophilin A1 binds directly to gephyrin and GABAAR subunits, and identifying the subdomains of Endophilin A1 that contribute to this effect. Overall, the authors state that their study places Endophilin A1 as a new regulator of GABAergic synapse function.

Strengths:

Overall, the topic of this manuscript is very timely, since there has been substantial recent interest in describing the mechanisms governing inhibitory synaptic transmission at GABAergic synapses. The study will therefore be of interest to a wide audience of neuroscientists studying synaptic transmission and its role in disease. The manuscript is well written and contains a substantial quantity of data. In the revised version of the manuscript, the authors have increased the number of samples analyzed and have significantly improved the statistical analysis, thereby substantially strengthening the conclusions of their study.

Comments on revised version:

The authors have addressed all of my concerns, and the manuscript has been substantially improved.

Reviewer #1 (Public review):

Summary:

The manuscript by Ross, Miscik, and others describes an intriguing series of observations made when investigating the requirement for podxl during hepatic development in zebrafish. Podxl morphants and CRISPants display a reduced number of hepatic stellate cells (HSCs), while mutants are either phenotypically wild type or display an increased number of HSCs.

The absence of observable phenotypes in genetic mutants could indeed be attributed to genetic compensation, as the authors postulate. However, in my opinion, the evidence provided in the manuscript at this point is insufficient to draw a firm conclusion. Furthermore, the opposite phenotype observed in the two deletion mutants is not readily explainable by genetic compensation and invokes additional mechanisms.

Major concerns:

(1) Considering discrepancies in phenotypes, the phenotypes observed in podxl morphants and CRISPants need to be more thoroughly validated. To generate morphants, authors use "well characterized and validated ATG Morpholino" (lines 373-374). However, published morphants, in addition to kidney malformations, display gross developmental defects including pericardial edema, yolk sack extension abnormalities, and body curvature at 2-3 dpf (reference 7 / PMID: 24224085). Were these gross developmental defects observed in the knockdown experiments performed in this paper? If yes, is it possible that the liver phenotype observed at 5 dpf is, to some extent, secondary to these preceding abnormalities? If not, why were they not observed? Did kidney malformations reproduce? On the CRISPant side, were these gross developmental defects also observed in sgRNA#1 and sgRNA#2 CRISPants? Considering that morphants and CRISPants show very similar effects on HSC development and assuming other phenotypes are specific as well, they would be expected to occur at similar frequencies. It would be helpful if full-size images of all relevant morphant and CRISPant embryos were displayed, as is done for tyr CRISPant in Figure S2. Finally, it is very important to thoroughly quantify the efficacy of podxl sgRNA#1 and sgRNA#2 in CRISPants. The HRMA data provided in Figure S1 is not quantitative in terms of the fraction of alleles with indels. Figure S3 indicates a very broad range of efficacies, averaging out at ~62% (line 100). Assuming random distribution of indels among cells and that even in-frame indels result in complete loss of function (possible for sgRNA#1 due to targeting the signal sequence), only ~38% (.62*.62) of all cells will be mutated bi-allelically. That does not seem sufficient to reliably induce loss-of-function phenotypes. My guess is that the capillary electrophoresis method used in Figure S3 underestimates the efficiency of mutagenesis, and that much higher mutagenesis rates would be observed if mutagenesis were assessed by amplicon sequencing (ideally NGS but Sanger followed by deconvolution analysis would suffice). This would strengthen the claim that CRISPant phenotypes are specific.

(2) In addition to confidence in morphant and CRISPant phenotypes, the authors' claim of genetic compensation rests on the observation that podxl (Ex1(p)_Ex7Δ) mutants are resistant to CRISPant effect when injected with sgRNA#1 (Figure 3L). Considering the issues raised in the paragraph above, this is insufficient. There is a very straightforward way to address both concerns, though. The described podxl(-194_Ex7Δ) and podxl(-319_ex1(p)Δ) deletions remove the binding site for the ATG morpholino. Therefore, deletion mutants should be refractive to the Morpholino (specificity assessment recommended in PMID: 29049395, see also PMID: 32958829). Furthermore, both deletion mutants should be refractive to sgRNA#1 CRISPant phenotypes, with the first being refractive to sgRNA#2 as well.

Reviewer #1 (Public review):

This study presents cryoEM-derived structures of the Trypanosome aquaporin AQP2, in complex with its natural ligand, glycerol, as well as two trypanocidal drugs, pentamidine and melarsoprol, which use AQP2 as an uptake route. The structures are high quality, and the density for the drug molecules is convincing, showing a binding site in the centre of the AQP2 pore.

The authors then continue to study this system using molecular dynamics simulations. Their simulations indicate that the drugs can pass through the pore and identify a weak binding site in the centre of the pore, which corresponds with that identified through cryoEM analysis. They also simulate the effect of drug resistance mutations, which suggests that the mutations reduce the affinity for drugs and therefore might reduce the likelihood that the drugs enter into the centre of the pore, reducing the likelihood that they progress through into the cell.

While the cryoEM and MD studies are well conducted, it is a shame that the drug transport hypothesis was not tested experimentally. For example, did they do cryoEM with AQP2 with drug resistance mutations and see if they could see the drugs in these maps? They might not bind, but another possibility is that the binding site shifts, as seen in Chen et al. Do they have an assay for measuring drug binding? I think that some experimental validation of the drug binding hypothesis would strengthen this paper. Without this, I would recommend the authors to soften the statement of their hypothesis (i.e, lines 65-68) as this has not been experimentally validated.

Reviewer #1 (Public review):

The manuscript by Chiu et al describes the modification of the Zwitch strategy to efficiently generate conditional knockouts of zebrafish betapix. They leverage this system to identify a surprising glia-exclusive function of betapix in mediating vascular integrity and angiogenesis. Betapix has been previously associated with vascular integrity and angiogenesis in zebrafish, and betapix function in glia has also been proposed. However, this study identifies glial betapix in vascular stability and angiogenesis for the first time.

The study derives its strength from the modified CRISPR-based Zwitch approach to identify the specific role of glial betapix (and not neuronal, mural, or endothelial). Using RNA-in situ hybridization and analysis of scRNA-Seq data, they also identify delayed maturation of neurons and glia and implicate a reduction in stathmin levels in the glial knockouts in mediating vascular homeostasis and angiogenesis. The study also implicates a betapix-zfhx3/4-vegfa axis in mediating cerebral angiogenesis.

There is both technical (the generation of conditional KOs) and knowledge-related (the exclusive role of glial betapix in vascular stability/angiogenesis) novelty in this work that is going to benefit the community significantly.<br /> While the text is well written, it often elides details of experiments and relies on implicit understanding on the part of the reader. Similarly, the figure legends are laconic and often fail to provide all the relevant details.

Specific comments:

(1) While the evidence from cKO's implicating glial betapix in vascular stability/angiogenesis is exciting, glia-specific rescue of betapix in the global KOs/mutants (like those performed for stathmin) would be necessary to make a water-tight case for glial betapix.

(2) Splice variants of betapix have been shown to have differential roles in haemorrhaging (Liu, 2007). What are the major glial isoforms, and are there specific splice variants in the glial that contribute to the phenotypes described?

(3) Liu et al, 2012 demonstrated reduced proliferation of endothelial cells in bbh fish and linked it to deficits in angiogenesis. Are there proliferation/survival defects in endothelial cells in the glial KOs?

Reviewer #1 (Public review):

Summary:

This study employs spatial transcriptomics to explore the molecular architecture of the adult mouse adrenal gland and the adjacent adipose tissue. The research aimed to identify zonation-specific genetic markers, elucidate cellular differentiation patterns, and investigate inter- and intra-zone communication within the adrenal gland. The findings support the centripetal differentiation model, highlighting the transition of cell populations across different cortical zones. The study also integrates ligand-receptor interaction analysis to uncover the adrenal gland's role in endocrine and neuroendocrine signaling, particularly in stress response. This high-resolution spatial transcriptomic map provides novel insights into adrenal gland biology and is a resource for further investigations.

Strengths:

The study, using the latest technologies and methods such as Visium CytAssist technology, UMAP & Seurat analysis, Gene Ontology (GO) & KEGG pathway enrichment analysis, Monocle3, and CellChat analysis, performed three-dimensional analysis, which has been challenging to achieve using the two-dimensional transcriptomics that have been commonly used up until now.

The unique gene expression patterns were demonstrated for each adrenal zone. Spatial transcriptomics confirmed unique gene expression patterns for each adrenal zone (ZG, ZF, ZX, medulla). The centripetal differentiation model shows the migration of the progenitor cells from the adrenal capsule towards the inner cortex. Key genetic markers were identified in each adrenal zone and adjacent adipose tissues. In addition, CellChat analysis identified major signaling pathways, including Wnt signaling, Hedgehog signaling, IGF2-IGF2R interactions, and Neuropeptide Y (NPY) signaling in the medulla. All these results offer a valuable dataset for future adrenal biology research, with potential applications in disease modeling and therapeutic target identification.

The results, high-resolution mapping of adrenal gland zonation, validation of the centripetal differentiation model, perspective on cell-cell communication, and potential translational impact on human adrenal gland function and disorders, are quite noble.

Weaknesses:

The reviewer requests that the following issues be addressed in the text:

(1) The study focuses only on adult male mice, which limits insights into developmental and sex-specific differences. What do the authors predict about the gender and age difference?

(2) Despite advanced methodologies, single-cell heterogeneity may not be fully captured, as Visium technology has limited spatial resolution.

(3) While the study suggests that ZX might have a role in androgen synthesis, further functional validation is required.

(4) The study is primarily descriptive, lacking in-depth mechanistic experiments to validate cell-cell communication interactions. It is quite interesting to suggest cell-cell communication, but the authors are still required to provide some evidence to support it.

(5) The data supports the conclusions, particularly in validating the centripetal differentiation model using Monocle3 trajectory analysis. However, functional validation experiments (e.g., gene knockout studies) would strengthen the findings, especially regarding ZX function and ligand-receptor interactions.

Reviewer #1 (Public review):

Summary:

This study addresses the issue of rapid skill learning and whether individual sequence elements (here: finger presses) are differentially represented in human MEG data. The authors use a decoding approach to classify individual finger elements, and accomplish an accuracy of around 94%. A relevant finding is that the neural representations of individual finger elements dynamically change over the course of learning. This would be highly relevant for any attempts to develop better brain machine interfaces - one now can decode individual elements within a sequence with high precision, but these representations are not static but develop over the course of learning.

Strengths:

The work follows from a large body of work from the same group on the behavioural and neural foundations of sequence learning. The behavioural task is well established a neatly designed to allow for tracking learning and how individual sequence elements contribute. The inclusion of short offline rest periods between learning epochs has been influential because it has revealed that a lot, if not most of the gains in behaviour (ie speed of finger movements) occur in these so-called micro-offline rest periods.

The authors use a range of new decoding techniques, and exhaustively interrogate their data in different ways, using different decoding approaches. Regardless of the approach, impressively high decoding accuracies are observed, but when using a hybrid approach that combines the MEG data in different ways, the authors observe decoding accuracies of individual sequence elements from the MEG data of up to 94%.

Reviewer #1 (Public review):

Summary:

Diarrheal diseases represent an important public health issue. Among the many pathogens that contribute to this problem, Salmonella enterica serovar Typhimurium is an important one. Due to the rise in antimicrobial resistance and the problems associated with widespread antibiotic use, the discovery and development of new strategies to combat bacterial infections is urgently needed. The microbiome field is constantly providing us with various health-related properties elicited by the commensals that inhabit their mammalian hosts. Harnessing the potential of these commensals for knowledge about host-microbe interactions as well as useful properties with therapeutic implications will likely to remain a fruitful field for decades to come. In this manuscript, Wang et al use various methods, encompassing classic microbiology, genomics, chemical biology, and immunology, to identify a potent probiotic strain that protects nematode and murine hosts from S. enterica infection. Additionally, authors identify gut metabolites that are correlated with protection, and show that a single metabolite can recapitulate the effects of probiotic administration.

Strengths:

The utilization of varied methods by the authors, together with the impressive amount of data generated, to support the claims and conclusions made in the manuscript is a major strength of the work. Also, the ability the move beyond simple identification of the active probiotic, also identifying compounds that are at least partially responsible for the protective effects, is commendable.

Weaknesses:

No major weaknesses noted.

Reviewer #1 (Public review):

Summary:

Walton et al. set out to isolate new phages targeting the opportunistic pathogen Pseudomonas aeruginosa. Using a double ∆fliF ∆pilA mutant strain, they were able to isolate 4 new phages, CLEW-1. -3, -6 and -10, that were unable to infected the parental PAO1F Wt strain. Further experiments showed that the 4 phages were only able to infect a ∆fliF strain, indicating a role of the MS-protein in the flagellum complex. Through further mutational analysis of the flagellum apparatus, the authors were able to identify the involvement of c-di-GMP in phage infection. Depletion of c-di-GMP levels by an inducible phosphodiesterase render the bacteria resistant to phage infection, while elevation of c-di-GMP through the Wsp system made the cells sensitive to infection by CLEW-1. Using TnSeq, the authors were able to not only reaffirm the involvement of c-di-GMP in phage infection but also able to identify the exopolysaccharide PSL as a downstream target for CLEW-1. C-di-GMP is a known regulator of PSL biosynthesis. The authors show that CLEW-1 binds directly to PSL on the cell surface and that deletion of the pslC gene resulted in complete phage resistance. The authors also provide evidence that the phage - PSL interaction happens during the biofilm mode of growth and that the addition of the CLEW-1 phage specifically resulted in a significant loss of biofilm biomass. Lastly, the authors set out to test if CLEW-1 could be used to resolve a biofilm infection using a mouse keratitis model. Unfortunately, while the authors noted a reduction in bacterial load assessed by GFP fluorescence, the keratitis did not resolve under the tested parameters.

Strengths:

The experiments carried out in this manuscript are thoughtful and rational, and sufficient explanation is provided for why the authors chose each specific set of experiments. The data presented strongly supports their conclusions and they give present compelling explanations for any deviation. The authors have not only developed a new technique for screening for phages targeting P. aeruginosa, but also highlights the importance of looking for phages during the biofilm mode of growth, as opposed to the more standard techniques involving planktonic cultures.

Weaknesses:

The authors did not include host-range testing or resistance development in this study, which would have strengthened the paper. Additionally, further characterisation of the CLEW-1 interaction with PSL at the molecular level would also have been welcomed. However, this will likely be the subject of future studies.

Reviewer #1 (Public review):

Summary:

In this paper, Behruznia and colleagues use long-read sequencing data for 339 strains of the Mycobacterium tuberculosis complex to study genome evolution in this clonal bacterial pathogen. They use both a "classical" pangenome approach that looks at the presence and absence of genes, and a pangenome graph based on whole genomes in order to investigate structural variants in non-coding regions. The comparison of the two approaches is informative and shows that much is missed when focussing only on genes. The two main biological results of the study are that 1) the MTBC has a small pangenome with few accessory genes, and that 2) pangenome evolution is driven by genome reduction. In the revised article, the description of the data set and the methods is much improved, and the comparison of the two pangenome approaches is more consistent. I still think, however, that the discussion of genome reduction suffers from a basic flaw, namely the failure to distinguish clearly between orthologs and homologs/paralogs.

Strengths:

The authors put together the so-far largest data set of long-read assemblies representing most lineages of the Mycobacterium tuberculosis context, and covering a large geographic area. They sequenced and assembled genomes for strains of M. pinnipedi, L9, and La2, for which no high-quality assemblies were available previously. State-of-the-art methods are used to analyze gene presence-absence polymorphisms (Panaroo) and to construct a pangenome graph (PanGraph). Additional analysis steps are performed to address known problems with misannotated or misassembled genes.

Weaknesses:

The revised manuscript has gained much clarity and consistency. One previous criticism, however, has in my opinion not been properly addressed. I think the problem boils down to not clearly distinguishing between orthologs and paralogs/homologs. As this problem affects a main conclusion - the prevalence of deletions over insertions in the MTBC - it should be addressed, if not through additional analyses, then at least in the discussion.

Insertions and deletions are now distinguished in the following way: "Accessory regions were further classified as a deletion if present in over 50% of the 192 sub-lineages or an insertion/duplication if present in less than 50% of sub-lineages." The outcome of this classification is suspicious: not a single accessory region was classified as an insertion/duplication. As a check of sanity, I'd expect at least some insertions of IS6110 to show up, which has produced lineage- or sublineage-specific insertions (Roychowdhury et al. 2015, Shitikov et al. 2019). Why, for example, wouldn't IS6110 insertions in the single L8 strain show up here?

In a fully clonal organism, any insertion/duplication will be an insertion/duplication of an existing sequence, and thus produce a paralog. If I'm correctly understanding your methods section, paralogs are systematically excluded in the pangraph analysis. Genomic blocks are summarized at the sublineage levels as follows (l.184 ): "The DNA sequences from genomic blocks present in at least one sub-lineage but completely absent in others were extracted to look for long-term evolution patterns in the pangenome." I presume this is done using blastn, as in other steps of the analysis.

So a sublineage-specific copy of IS6110 would be excluded here, because IS6110 is present somewhere in the genome in all sublineages. However, the appropriate category of comparison, at least for the discussion of genome reduction, is orthology rather than homology: is the same, orthologous copy of IS6110, at the same position in the genome, present or absent in other sublineages? The same considerations apply to potential sublineage-specific duplicates of PE, PPE, and Esx genes. These gene families play important roles in host-pathogen interactions, so I'd argue that the neglect of paralogs is not a finicky detail, but could be of broader biological relevance.

Reviewer #2 (Public review):

Summary:

The authors aimed to explore the role of climbing fibers (CFs) in cerebellar learning, with a focus on optokinetic reflex (OKR) adaptation. Their goal was to understand how CF activity influences memory acquisition, memory consolidation, and memory retrieval by optogenetically suppressing CF inputs at various stages of the learning process.

Strengths:

The study addresses a significant question in the cerebellar field by focusing on the specific role of CFs in adaptive learning. The authors use optogenetic tools to manipulate CF activity. This provides a direct method to test the causal relationship between CF activity and learning outcomes.

Weaknesses:

Despite shedding light on the potential role of CFs in cerebellar learning, the study is hampered by significant methodological issues that question the validity of its conclusions. The absence of detailed evidence on the effectiveness of CF suppression and concerns over tissue damage from optogenetic stimulation weaken the argument that CFs are not essential for memory consolidation. These challenges make it difficult to confirm whether the study's objectives were fully met or if the findings conclusively support the authors' claims. The research commendably attempts to unravel the temporal involvement of CFs in learning but also underscores the difficulties in pinpointing specific neural mechanisms that underlie the phases of learning. Addressing these methodological issues, investigating other signals that might instruct consolidation, and understanding CFs' broader impact on various learning behaviors are crucial steps for future studies.

Comments on revisions:

In this revision, the authors provide new data regarding the effect of eNpHR on CF-evoked complex spiking in vivo but fails to address overall concerns showing the functional effect that explains their causal results. Additionally, the paper has a narrow "CF-or-nothing" framing that leaves unanswered the central question of which signal instructs consolidation if CFs do not. Substantial new experiments and tighter logic are required before the work can serve as a definitive test of CF involvement in different memory processes.

Reviewer #1 (Public review):

This paper describes technically-impressive measurements of calcium signals near synaptic ribbons in goldfish bipolar cells. The data presented provides high spatial and temporal resolution information about calcium concentrations along the ribbon at various distances from the site of entry at the plasma membrane. This is important information. Important gaps in the data presented mean that the evidence for the main conclusions is currently inadequate.

Strengths

• The technical aspects of the measurements are impressive. The authors use calcium indicators bound to the ribbon and high speed line scans to resolve changes with a spatial resolution of ~250 nm and temporal resolution of less than 10 ms. These spatial and temporal scales are much closer to those relevant for vesicle release than previous measurements.

• The use of calcium indicators with very different affinities and of different intracellular calcium buffers helps provide confirmation of key results.

Weaknesses

• Multiple key points of the paper lack a statistical test or summary data from populations of cells. For example, the text states that the proximal and distal calcium kinetics in Figure 2A differ. This is not clear from the inset to Figure 2A - where the traces look like scaled versions of each other. Values for time to half-maximal peak fluorescence are given for one example cell but no statistics or summary are provided. Figure 8 shows examples from one cell with no summary data. This issue comes up in other places as well.

• The rise time measurements in Figure 2 are very different for low and high affinity indicators, but no explanation is given for this difference. Similarly, the measurements of peak calcium concentration in Figure 4 are very different with the two indicators. That might suggest that the high affinity indicator is strongly saturated, which raises concerns about whether that is impacting the kinetic measurements.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors attempt to reconstitute some active zone properties by introducing synaptic ribbon proteins into HEK cells. This "ground-up" approach can be valuable for assessing the necessity of specfic proteins in synaptic function. Here, the authors co-transfect a membrane-targeted bassoon, RBP2, calcium channel subunits and Ribeye to generate what they call "synthetic ribbons". The resultant structures show an ability to cluster calcium channels (Figure 4B) and a modest ability to concentrate calcium entry locations (figure 7J). At the light level, the ribeye aggregates look spherical and localize to the membrane through its interaction with the membrane-targeted bassoon and at the EM level the structures resemble those observed when Ribeye is overexpressed alone. It is a nice proof-of-principle in establishing a useful experimental system for studying calcium channel localization and with expression of other proteins perhaps a means to understanding structure and function of the ribbon. The paper does establish that previously described protein-interactions can be reconstituted in a heterologous system to and that the addition of Ribeye can increase the size of calcium channel patches via indirect interactions.

Strengths:

(1) The authors establish a new experimental system for the study of calcium channel localization to active zones.<br /> (2) The clustering of calcium channels to bassoon via RBP2 is a nice confirmation of a previously-described interaction between bassoon and calcium channels in a cell-based system<br /> (3) The "ground-up" approach is an attractive one and theoretically allows one learn a lot about the essential interactions for building a ribbon structure.<br /> (4) The finding that introducing Ribeye can enhance the size of calcium channel patches is a novel finding that is interesting.

Weaknesses:

(1) The addition of EM is welcome, but the structures seem to resemble those created by overexpression of Ribeye alone, albeit at the membrane. It is unclear to me whether the interaction with Bsn or indirect interactions with other proteins has any effect on these structures. Also, while the abstract mentions that the size and shape are similar to ribbons, the EM seems to show that the size and shape are quite variable.<br /> (2) The clustering of channels is accomplished by taking advantage of previously described interactions between RBP2, Ca channels and bassoon. While it is nice to see that it can be reconstituted in a naive cell, the interactions were previously described. The localization of Ribeye to bassoon takes advantage of a previously described interaction between the two and the membrane localization of the complexes required introduction of a membrane-anchoring motif. These factors limit the novelty of the findings.<br /> (3) The difference in Ca imaging between SyRibbons and other locations is subtle. While there are reasonable explanations for why this could be the case, it may limit the utility of this system for studying Ca-channel-ribbon dynamics moving forward.

Reviewer #1 (Public review):

Summary:

The authors investigated the elasticity of controllability by developing a task that manipulates the probability of achieving a goal with a baseline investment (which they refer to as inelastic controllability) and the probability that additional investment would increase the probability of achieving a goal (which they refer to as elastic controllability). They found that a computational model representing the controllability and elasticity of the environment accounted better for the data than a model representing only the controllability. They also found that prior biases about the controllability and elasticity of the environment was associated with a composite psychopathology score. The authors conclude that elasticity inference and bias guide resource allocation.

Strengths:

This research takes a novel theoretical and methodological approach to understanding how people estimate the level of control they have over their environment and how they adjust their actions accordingly. The task is innovative and both it and the findings are well-described (with excellent visuals). They also offer thorough validation for the particular model they develop. The research has the potential to theoretically inform understanding of control across domains, which is a topic of great importance.

Weaknesses:

In its revised form, the manuscript addresses most of my previous concerns. The main remaining weakness pertains to the analyses aimed at addressing my suggesting of Bayesian updating as an alternative to the model proposed by the authors. My suggestion was to assume that people perform a form of function approximation to relate resource expenditure to success probability. The authors performed a version of this where people were weighing evidence for a few canonical functions (flat, step, linear), and found that this model underperformed theirs. However, this Bayesian model is quite constrained in its ability to estimate the function relating resources. A more robust test would be to assume a more flexible form of updating that is able to capture a wide range of distributions (e.g., using basis functions, gaussian processes, or nonparametric estimators); see, e.g., work by Griffiths on human function learning). The benefit of testing this type of model is that it would make contact with a known form of inference that individuals engage in across various settings and therefore could offer a more parsimonious and generalizable account of function learning, whereby learning of resource elasticity is a special case. I defer to the authors as to whether they'd like to pursue this direction, but if not I think it's still important that they acknowledge that they are unable to rule out a more general process like this as an alternative to their model. This pertains also to inferences about individual differences, which currently hinge on their preferred model being the most parsimonious.

Reviewer #1 (Public review):

Summary:

This is an interesting study characterizing and engineering so-called bathy phytochromes, i.e., those that respond to near infrared (NIR) light in the ground state, for optogenetic control of bacterial gene expression. Previously, the authors have developed a structure-guided approach to functionally link several light-responsive protein domains to the signaling domain of the histidine kinase FixL, which ultimately controls gene expression. Here, the authors use the same strategy to link bathy phytochrome light-responsive domains to FixL, resulting in sensors of NIR light. Interestingly, they also link these bathy phytochrome light-sensing domains to signaling domains from the tetrathionate-sensing SHK TtrS and the toluene-sensing SHK TodS, demonstrating the generality of their protein engineering approach more broadly across bacterial two-component systems.

This is an exciting result that should inspire future bacterial sensor design. They go on to leverage this result to develop what is, to my knowledge, the first system for orthogonally controlling the expression of two separate genes in the same cell with NIR and Red light, a valuable contribution to the field.

Finally, the authors reveal new details of the pH-dependent photocycle of bathy phytochromes and demonstrate that their sensors work in the gut - and plant-relevant strains E. coli Nissle 1917 and A. tumefaciens.

Strengths:

(1) The experiments are well-founded, well-executed, and rigorous.

(2) The manuscript is clearly written.

(3) The sensors developed exhibit large responses to light, making them valuable tools for ontogenetic applications.

(4) This study is a valuable contribution to photobiology and optogenetics.

Weaknesses:

(1) As the authors note, the sensors are relatively insensitive to NIR light due to the rapid dark reversion process in bathy phytochromes. Though NIR light is generally non-phototoxic, one would expect this characteristic to be a limitation in some downstream applications where light intensities are not high (e.g., in vivo).

(2) Though they can be multiplexed with Red light sensors, these bathy phytochrome NIR sensors are more difficult to multiplex with other commonly used light sensors (e.g., blue) due to the broad light responsivity of the Pfr state. This challenge may be overcome by careful dosing of blue light, as the authors discuss, but other bacterial NIR sensing systems with less cross-talk may be preferred in some applications.

Reviewer #1 (Public review):

Summary:

Can a plastic RNN serve as a basis function for learning to estimate value. In previous work this was shown to be the case, with a similar architecture to that proposed here. The learning rule in previous work was back-prop with an objective function that was the TD error function (delta) squared. Such a learning rule is non-local as the changes in weights within the RNN, and from inputs to the RNN depends on the weights from the RNN to the output, which estimates value. This is non-local, and in addition, these weights themselves change over learning. The main idea in this paper is to examine if replacing the values of these non-local changing weights, used for credit assignment, with random fixed weights can still produce similar results to those obtained with complete bp. This random feedback approach is motivated by a similar approach used for deep feed-forward neural networks.

This work shows that this random feedback in credit assignment performs well but is not as well as the precise gradient-based approach. When more constraints due to biological plausibility are imposed performance degrades. These results are consistent with previous results on random feedback.

Strengths:

• The authors show that random feedback can approximate well a model trained with detailed credit assignment.<br /> • The authors simulate several experiments including some with probabilistic reward schedules and show results similar to those obtained with detailed credit assignments as well as in experiments.<br /> • The paper examines the impact of more biologically realistic learning rules and the results are still quite similar to the detailed back-prop model.

Weaknesses:

• The impact of the article is limited by using a network with discrete time-steps, and only a small number of time steps from stimulus to reward. They assume that each time step is on the order of hundreds of ms. They justify this by pointing to some slow intrinsic mechanisms, but they do not implement these slow mechanisms is a network with short time steps, instead they assume without demonstration that these could work as suggested. This is a reasonable first approximation, but its validity should be explicitly tested.

• As the delay between cue and reward increases the performance decreases. This is not surprising given the proposed mechanism, but is still a limitation, especially given that we do not really know what a is the reasonable value of a single time step.

Reviewer #2 (Public review):

Summary:

In this study, the authors characterized population genetic variation in the MHC locus across primates and looked for signals of long-term balancing selection (specifically trans-species polymorphism, TSP) in this highly polymorphic region. To carry out these tasks, they used Bayesian methods for phylogenetic inference (i.e. BEAST2) and applied a new Bayesian test to quantify evidence supporting monophyly vs. transspecies polymorphism for each exon across different species pairs. Their results, although mostly confirmatory, represent the most comprehensive analyses of primate MHC evolution to date and novel findings or possible discrepancies are clearly pointed out. However, as the authors discuss, the available data are insufficient to fully capture primates' MHC evolution.

Strengths of the paper include: using appropriate methods and statistically rigorous analyses; very clear figures and detailed description of the results methods that make it easy to follow despite the complexity of the region and approach; a clever test for TSP that is then complemented by positive selection tests and the protein structures for a quite comprehensive study.

That said, weaknesses include: lack of information about how many sequences are included and whether uneven sampling across taxa might results in some comparisons without evidence for TSP; frequent reference to the companion paper instead of summarizing (at least some of) the critical relevant information (e.g., how was orthology inferred?); no mention of the quality of sequences in the database and whether there is still potential effects of mismapping or copy number variation affecting the sequence comparison.

Comments on revisions:

The authors have sufficiently addressed the reviewers' comments or provided additional details justifying their work. In particular, expansion of the discussion section on limitations of the analysis and clearer reference to how this relates to their companion paper represent improvements. Remaining suggestions are to still make clearer how much sparsity of sequences in the database may impact the conclusions (e.g., is this more of a problem for some genes or taxa than others? Is it a small problem or a large problem?). The data summary tables are a bit hard to read and seem to contain some information not used in the article - maybe the presentation of these could be improved or the full details, or a shorter table summer in the main paper and full details only in the supplement.

Reviewer #1 (Public review):

Summary and Strengths:

This work shows that the gene encoding Layilin is expressed preferentially in human skin Tregs, and that the fraction of Tregs expressing Layilin may overexpress genes related to T cell activation and adhesion. Expression of Layilin on Tregs would have no impact on activation markers or in vitro suppressive function. However, activation of Layilin either with a cross-linking antibody or collagen IV, its natural ligand, would promote cell adhesion via LFA1 activation. The in vivo functional role of Layilin in Tregs is studied in a conditional KO mouse model in a model of skin inflammation. Deletion of Layilin in Tregs led to an attenuation of the disease score and a reduction in the cutaneous lymphocyte infiltrate. This work is clearly innovative, but a number of major points limit its interest.

Weakness and major points:

(1) The number of panels and figures suggests that this story is quite complete but several data presented in the main figures do not provide essential information for a proper understanding of Layilin's role in Tregs.

Figures 1I, 1J, and the whole of Figure 2 could be placed as supplementary figures. Also, for Figure 3E, it would be preferable to show the percentage of cells expressing cytokines rather than their absolute numbers. In fact, the drop in the numbers of cytokine-producing cells is probably due solely to the drop in total cell numbers and not to a decrease in the proportion of cells expressing cytokines. If this is the case, these data should be shown in supplementary figures. Finally, Figures 4 and 5 could be merged.

(2) Some important data are not shown or not mentioned.

(a) It would be important to show the proportion of Treg, Tconv, and CD8 expressing Layilin in healthy skin and in patients developing psoriasis, as well as in the blood of healthy subjects.<br /> (b) We lack information to be convinced that there is enrichment for migration and adhesion genes in Layilin+ Tregs in the GSEA data. The authors should indicate what geneset libraries they used. Indeed, it is tempting to show only the genesets that give results in line with the message you want to get across. If these genesets come from public banks, the bank used should be indicated, and the results of all gene sets shown in an unbiased way. In addition, it should be indicated whether the analyses were performed on untransformed or pseudobulk scRNAseq data analyses. Finally, it would be preferable to confirm the GSEA data with z-score analyses, as Ingenuity does, for example. Indeed, in GSEA-type analyses, there are genes that have activating but also inhibiting effects on a pathway in a given gene set.<br /> (c) For all FACS data, the raw data should be shown as histograms or dot plots for representative samples.<br /> (d) For Figure 5B, the number of samples analyzed is insufficient to draw clear conclusions.

(3) For Figs. 4 and 5, the design of the experiment poses a problem. Indeed, the comparison between Layn+ and Layn- cells may, in part, not be directly linked to the expression or absence of expression of this protein. Indeed, Layn+ and Layn- Tregs may constitute populations with different biological properties, beyond the expression of Layn. However, in the experiment design used here, a significant fraction of the sorted Layn- Tregs will be cells belonging to the population that has never expressed this protein. It would have been preferable to sort first the Layn+ Tregs, then knock down this protein and re-sort the Layn- Tregs and Layn+ Tregs. If this experiment is too cumbersome to perform, I agree that the authors should not do it. However, it would be important to mention the point I have just made in the text.

Reviewer #1 (Public review):

Summary:

Aicardi-Goutières Syndrome (AGS) is a genetic disorder that primarily affects the brain and immune system through excessive interferon production. The authors sought to investigate the role of microglia in AGS by first developing bone-marrow-derived progenitors in vitro that carry the estrogen-regulated (ER) Hoxb8 cassette, allowing them to expand indefinitely in the presence of estrogen and differentiate into macrophages when estrogen is removed. When injected into the brains of Csf1r-/- mice, which lack microglia, these cells engraft and resemble wild-type (WT) microglia in transcriptional and morphological characteristics, although they lack Sall1 expression. The authors then generated CRISPR-Cas9 Adar1 knockout (KO) ER-Hoxb8 macrophages, which exhibited increased production of inflammatory cytokines and upregulation of interferon-related genes. This phenotype could be rescued using a Jak-Stat inhibitor or by concurrently mutating Ifih1 (Mda5). However, these Adar1-KO macrophages fail to successfully engraft in the brain of both Csf1r-/- and Cx3cr1-creERT2:Csf1rfl/fl mice. To overcome this, the authors used a mouse model with a patient-specific Adar1 mutation (Adar1 D1113H) to derive ER-Hoxb8 bone marrow progenitors and macrophages. They discovered that Adar1 D1113H ER-Hoxb8 macrophages successfully engraft the brain, although at lower levels than WT-derived ER-Hoxb8 macrophages, leading to increased production of Isg15 by neighboring cells. These findings shed new light on the role of microglia in AGS pathology.

Strengths:

The authors convincingly demonstrate that ER-Hoxb8 differentiated macrophages are transcriptionally and morphologically similar to bone marrow-derived macrophages. They also show evidence that when engrafted in vivo, ER-Hoxb8 microglia are transcriptomically similar to WT microglia. Furthermore, ER-Hoxb8 macrophages engraft the Csf1r-/- brain with high efficiency and rapidly (2 weeks), showing a homogenous distribution. The authors also effectively use CRISPR-Cas9 to knock out TLR4 in these cells with little to no effect on their engraftment in vivo, confirming their potential as a model for genetic manipulation and in vivo microglia replacement.

Overall, this paper demonstrates an innovative approach to manipulating microglia using ER-Hoxb8 cells as surrogates. The authors present convincing evidence of the model's efficacy and potential for broader application in microglial research, given its ease of production and rapid brain engraftment potential in microglia-deficient mice. Using mouse-derived cells for transplantation reduces complications that can come with the use of human cell lines, highlighting the utility of this system for research in mouse models.

Reviewer #2 (Public review):

In the manuscript, Ruhling et al propose a rapid uptake pathway that is dependent on lysosomal exocytosis, lysosomal Ca2+ and acid sphingomyelinase, and further suggest that the intracellular trafficking and fate of the pathogen is dictated by the mode of entry. Overall, this is manuscript argues for an important mechanism of a 'rapid' cellular entry pathway of S.aureus that is dependent on lysosomal exocytosis and acid sphingomyelinase and links the intracellular fate of bacterium including phagosomal dynamics, cytosolic replication and host cell death to different modes of uptake.

Key strength is the nature of the idea proposed, while continued reliance on inhibitor treatment combined with lack of phenotype for genetic knock out is a major weakness. While the authors argue a role for undetectable nano-scale Cer platforms on the cell surface caused by ASM activity, results do not rule out a SM independent role in the cellular uptake phenotype of ASM inhibitors.

The authors have attempted to address many of the points raised in the previous revision. While the new data presented provide partial evidence, the reliance on chemical inhibitors and lack of clear results directly documenting release of lysosomal Ca2+, or single bacterial tracking, or clear distinction between ASM dependent and independent processes dampen the enthusiasm.

I acknowledge the author's argument of different ASM inhibitors showing similar phenotypes across different assays as pointing to a role for ASM, but the lack of phenotype in ASM KO cells is concerning. The author's argument that altered lipid composition in ASM KO cells could be overcoming the ASM-mediated infection effects by other ASM-independent mechanisms is speculative, as they acknowledge, and moderates the importance of ASM-dependent pathway. The SM accumulation in ASM KO cells does not distinguish between localized alterations within the cells. If this pathway can be compensated, how central is it likely to be ?

The authors allude to lower phagosomal escape rate in ASM KO cells compared to inhibitor treatment, which appears to contradict the notion of uptake and intracellular trafficking phenotype being tightly linked. As they point out, these results might be hard to interpret. Could an inducible KD system recapitulate (some of) the phenotype of inhibitor treatment ? If S. aureus does not escape phagosome in macrophages, could it provide a system to potentially decouple the uptake and intracellular trafficking effects by ASM (or its inhibitor treatment) ?

The role of ASM on cell surface remains unclear. The hypothesis proposed by the authors that the localized generation of Cer on the surface by released ASM leads to generation of Cer-enriched platforms could be plausible, but is not backed by data, technical challenges to visualize these platforms notwithstanding. These results do not rule out possible SM independent effects of ASM on the cell surface, if indeed the role of ASM is confirmed by controlled genetic depletion studies.

The reviewer acknowledges technical challenges in directly visualizing lysosomal Ca2+ using the methods outlined. Genetically encoded lysosomal Ca2+ sensor such as Gcamp3-ML1 might provide better ways to directly visualize this during inhibitor treatment, or S. aureus infection.

Reviewer #1 (Public review):

Summary:

This important study by Chen et. al. describes a novel approach for optogentically evoking seizures in an etiologically relevant mouse model of epilepsy. The authors developed a model that can trigger seizures "on demand" using optogenetic stimulation of CA1 principal cells in mice rendered epileptic by an intra-hippocampal kainate (IHK) injection into CA3. The authors discuss their model in the context of the limitations of current animal models used in epilepsy drug development. In particular, their model addresses concerns regarding existing models where testing typically involves inducing acute seizures in healthy animals or waiting on infrequent, spontaneous seizures in epileptic animals.

Strengths:

A strength of this manuscript is that this approach may facilitate the evaluation of novel therapeutics since these evoked seizures, despite having some features that were significantly different from spontaneous seizures, are suggested to be sufficiently similar to spontaneous seizures which are more laborious to analyze. The data demonstrating the commonality of pharmacology and EEG features between evoked seizures and spontaneous seizures in epileptic mice, while also being different from evoked seizures in naïve mice, are convincing. The structural, functional, and behavioral differences between a seizure-naïve and epileptic mouse, which emerge due to the enduring changes occurring during epileptogenesis, are complex and important. Accordingly, this study highlights the importance of using mice that have underwent epileptogenesis as model organisms for testing novel therapeutics. Furthermore, this study positively impacts the wider epilepsy research community by investigating seizure semiology in these populations.

Weaknesses:

This study convincingly demonstrates that the feature space measurements for stimulus-evoked seizures in epileptic mice were significantly different from those in naïve mice; this result allows the authors to conclude that "seizures induced in chronically epileptic animals differed from those in naïve animals". However, the authors also conclude that "induced seizures resembled naturally occurring spontaneous seizures in epileptic animals" despite their own data demonstrating similar, albeit fewer, significant differences in feature space measurements. It is unclear if and what the threshold is whereby significant differences in these feature space measurements lead to the conclusion that the differences are meaningful, as in the comparison of epileptic and naïve mice, or not meaningful, as in the comparison of evoked and spontaneous seizures.

Reviewer #1 (Public review):

Strengths:

This work adds another mouse model for LAMA2-MD that re-iterates the phenotype of previously published models. Such as dy3K/dy3K; dy/dy and dyW/dyW mice. The phenotype is fully consistent with the data from others.

One of the major weaknesses of the manuscript initially submitted was the overinterpretation and the overstatements. The revised version is clearly improved as the authors toned-down their interpretation and now also cite the relevant literature of previous work.

Comments on revisions:

This is the second revision of a paper focusing on the generation of a CRISPR/Cas9-engineered mouse model for LAMA2-MD. I have reviewed the initial submission, the first revision, and now this second revision. While there have been improvements, several issues still need to be addressed by the authors. I will outline these points without dividing them into major and minor categories:

Introduction:

The statement regarding existing mouse models requires correction: The claim, "They were established in the pre-gene therapy era, leaving trace of engineering, such as bacterial elements in the Lama2 gene locus, thus unsuitable for testing various gene therapy strategies," is inaccurate. Current mouse models can indeed be used for testing gene therapy strategies, regardless of whether they contain elements in the Lama2 locus. The primary consideration is whether or not they express laminin-alpha2. Please revise this statement.<br /> Results Section:

scRNA-seq:

The authors note that they analyzed "a total of 8,111 cells from the dyH/dyH mouse brain and 8,127 cells from the WT mouse brain were captured using the 10X Genomics platform (Figure supplement 4A, B)." This is too few cells to support firm conclusions. Furthermore, there is a discrepancy in the referred figure S4, which indicates that 10,094 cells were analyzed for dyH/dyH mice and 10,496 for wild-type mice. Please correct this inconsistency.

Figure 5C displays differences in cell populations between wild-type and dyH/dyH mice. Given the low number of cells analyzed and the lack of replicates, these differences cannot be considered reliable. More samples should be analyzed to support these findings.

The data suggest a defect in the BBB for dyH/dyH mice, but this conclusion is based on minimal cell counts and remains purely correlative. If BBB issues exist, experimental validation is necessary, such as injecting dyes into the bloodstream to detect any leakage. I have previously highlighted this in my comments on earlier manuscript versions.

Bulk RNA-seq:

The number of samples analyzed here is substantial, making the data potentially more robust. These data could serve as a valuable resource for other researchers. However, it is important to note that all data are correlative and do not provide functional insights.

Overall:

The manuscript still lacks significant insights, partly because existing mouse models for LAMA2-MD have been extensively analyzed. While the bulk RNA-seq data offer some value as a resource, I recommend that the authors re-assess their writing and further temper their interpretations of the findings.

Reviewer #1 (Public review):

Translating discoveries from model organisms to humans is often challenging, especially in neuropsychiatric diseases, due to the vast gaps in the circuit complexities and cognitive capabilities. Kajtor et al. propose to bridge this gap in the fly models of Parkinson's disease (PD) by developing a new behavioural assay where flies respond to a moving shadow by modifying their locomotor activities. The authors believe the flies' response to the shadow approximates their escape response to an approaching predator. To validate this argument, they tested several PD-relevant transgenic fly lines and showed that some of them indeed have altered responses in their assay.

Strengths:

This single-fly-based assay is easy and inexpensive to set up, scalable and provides sensitive, quantitative estimates to probe flies' optomotor acuity. The behavioural data is detailed, and the analysis parameters are well-explained.

Weaknesses:

The authors have yet to link cellular physiology to behaviour. It will be interesting to see how future use of this assay helps uncover connections between cellular pathology and behavioural changes.

Reviewer #1 (Public review):

Summary:

This manuscript explores the multiple cell types present in the wall of murine collecting lymphatic vessels with the goal of identifying cells that initiate the autonomous action potentials and contractions needed to drive lymphatic pumping. Through the use of genetic models to delete individual genes or detect cytosolic calcium in specific cell types, the authors convincingly determine that lymphatic muscle cells are the origin of the action potential that triggers lymphatic contraction.

Strengths:

The experiments are rigorously performed, the data justify the conclusions and the limitations of the study are appropriately discussed.

There is a need to identify therapeutic targets to improve lymphatic contraction and this work helps identify lymphatic muscle cells as potential cellular targets for intervention.

Comments on revisions: The authors have addressed all of the reviewer comments. They should be congratulated on their precise and comprehensive study.

Reviewer #2 (Public review):

Summary:

Sukhina et al. uses a chronic murine dietary restriction model to investigate the cellular mechanisms underlying nutritionally acquired immunodeficiency as well as the consequences of a refeeding intervention. The authors report a substantial impact of undernutrition to the myeloid compartment, which is not rescued by refeeding despite rescue of other phenotypes including lymphocyte levels, and which is associated with maintained partial susceptibility to bacterial infection.

Strengths:

Overall, this is a nicely executed study with an appropriate number of mice, robust phenotypes, and interesting conclusions, and the text is very well written. The authors' conclusions are generally well-supported by their data.

Weaknesses:

There is little evaluation of known critical drivers of myelopoiesis (e.g. PMID 20535209, 26072330, 29218601) over the course of the 40% diet, which would be of interest with regard to comparing this chronic model to other more short-term models of undernutrition.

Further, the microbiota, well-established to be regulated by undernutrition (e.g. PMID 22674549, 27339978, etc.), and also well-established to be a critical regulator of hematopoiesis/myelopoiesis (e.g. PMID 27879260, 27799160, etc.), should be studied in any future explorations using this model.

The authors have recognized these limitations to the study in their discussion.

Reviewer #1 (Public review):

Summary:

Desingu et al. show that JEV infection reduces SIRT2 expression. Upon JEV infection, 10-day-old SIRT2 KO mice showed increased viral titer, more severe clinical outcomes, and reduced survival. Conversely, SIRT2 overexpression reduced viral titer, clinical outcomes, and improved survival. Transcriptional profiling shows dysregulation of NF-KB and expression of inflammatory cytokines. Pharmacological NF-KB inhibition reduced viral titer. The authors conclude that SIRT2 is a regulator of JEV infection.

Strengths:

This paper is novel because sirtuins have been primarily studied for aging, metabolism, stem cells/regeneration. Their role in infection has not been explored until recently. Indeed, Barthez et al. showed that SIRT2 protects aged mice from SARS-CoV-2 infection (Barthez, Cell Reports 2025). Therefore, this is a timely and novel research topic. Mechanistically, the authors showed that SIRT2 suppresses the NF-KB pathway. Interestingly, SIRT2 has also been shown recently to suppress other major inflammatory pathways, such as cGAS-STING (Barthez, Cell Reports 2025) and the NLRP3 inflammasome (He, Cell Metabolism 2020; Luo, Cell Reports 2019). Together, these findings support the emerging concept that SIRT2 is a master regulator of inflammation.

Weaknesses:

(1) Figures 2 and 3. Although SIRT2 KO mice showed increased viral titer, more severe clinical outcomes, and reduced survival upon JEV infection, the difference is modest because even WT mice exhibited very severe disease at this viral dose. The authors should perform the experiment using a sub-lethal viral dose for WT mice, to allow the assessment of increased clinical outcomes and reduced survival in KO mice.

(2) Figure 5K-N, the authors examined the expression of inflammatory cytokines in WT and SIRT2 KO cells upon JEV infection, in line with the dysregulation of NF-kB. It has been shown recently that SIRT2 also regulates the cGAS-STING pathway (Barthez, Cell Reports 2025) and the NLRP3 inflammasome (He, Cell Metabolism 2020; Luo, Cell Reports 2019). Do you also observe increased IFNb, IL1b, and IL18 in SIRT2 KO cells upon JEV infection? This may indicate that SIRT2 regulates systemic inflammatory responses and represents a potent protection upon viral infection. This is particularly important because in Figure 7F, the authors showed that SIRT2 overexpression reduced viral load even when NF-KB is inhibited, suggesting that NF-KB is not the only mediator of SIRT2 to suppress viral infection.

Reviewer #1 (Public review):

Fombellida-Lopez and colleagues describe the results of an ART intensification trial in people with HIV infection (PWH) on suppressive ART to determine the effect of increasing the dose of one ART drug, dolutegravir, on viral reservoirs, immune activation, exhaustion, and circulating inflammatory markers. The authors hypothesize that ART intensification will provide clues about the degree to which low-level viral replication is occurring in circulation and in tissues despite ongoing ART, which could be identified if reservoirs decrease and/or if immune biomarkers change. The trial design is straightforward and well-described, and the intervention appears to have been well tolerated. The investigators observed an increase in dolutegravir concentrations in circulation, and to a lesser degree in tissues, in the intervention group, indicating that the intervention has functioned as expected (ART has been intensified in vivo). Several outcome measures changed during the trial period in the intervention group, leading the investigators to conclude that their results provide strong evidence of ongoing replication on standard ART. The results of this small trial are intriguing, and a few observations in particular are hypothesis-generating and potentially justify further clinical trials to explore them in depth. However, I am concerned about over-interpretation of results that do not fully justify the authors' conclusions.

(1) Trial objectives: What was the primary objective of the trial? This is not clearly stated. The authors describe changes in some reservoir parameters and no changes in others. Which of these was the primary outcome? No a priori hypothesis / primary objective is stated, nor is there explicit justification (power calculations, prior in vivo evidence) for the small n, unblinded design, and lack of placebo control. In the abstract (line 36, "significant decreases in total HIV DNA") and conclusion (lines 244-246), the authors state that total proviral DNA decreased as a result of ART intensification. However, in Figures 2A and 2E (and in line 251), the authors indicate that total proviral DNA did not change. These statements are confusing and appear to be contradictory. Regarding the decrease in total proviral DNA, I believe the authors may mean that they observed transient decrease in total proviral DNA during the intensification period (day 28 in particular, Figure 2A), however this level increases at Day 56 and then returns to baseline at Day 84, which is the source of the negative observation. Stating that total proviral DNA decreased as a result of the intervention when it ultimately did not is misleading, unless the investigators intended the day 28 timepoint as a primary endpoint for reservoir reduction - if so, this is never stated, and it is unclear why the intervention would then be continued until day 84? If, instead, reservoir reduction at the end of the intervention was the primary endpoint (again, unstated by the authors), then it is not appropriate to state that the total proviral reservoir decreased significantly when it did not.

(2) Intervention safety and tolerability: The results section lacks a specific heading for participant safety and tolerability of the intervention. I was wondering about clinically detectable viremia in the study. Were there any viral blips? Was the increased DTG well tolerated? This drug is known to cause myositis, headache, CPK elevation, hepatotoxicity, and headache. Were any of these observed? What is the authors' interpretation of the CD4:8 ratio change (line 198)? Is this a significant safety concern for a longer duration of intensification? Was there also a change in CD4% or only in absolute counts? Was there relative CD4 depletion observed in the rectal biopsy samples between days 0 and 84? Interestingly, T cells dropped at the same timepoints that reservoirs declined... how do the authors rule out that reservoir decline reflects transient T cell decline that is non-specific (not due to additional blockade of replication)?

(3) The investigators describe a decrease in intact proviral DNA after 84 days of ART intensification in circulating cells (Figure 2D), but no changes to total proviral DNA in blood or tissue (Figures 2A and 2E; IPDA does not appear to have been done on tissue samples). It is not clear why ART intensification would result in a selective decrease in intact proviruses and not in total proviruses if the source of these reservoir cells is due to ongoing replication. These reservoir results have multiple interpretations, including (but not limited to) the investigators' contention that this provides strong evidence of ongoing replication. However, ongoing replication results in the production of both intact and mutated/defective proviruses that both contribute to reservoir size (with defective proviruses vastly outnumbering intact proviruses). The small sample size and well-described heterogeneity of the HIV reservoir (with regard to overall size and composition) raise the possibility that the study was underpowered to detect differences over the 84-day intervention period. No power calculations or prior studies were described to justify the trial size or the duration of the intervention. Readers would benefit from a more nuanced discussion of reservoir changes observed here.

(4) While a few statistically significant changes occurred in immune activation markers, it is not clear that these are biologically significant. Lines 175-186 and Figure 3: The change in CD4 cells + for TIGIT looks as though it declined by only 1-2%, and at day 84, the confidence interval appears to widen significantly at this timepoint, spanning an interquartile range of 4%. The only other immune activation/exhaustion marker change that reached statistical significance appears to be CD8 cells + for CD38 and HLA-DR, however, the decline appears to be a fraction of a percent, with the control group trending in the same direction. Despite marginal statistical significance, it is not clear there is any biological significance to these findings; Figure S6 supports the contention that there is no significant change in these parameters over time or between groups. With most markers showing no change and these two showing very small changes (and the latter moving in the same direction as the control group), these results do not justify the statement that intensifying DTG decreases immune activation and exhaustion (lines 38-40 in the abstract and elsewhere).

(5) There are several limitations of the study design that deserve consideration beyond those discussed at line 327. The study was open-label and not placebo-controlled, which may have led to some medication adherence changes that confound results (authors describe one observation that may be evidence of this; lines 146-148). Randomized/blinded / cross-over design would be more robust and help determine signal from noise, given relatively small changes observed in the intervention arm. There does not seem to be a measurement of key outcome variables after treatment intensification ceased - evidence of an effect on replication through ART intensification would be enhanced by observing changes once intensification was stopped. Why was intensification maintained for 84 days? More information about the study duration would be helpful. Table 1 indicates that participants were 95% male. Sex is known to be a biological variable, particularly with regard to HIV reservoir size and chronic immune activation in PWH. Worldwide, 50% of PWH are women. Research into improving management/understanding of disease should reflect this, and equal participation should be sought in trials. Table 1 shows differing baseline reservoir sizes betweenthe control and intervention groups. This may have important implications, particularly for outcomes where reservoir size is used as the denominator.

(6) Figure 1: the increase in DTG levels is interesting - it is not uniform across participants. Several participants had lower levels of DTG at the end of the intervention. Though unlikely to be statistically significant, it would be interesting to evaluate if there is a correlation between change in DTG concentrations and virologic / reservoir / inflammatory parameters. A positive relationship between increasing DTG concentration and decreased cell-associated RNA, for example, would help support the hypothesis that ongoing replication is occurring.

(7) Figure 2: IPDA in tissue- was this done? scRNA in blood (single copy assay) - would this be expected to correlate with usCaRNA? The most unambiguous result is the decrease in cell-associated RNA - accompanying results using single-copy assay in plasma would be helpful to bolster this result. The use of the US RNA / Total DNA ratio is not helpful/difficult to interpret since the control and intervention arms were unmatched for total DNA reservoir size at study entry.

Reviewer #1 (Public review):

Summary:

The authors combine PSMC and habitat modeling to try to connect habitat change during the Last Glacial Period to changes in Ne.

Strengths:

Observing how tropical single-island endemic bird species responded to habitat change in the past may help inform conservation interventions for these particularly vulnerable species. The combination of genomics and habitat modeling is a good idea - this sort of interdisciplinary thinking is what is needed to tackle these complex questions. Additionally, the use of PSMC makes it possible to perform this analysis on poorly-studied species with only a single genome available.

Room for Improvement:

Why coalescent Ne is a better predictor of extinction risk than current genomic diversity, or current Ne, isn't explicitly explained. PSMC in particular has many caveats, and some are not acknowledged or adequately addressed by the authors. For example, the authors note that population structure is a confounding factor with PSMC, but that it is not a problem in this instance. They do not provide compelling evidence for why this would be the case, they simply state that the species studied are all single-island endemics. However, single-island endemic species are not necessarily panmictic; this is even less likely to be true for species studied here that inhabit a large geographic area (ie, Australian species). Differing PSMC parameters may also impact results: the differences between passerines and non-passerines were one of their main results, but they do not provide any analysis to show that this difference was not driven by the different mutation rates used for the two groups.

Parameters for many steps are not described, and choices that are described (such as the PSMC parameters) are not always fully explained. It is unclear why all data was mapped to the autosomes rather than removing reads that map to the sex chromosomes first. Using all the data, the reads belonging to the sex chromosomes could potentially map to other areas of the genome. It does not seem like a mapping quality filter was used, so these potential spurious alignments would not have been removed prior to analysis.

There are points where the results are described in ways that appear to potentially differ from the supplementary figures. The authors state that even for species where PSMC results differed between models, "trends of Ne increase or decrease from the LIG to LGM were robust across all three PSMC models considered." The figures in the supplement for Pachycephala philippinensis, Rhynochetos jubatus, and Zosterops hypoxanthus appear to potentially contradict this statement, but it is difficult to tell, as the time period observed is not clearly marked on the graphs. How this robustness of trends was determined is not explained, leaving the precision of the analysis unclear.

Table 1 also includes some information that contradicts what is in the Supplementary Tables, leading to a lack of clarity. Centropus unirufus, Chaetorhynchus papuensis, and Cnemophilus loriae are not included in Supplementary Table 4. Table 1 says Eulacestoma nigropectus, Paradisaea rubra, and Parotia lawesii did not undergo PSMC analysis, but Supplementary Table 4 says PSMC and modeling trends matched for these species. Table 1 says Rhagologus leucostigma underwent both PSMC and climate modeling, but Supplementary Table 4 says "NA" as if it was missing one of these analyses.

Additionally, some of the results appear to contradict each other. For example, they show that there is no impact of habitat change in larger-bodied species, but also that larger-bodied species saw a decrease in Ne during the LGP. In another example, they state that when a species saw an increase in habitat during the LGP, they also had an increase in Ne. However, they also state that this was not the case for non-passerines.

Ecosystems are highly complex; there may also be other variables influencing past demographic change other than those explored here. Results should be interpreted with caution.

Reviewer #1 (Public review):

Summary:

This manuscript investigates how cellular NAD/NADH ratios are controlled in cancer cell lines in vitro. The authors build on previous work, which shows that serine synthesis is sensitive to NAD/NADH ratios and PHGDH expression. Here, the authors demonstrate that serine synthesis is variable across a panel of cell lines, even when controlling for expression of serine synthesis enzymes such as PHGDH. The authors show that cellular NAD/NADH ratios correlate with the ability to synthesize serine and grow in serine-deprived environments when PHGDH levels remain constant. Investigating this variability in NAD/NADH ratios, the authors find that the cells that can positively respond to serine deprivation are able to increase oxygen consumption and cellular NAD/NADH ratios. Cells that do not increase oxygen consumption in response to serine deprivation do not increase NAD/NADH ratios and cannot grow well without serine. The authors go on to show that in cells with the ability to increase oxygen consumption upon serine deprivation, PHGDH expression alone is sufficient to fully restore growth-serine; in cells that cannot increase oxygen consumption, both PHGDH expression and interventions to increase NAD/NADH ratios are required to increase growth. Thus, cells need both PHGDH and NAD/NADH increases to maximize serine synthesis in response to serine deprivation. The authors previously showed that lipid synthesis likewise requires NAD regeneration. Interestingly, one cell line that does not increase oxygen consumption in response to serine limitation tends to increase oxygen consumption in response to lipid deprivation; accordingly, depriving this cell line of lipids increases the synthesis of serine. Together, these findings show that how cells respond to nutrient deprivation is highly variable and that the response to nutrient deprivation (for example, whether or not oxygen consumption is increased) will determine how well cells tolerate depletion of nutrients with related biosynthetic constraints. This work sheds light on the complexity of cancer cell metabolism and helps to explain why it is difficult to predict which nutrients will be limiting to any cancer cell type or environment.

Strengths:

(1) The authors use multiple interventions to manipulate NAD/NADH ratios in cells.

(2) Experiments are well controlled and appropriately interpreted.

Weaknesses:

Overall the data support the conclusions of the manuscript. I have only two minor comments and suggestions:.

(1) Figure 2B/C: data are presented as relative to +serine, which shows how some cells respond to -serine, but may also be of interest to see how absolute (not relative) NAD/NADH levels correlate with serine synthesis and serine-independent proliferation. In other words, is it the dynamic increase in the ratio that is most important, or the absolute level of the ratio?

(2) Line 177-178: the authors write, "We hypothesized that the elevated NAD+/NADH ratio represented a cellular response to make the NAD+/NADH ratio more oxidized to enable serine synthesis". I recommend modest edits to avoid anthropomorphizing. It is possible that the ratio responds for reasons yet to be determined and not necessarily because the cell is deliberately trying to enable serine synthesis.

Reviewer #1 (Public review):

Summary:

Weiss et. al. seek to delineate the mechanisms by which antigen-specific CD8+ T cells outcompete bystanders in the epidermis when active TGF-b is limiting, resulting in selective retention of these cells and more complete differentiation into the TRM phenotype.

Strengths:

They begin by demonstrating that at tissue sites where cognate antigen was expressed, CD8+ T cells adopt a more mature TRM transcriptome than cells at tissue sites where cognate antigen was never expressed. By integrating their scRNA-Seq data on TRM with the much more comprehensive ImmGenT atlas, the authors provide a very useful resource for future studies in the field. Furthermore, they conclusively show that these "local antigen-experienced" TRM have increased proliferative capacity and that TCR avidity during TRM formation positively correlates with their future fitness. Finally, using an elegant experimental strategy, they establish that TCR signaling in CD8+ T cells in epidermis induces TGFBRIII expression, which likely contributes to endowing them with a competitive advantage over antigen-inexperienced TRM.

Weaknesses:

The main weakness in this paper lies in the authors' reliance on a single model to derive conclusions on the role of local antigen during the acute phase of the response by comparing T cells in model antigen-vaccinia virus (VV-OVA) exposed skin to T cells in contralateral skin exposed to DNFB 5 days after the VV-OVA exposure. In this setting, antigen-independent factors may contribute to the difference in CD8+ T cell number and phenotype at the two sites. For example, it was recently shown that very early memory precursors (formed 2 days after exposure) are more efficient at seeding the epithelial TRM compartment than those recruited to skin at later times (Silva et al, Sci Immunol, 2023). DNFB-treated skin may therefore recruit precursors with reduced TRM potential. In addition, TRM-skewed circulating memory precursors have been identified (Kok et al, JEM, 2020), and perhaps VV-OVA exposed skin more readily recruits this subset compared to DNFB-exposed skin. Therefore, when the DNFB challenge is performed 5 days after vaccinia virus, the DNFB site may already be at a disadvantage in the recruitment of CD8+ T cells that can efficiently form TRM. In addition, CD8+ T cell-extrinsic mechanisms may be at play, such as differences in myeloid cell recruitment and differentiation or local cytokine and chemokine levels in VV-infected and DNFB-treated skin that could account for differences seen in TRM phenotype and function between these two sites. Although the authors do show that providing exogenous peptide antigen at the DNFB-site rescues their phenotype in relation to the VV-OVA site, the potential antigen-independent factors distinguishing these two sites remain unaddressed. In addition, there is a possibility that peptide treatment of DNFB-treated initiates a second phase of priming of new circulatory effectors in the local-draining lymph nodes that are then recruited to form TRM at the DFNB-site, and that the effect does not solely rely on TRM precursors at the DNFB-treated skin site at the time of peptide treatment.

Secondly, although the authors conclusively demonstrate that TGFBRIII is induced by TCR signals and required for conferring increased fitness to local-antigen-experienced CD8+ TRM compared to local antigen-inexperienced cells, this is done in only one experiment, albeit repeated 3 times. The data suggest that antigen encounter during TRM formation induces sustained TGFBRIII expression that persists during the antigen-independent memory phase. It remains unclear why only the antigen encounter in skin, but not already in the draining lymph nodes, induces sustained TGFBRIII expression. Further characterizing the dynamics of TGFBRIII expression on CD8+ T cells during priming in draining lymph nodes and over the course of TRM formation and persistence may shed more light on this question. Probing the role of this mechanism at other sites of TRM formation would also further strengthen their conclusions and enhance the significance of this finding.

Reviewer #1 (Public review):

Summary

The manuscript presents EIDT, a framework that extracts an "individuality index" from a source task to predict a participant's behaviour in a related target task under different conditions. However, the evidence that it truly enables cross-task individuality transfer is not convincing.

Strengths

The EIDT framework is clearly explained, and the experimental design and results are generally well-described. The performance of the proposed method is tested on two distinct paradigms: a Markov Decision Process (MDP) task (comparing 2-step and 3-step versions) and a handwritten digit recognition (MNIST) task under various conditions of difficulty and speed pressure. The results indicate that the EIDT framework generally achieved lower prediction error compared to baseline models and that it was better at predicting a specific individual's behaviour when using their own individuality index compared to using indices from others.

Furthermore, the individuality index appeared to form distinct clusters for different individuals, and the framework was better at predicting a specific individual's behaviour when using their own derived index compared to using indices from other individuals.

Weaknesses

(1) Because the "source" and "target" tasks are merely parameter variations of the same paradigm, it is unclear whether EIDT achieves true cross-task transfer. The manuscript provides no measure of how consistent each participant's behaviour is across these variants (e.g., two- vs three-step MDP; easy vs difficult MNIST). Without this measure, the transfer results are hard to interpret. In fact, Figure 5 shows a notable drop in accuracy when transferring between the easy and difficult MNIST conditions, compared to transfers between accuracy-focused and speed-focused conditions. Does this discrepancy simply reflect larger within-participant behavioural differences between the easy and difficult settings? A direct analysis of intra-individual similarity for each task pair - and how that similarity is related to EIDT's transfer performance - is needed.

(2) Related to the previous comment, the individuality index is central to the framework, yet remains hard to interpret. It shows much greater within-participant variability in the MNIST experiment (Figure S1) than in the MDP experiment (Figure 3). Is such a difference meaningful? It is hard to know whether it reflects noisier data, greater behavioural flexibility, or limitations of the model.

(3) The authors suggests that the model's ability to generalize to new participants "likely relies on the fact that individuality indices form clusters and individuals similar to new participants exist in the training participant pool". It would be helpful to directly test this hypothesis by quantifying the similarity (or distance) of each test participant's individuality index to the individuals or identified clusters within the training set, and assessing whether greater similarity (or closer proximity) to the clusters in the training set is associated with higher prediction accuracy for those individuals in the test set.

Reviewer #1 (Public review):

Summary:

This work presents a formalism for the relationship between neural signals and pooled signals (e.g., voxel estimates in fMRI) and explores why correlation-based and mean-removed Euclidean RDMs perform well in practice. The key assumption is that the pooled estimates are weighted averages, with i.i.d. non-negative weights. Two sets of simulations are used to support the theoretical findings: one based on fully simulated neural data and another that reverse-engineers neural data from an RDM estimated from real macaque data. The authors also discuss limitations of their simulations, particularly concerning the i.i.d. assumption of the weights.

Strengths:

The strengths of this work include its mathematical rigor and the clear connection that is drawn between the derivations and empirical observations. The simulations were well-designed and easy to follow. One small suggestion: a brief explanation of what is meant by "sparse" in Figure 3 would help orient the reader without requiring them to jump ahead to the methods. Overall, I found the work engaging and insightful.

Weaknesses:

Although I appreciate the effort to explore *why* certain dissimilarity measures perform well, it wasn't clear how these findings would inform the practical choices of researchers conducting RDM-based analyses. Many researchers likely already use correlation-based or mean-removed Euclidean distance measures, given their popularity. In that case, how do these results provide additional value or guidance beyond current practice?

Another aspect that could benefit from further clarification is the core assumption underlying the work - that channel-based activity reflects a non-negative weighted average of neural activity. Is this widely accepted as the most plausible model, or are there alternative relationships that researchers should consider? While this may seem intuitive, it's not something I would expect all readers to be familiar with, and only a single reference was provided to support it (which I unfortunately didn't have time to read). That said, I did appreciate the discussion of the i.i.d. assumption in the discussion section. Can more be said to educate researchers as to when the i.i.d. assumption might be violated?

I didn't find the "Simulations based on neural data" section added much, and it risks being misinterpreted. The main difference here is that neural data were reverse-engineered from a macaque RDM and then used in simulations similar to those in the previous section. What is the added value of using a real RDM to generate simulated data? Were the earlier simulations lacking in some way? There's also a risk of readers mistakenly inferring that human dissimilarities have been reconstructed from macaque data, an assumption that goes beyond the paper's core message, which focuses on linking neural and channel-based signals from the *same* source. If this section is retained, the motivation should be clarified, and the implied parallel in Figure 6, between the human data and simulated data, should be reconsidered.

Reviewer #1 (Public review):

Kaller et al. (2025) explore the impact of environmental enrichment (EE) on the developing mouse brain, specifically during the perinatal period. The authors use high-resolution MRI to examine structural brain changes in neonates (postnatal day 7, P7) and compare these changes to those observed in adulthood. A key aspect of the study is the investigation of maternal care as a potential mediating factor in the effects of perinatal EE on neonatal brain development.

The work exhibits the following notable strengths:

(1) The study addresses a significant gap in the literature by investigating the effects of perinatal EE on whole-brain structure in neonates. Previous research has primarily focused on the effects of EE on the adult brain or specific aspects of early development, such as the visual system.

(2) The authors employ a combination of high-resolution MRI and behavioral analysis of maternal care, providing a comprehensive view of the effects of EE.

(3) The study reveals that EE affects brain structure as early as P7, with distinct regional changes compared to adulthood. The finding that maternal care influences neonatal brain structure and correlates with the effects of EE is particularly noteworthy.

(4) The paper is clearly written, well-organized, and easy to follow. The figures and tables are informative and effectively illustrate the key findings.

However, some weaknesses should be addressed to improve the quality of this study:

(1) While the study includes an assessment of maternal care, the observational period is relatively short. A more extended or continuous assessment of maternal behavior could provide a more comprehensive understanding of its role in mediating the effects of EE.

(2) The study primarily focuses on structural brain changes. Investigating the functional consequences of these changes could provide further insights into the long-term impact of perinatal EE.

(3) The study demonstrates a correlation between maternal care and neonatal brain structure but does not elucidate the underlying mechanisms. Future studies could explore potential molecular or cellular mechanisms involved in these effects.

Reviewer #1 (Public review):

Summary:

The authors present an interesting study using RL and Bayesian modelling to examine differences in learning rate adaptation in conditions of high and low volatility and noise respectively. Through "lesioning" an optimal Bayesian model, they reveal that apparently suboptimal adaptation of learning rates results from incorrectly detecting volatility in the environment when it is not in fact present.

Strengths:

The experimental task used is cleverly designed and does a good job of manipulating both volatility and noise. The modelling approach takes an interesting and creative approach to understand the source of apparently suboptimal adaptation of learning rates to noise, through carefully "lesioning" and optimal Bayesian model to determine which components are responsible for this behaviour.

Weaknesses:

The model space could be more extensive, although the authors have covered the most relevant models for the question at hand.

Comments on revisions: I have no further recommendations for the authors, they have addressed my previous comments very well.

Reviewer #1 (Public review):

The chromophore molecule of animal and microbial rhodopsins is retinal which forms a Schiff base linkage with a lysine in the 7-th transmembrane helix. In most cases, the chromophore is positively charged by protonation of the Schiff base, which is stabilized by a negatively charged counterion. In animal opsins, three sites have been experimentally identified, Glu94 in helix 2, Glu113 in helix 3, and Glu181 in extracellular loop 2, where a glutamate acts as the counterion by deprotonation. In this paper, Sakai et al. investigated molecular properties of anthozoan-specific opsin II (ASO-II opsins), as they lack these glutamates. They found an alternative candidate, Glu292 in helix 7, from the sequences. Interestingly, the experimental data suggested that Glu292 is not the direct counterion in ASO-II opsins. Instead, they found that ASO-II opsins employ a chloride ion as the counterion. In case of microbial rhodopsin, a chloride ion serves as the counterion of light-driven chloride pumps. This paper reports the first observation of a chloride ion as the counterion in animal rhodopsin. Theoretical calculation using a QM/MM method supports their experimental data. The authors also revealed the role of Glu292, which serves as the counterion in the photoproduct and is involved in G protein activation.

The conclusions of this paper are well supported by data.

Reviewer #1 (Public review):

Summary:

This useful work extends a prior study from the authors to observe distance changes within the CNBD domains of a full length CNG channel based on changes in single photon lifetimes due to tmFRET between a metal at an introduced chelator site and a fluorescent non canonical amino acid at another site. The data are excellent and convincingly support the authors' conclusions. In addition to the methodology being of general use for other proteins, the authors show that coupling of the CNBDs to the rest of the channel stabilizes the CNBDs in their active state relative to an isolated CNBD construct.

Strengths:

The manuscript is very well written and clear.

Reviewer #1 (Public review):

This is a convincing description of approximately ten years of funding from the NIH BRAIN initiative. It is of particular value at this moment in history, given the cataclysmic changes in the US government structure and function occurring in early 2025.

The paper contains a fair bit of documentation so that the curious reader can actually parse what this BRAIN program funded. The authors are able to draw on a wealth of real-life experience reviewing, funding, and administering large team projects, and assessing how well they achieve their goals. In revision, the paper has been improved with respect to clarity and by bringing together two separate papers into one stronger piece.

Reviewer #1 (Public review):

Summary:

The study investigated how individuals living in urban slums in Salvador, Brazil, interact with environmental risk factors, particularly focusing on domestic rubbish piles, open sewers, and a central stream. The study makes use of the step selection functions using telemetry data, which is a method to estimate how likely individuals move towards these environmental features, differentiating among groups by gender, age, and leptospirosis serostatus. The results indicated that women tended to stay closer to the central stream while avoiding open sewers more than men. Furthermore, individuals who tested positive for leptospirosis tended to avoid open sewers, suggesting that behavioral patterns might influence exposure to risk factors for leptospirosis, hence ensuring more targeted interventions.

Strengths:

(1) The use of step selection functions to analyze human movement represents an innovative adaptation of a method typically used in animal ecology. This provides a robust quantitative framework for evaluating how people interact with environmental risk factors linked to infectious diseases (in this case, leptospirosis).

(2) Detailed differentiation by gender and serological status allows for nuanced insights, which can help tailor targeted interventions and potentially improve public health measures in urban slum settings.

(3) The integration of real-world telemetry data with epidemiological risk factors supports the development of predictive models that can be applied in future infectious disease research, helping to bridge the gap between environmental exposure and health outcomes.

Weaknesses:

(1) The sample size for the study was not calculated, although it was a nested cohort study.

(2) The step‐selection functions, though a novel method, may face challenges in fully capturing the complexity of human decision-making influenced by socio-cultural and economic factors that were not captured in the study.

(3) The study's context is limited to a specific urban slum in Salvador, Brazil, which may reduce the generalizability of its findings to other geographical areas or populations that experience different environmental or socio-economic conditions.

(4) The reliance on self-reported or telemetry-based movement data might include some inaccuracies or biases that could affect the precision of the selection coefficients obtained, potentially limiting the study's predictive power.

(5) Some participants with less than 50 relocations within the study area were excluded without clear justification, see line 149.

(6) Some figures are not clear (see Figure 4 A & B).

(7) No statement on conflict of interest was included, considering sponsorship of the study.

Reviewer #1 (Public review):

In this study, Nishi et al. claim that the ratio of long-term hematopoietic stem cell (LT-HSC) versus short-term HSC (ST-HSC) determines the lineage output of HSCs and reduced ratio of ST-HSC in aged mice causes myeloid-biased hematopoiesis. Authors used Hoxb5 reporter mice to isolated LT-HSC and ST-HSC and performed molecular analyses and transplantation assays to support their arguments. How hematopoietic system becomes myeloid-biased upon aging is an important question with many implications in disease context as well. However, this study needs more definitive data.

(1) Authors' experimental designs have some caveats to definitely support their claims. Authors claimed that aged LT-HSCs have no myeloid-biased clone expansion using transplantation assays. In these experiments, authors used 10 HSCs and young mice as recipients. Given the huge expansion of old HSC by number and known heterogeneity in immunophenotypically defined HSC populations, it is questionable how 10 out of so many old HSCs (an average of 300,000 up to 500,000 cells per mouse; Mitchell et al., Nature Cell Biology, 2023) can faithfully represent old HSC population. The Hoxb5+ old HSC primary and secondary recipient mice data (Fig. 2C and D) support this concern. In addition, they only used young recipients. Considering the importance of inflammatory aged niche in the myeloid-biased lineage output, transplanting young vs old LT-HSCs into aged mice will complete the whole picture.

In response to the above comments, the authors calculated the required sample size as approximately 384 cells to represent 500,000 HSCs per old mouse. Based on the total 1260 cells used throughout the whole manuscript (Figures 2, 3, 5, 6, S3, and S6), the authors claimed that the data is reflecting old HSC behavior. However, 384 cells represent HSCs from one old mouse. Following the authors' logic, they did only 3.2 mice (1260/384) experiment for the whole manuscript to make their argument. N of 3 is not enough, especially for old mice experiments considering the heterogeneity of aged mice. Also, they did not address the comment regarding inflammatory aged niche effects.

(2) Authors' molecular data analyses need more rigor with unbiased approaches. They claimed that neither aged LT-HSCs nor aged ST-HSCs exhibited myeloid or lymphoid gene set enrichment but aged bulk HSCs, which are just a sum of LT-HSCs and ST-HSCs by their gating scheme (Fig. 4A), showed the "tendency" of enrichment of myeloid-related genes based on the selected gene set (Fig. 4D). Although the proportion of ST-HSCs is reduced in bulk HSCs upon aging, since ST-HSCs do not exhibit lymphoid gene set enrichment based on their data, it is hard to understand how aged bulk HSCs have more myeloid gene set enrichment compared to young bulk HSCs. This bulk HSC data rather suggest that there could be a trend toward certain lineage bias (although not significant) in aged LT-HSCs or ST-HSCs. Authors need to verify the molecular lineage priming of LT-HSCs and ST-HSCs using another comprehensive dataset.

(3) Although authors could not find any molecular evidence for myeloid-biased hematopoiesis from old HSCs (either LT or ST), they argued that the ratio between LT-HSC and ST-HSC causes myeloid-biased hematopoiesis upon aging based on young HSC experiments (Fig. 6). However, old ST-HSC functional data showed that they barely contribute to blood production unlike young Hoxb5- HSCs (ST-HSC) in the transplantation setting (Fig. 2). Is there any evidence that in unperturbed native old hematopoiesis, old Hoxb5- HSCs (ST-HSC) still contribute to blood production? To answer this question, authors performed additional experiments with increased cell number (Fig. S6). Although Fig. S6.D data has a statistical significance, it is questionable how biologically meaningful it is. More fundamental question is back to the representability. Can this cell number used in this experiment represent old HSC (either LT or ST) behavior?

Reviewer #1 (Public review):

Summary:

Taber et al report the biochemical characterization of 7 mutations in PHD2 that induce erythrocytosis. Their goal is to provide a mechanism for how these mutations cause the disease. PHD2 hydroxylates HIF1a in the presence of oxygen at two distinct proline residues (P564 and P402) in the "oxygen degradation domain" (ODD). This leads to the ubiquitylation of HIF1a by the VHL E3 ligase and its subsequent degradation. Multiple mutations have been reported in the EGLN1 gene (coding for PHD2), which are associated with pseudohypoxic diseases that include erythrocytosis. Furthermore, 3 mutations in PHD2 also cause pheochromocytoma and paraganglioma (PPGL), a neuroendocrine tumour. These mutations likely cause elevated levels of HIF1a, but their mechanisms are unclear. Here, the authors analyze mutations from 152 case reports and map them on the crystal structure. They then focus on 7 mutations, which they clone in a plasmid and transfect into PHD2-KO to monitor HIF1a transcriptional activity via a luciferase assay. All mutants show impaired activation. Some mutants also impaired stability in pulse chase turnover assays (except A228S, P317R, and F366L). In vitro purified PHD2 mutants display a minor loss in thermal stability and some propensity to aggregate. Using MST technology, they show that P317R is strongly impaired in binding to HIF1a and HIF2a, whereas other mutants are only slightly affected. Using NMR, they show that the PHD2 P317R mutation greatly reduces hydroxylation of P402 (HIF1a NODD), as well as P562 (HIF1a CODD), but to a lesser extent. Finally, BLI shows that the P317R mutation reduces affinity for CODD by 3-fold, but not NODD.

Strengths:

(1) Simple, easy-to-follow manuscript. Generally well-written.

(2) Disease-relevant mutations are studied in PHD2 that provide insights into its mechanism of action.

(3) Good, well-researched background section.

Weaknesses:

(1) Poor use of existing structural data on the complexes of PHD2 with HIF1a peptides and various metals and substrates. A quick survey of the impact of these mutations (as well as analysis by Chowdhury et al, 2016) on the structure and interactions between PHD2 peptides of HIF1a shows that the P317R mutation interferes with peptide binding. By contrast, F366L will affect the hydrophobic core, and A228S is on the surface, and it's not obvious how it would interfere with the stability of the protein.

(2) To determine aggregation and monodispersity of the PHD2 mutants using size-exclusion chromatography (SEC), equal quantities of the protein must be loaded on the column. This is not what was done. As an aside, the colors used for the SEC are very similar and nearly indistinguishable.

(3) The interpretation of some mutants remains incomplete. For A228S, what is the explanation for its reduced activity? It is not substantially less stable than WT and does not seem to affect peptide hydroxylation.

(4) The interpretation of the NMR prolyl hydroxylation is tainted by the high concentrations used here. First of all, there is a likely a typo in the method section; the final concentration of ODD is likely 0.18 mM, and not 0.18 uM (PNAS paper by the same group in 2024 reports using a final concentration of 230 uM). Here, I will assume the concentration is 180 uM. Flashman et al (JBC 2008) showed that the affinity of the NODD site (P402; around 10 uM) for PHD2 is 10-fold weaker than CODD (P564, around 1 uM). This likely explains the much faster kinetics of hydroxylation towards the latter. Now, using the MST data, let's say the P317R mutation reduces the affinity by 40-fold; the affinity becomes 400 uM for NODD (above the protein concentration) and 40 uM for CODD (below the protein concentration). Thus, CODD would still be hydroxylated by the P317R mutant, but not NODD.

(5) The discrepancy between the MST and BLI results does not make sense, especially regarding the P317R mutant. Based on the crystal structures of PHD2 in complex with the ODD peptides, the P317R mutation should have a major impact on the affinity, which is what is reported by MST. This suggests that the MST is more likely to be valid than BLI, and the latter is subject to some kind of artefact. Furthermore, the BLI results are inconsistent with previous results showing that PHD2 has a 10-fold lower affinity for NODD compared to CODD.

(6) Overall, the study provides some insights into mutants inducing erythrocytosis, but the impact is limited. Most insights are provided on the P317R mutant, but this mutant had already been characterized by Chowdhury et al (2016). Some mutants affect the stability of the protein in cells, but then no mechanism is provided for A228S or F366L, which have stabilities similar to WT, yet have impaired HIF1a activation.

Reviewer #1 (Public review):

Summary:

The authors have investigated the role of FMRP in the formation and function of RNA granules in mouse brain/cultured hippocampal neurons. Most of their results indicate that FMRP does not have a role in the formation or function of RNA granules with specific mRNAs, but may have some role in distal RNA granules in neurons and their response to synaptic stimulation. This is an important work (though the results are mostly negative) in understanding the composition and function of neuronal RNA granules. The last part of the work in cultured neurons is disjointed from the rest of the manuscript, and the results are neither convincing nor provide any mechanistic insight.

Strengths:

(1) The study is quite thorough, the methods and analysis used are robust, and the conclusion and interpretation are diligent.

(2) The comparative study of Rat and Mouse RNA granules is very helpful for future studies.

(3) The conclusion that the absence of FMRP does not affect the RNA granule composition and many of its properties in the system the authors have chosen to study is well supported by the results.

(4) The difference in the response to DHPG stimulation concerning RNA granules described here is very interesting and could provide a basis for further studies, though it has some serious technical issues.

Weaknesses:

(1) The system used for the study (P5 mouse brain or DIV 8-10 cultured neuron) is surprising, as the majority of defects in the absence of FMRP are reported in later stages (P30+ brain and DIV 14+ neurons). It is important to test if the conclusions drawn here hold good at different developmental stages.

(2) The term 'distal granules' is very vague. Since there is no structural or biochemical characterization of these granules, it is difficult to understand how they are different from the proximal granules and why FMRP has an effect only on these granules.

(3) Since the manuscript does not find any effect of FMRP on neuronal RNA granules, it does not provide any new molecular insight with respect to the function of FMRP

Reviewer #1 (Public review):

Summary:

ZMAT3 is a p53 target gene that the Lal group and others have shown is important for p53-mediated tumor suppression, and which plays a role in the control of RNA splicing. In this manuscript, Lal and colleagues perform quantitative proteomics of cells with ZMAT3 knockout and show that the enzyme hexokinase HKDC1 is the most upregulated protein. Mechanistically, the authors show that ZMAT3 does not appear to directly regulate the expression of HKDC1; rather, they show that the transcription factor c-JUN was strongly enriched in ZMAT3 pull-downs in IP-mass spec experiments, and they perform IP-western to demonstrate an interaction between c-JUN and ZMAT3. Importantly, the authors demonstrate, using ChIP-qPCR, that JUN is present at the HKDC1 gene (intron 1) in ZMAT3 WT cells and shows markedly enhanced binding in ZMAT3 KO cells. The data best fit a model whereby p53 transactivates ZMAT3, leading to decreased JUN binding to the HKDC1 promoter, and altered mitochondrial respiration.

Strengths:

The authors use multiple orthogonal approaches to test the majority of their findings.

The authors offer a potentially new activity of ZMAT3 in tumor suppression by p53: the control of mitochondrial respiration.

Weaknesses:

Some indication as to whether other c-JUN target genes are also regulated by ZMAT3 would improve the broad relevance of the authors' findings.

Reviewer #1 (Public review):

In this work, Neiswender and colleagues test the hypothesis that mutations in BicD2 that are associated with SMALED alter BicD2-cargo interactions. To do this, they first establish the WT BicD2 cargo interactome (using a proximity-dependent biotin ligase screen with Turbo-ID on the BicD2 C-terminus). In addition to known cargo interactors, they also identified many proteins in the HOPs complex. Interestingly, they find that the HOPs complex may interact with BicD2 in a different manner than other known cargos. The authors also show that while BicD2 is required for the HOPs complex localization, on average, depletion of BicD2 from HeLa and Cos7 cells causes HOPs and Lysosome mislocalization that is consistent with Kinesin-1 trafficking defects, rather than dynein. The authors also use proximity biotin ligase approaches to define the cargo interactome of three BicD2 variants associated with SMALED. One variant (R747C) has the most altered cargo interactome. The authors highlight one protein, in particular, GRAMD1A, that is only found in the R747C dataset and mislocalizes specifically when R747C is expressed.

The work in this manuscript is of a very high quality and contributes important findings to the field. I have a few questions that, if answered, could increase the impact of this work.

(1) I was surprised at the effect of BicD2 knockdown on LAMP (and VPS41) localization, which really suggests that in HeLa and Cos7 cells, BicD2 regulation of Kinesin-1 (rather than dynein) is the primary driver of lysosome localization. The KIF5B-knockout rescue of the BicD2-overexpression phenotype was a very powerful result that supports this conclusion. Have the authors looked at other cargos, eg, Golgi or centrosomes in G2? Can the authors include more discussion about what this result means or how they imagine dynein and kinesin-1's interaction with BicD2 is regulated?

(2) Have the authors examined if the SMALED mutants show diminished or increased binding to KIF5B? While the authors are correct that the mutations could hyperactivate dynein because they reduce BicD2 autoinhibition, it is possible that the SMALED mutants hyperactivate dynein because they no longer bind kinesin. This would be particularly interesting, given the complex relationship between BicD2 regulation of dynein and kinesin that the authors show in Figure 3.

(3) What is already known about the protein GRAMD1A? Did the authors choose to focus on GRAMD1A because it was the only novel interaction found in the SMALED mutant interactomes, or was this protein interesting for a different reason? Does the known function of GRAMD1A explain the potential dysfunction of cells expressing BICD2_R747C or patients who have this mutation? More discussion of this protein and why the authors focused on it would really strengthen the manuscript.

Reviewer #1 (Public review):

Summary:

In this study, the authors aim to understand how Rhino, a chromatin protein essential for small RNA production in fruit flies, is initially recruited to specific regions of the genome. They propose that asymmetric arginine methylation of histones, particularly mediated by the enzyme DART4, plays a key role in defining the first genomic sites of Rhino localization. Using a combination of inducible expression systems, chromatin immunoprecipitation, and genetic knockdowns, the authors identify a new class of Rhino-bound loci, termed DART4 clusters, that may represent nascent or transitional piRNA clusters.

Strengths:

One of the main strengths of this work lies in its comprehensive use of genomic data to reveal a correlation between ADMA histones and Rhino enrichment at the border of known piRNA clusters. The use of both cultured cells and ovaries adds robustness to this observation. The knockdown of DART4 supports a role for H3R17me2a in shaping Rhino binding at a subset of genomic regions.

Weaknesses:

However, Rhino binding at, and piRNA production from, canonical piRNA clusters appears largely unaffected by DART4 depletion, and spreading of Rhino from ADMA-rich boundaries was not directly demonstrated. Therefore, while the correlation is clearly documented, further investigation would be needed to determine the functional requirement of these histone marks in piRNA cluster specification.

The study identify piRNA cluster-like regions called DART4 clusters. While the model proposes that DART4 clusters represent evolutionary precursors of mature piRNA clusters, the functional output of these clusters remains limited. Additional experiments could help clarify whether low-level piRNA production from these loci is sufficient to guide Piwi-dependent silencing.

In summary, the authors present a well-executed study that raises intriguing hypotheses about the early chromatin context of piRNA cluster formation. The work will be of interest to researchers studying genome regulation, small RNA pathways, and the chromatin mechanisms of transposon control. It provides useful resources and new candidate loci for follow-up studies, while also highlighting the need for further functional validation to fully support the proposed model.

Reviewer #1 (Public review):

The manuscript by Shan et al seeks to define the role of the CHI3L1 protein in macrophages during the progression of MASH. The authors argue that the Chil1 gene is expressed highly in hepatic macrophages. Subsequently, they use Chil1 flx mice crossed to Clec4F-Cre or LysM-Cre to assess the role of this factor in the progression of MASH using a high-fat, high-fructose diet (HFFC). They found that loss of Chil1 in KCs (Clec4F Cre) leads to enhanced KC death and worsened hepatic steatosis. Using scRNA seq, they also provide evidence that loss of this factor promotes gene programs related to cell death. From a mechanistic perspective, they provide evidence that CHI3L serves as a glucose sink and thus loss of this molecule enhances macrophage glucose uptake and susceptibility to cell death. Using a bone marrow macrophage system and KCs they demonstrate that cell death induced by palmitic acid is attenuated by the addition of rCHI3L1. While the article is well written and potentially highlights a new mechanism of macrophage dysfunction in MASH, there are some concerns about the current data that limit my enthusiasm for the study in its current form. Please see my specific comments below.

Major:

(1) The authors' interpretation of the results from the KC ( Clec4F) and MdM KO (LysM-Cre) experiments is flawed. For example, in Figure 2 the authors present data that knockout of Chil1 in KCs using Clec4f Cre produces worse liver steatosis and insulin resistance. However, in supplemental Figure 4, they perform the same experiment in LysM-Cre mice and find a somewhat different phenotype. The authors appear to be under the impression that LysM-Cre does not cause recombination in KCs and therefore interpret this data to mean that Chil1 is relevant in KCs and not MdMs. However, LysM-Cre DOES lead to efficient recombination in KCs and therefore Chil1 expression will be decreased in both KCs and MdM (along with PMNs) in this line.

Therefore, a phenotype observed with KC-KO should also be present in this model unless the authors argue that loss of Chil1 from the MdMs has the opposite phenotype of KCs and therefore attenuates the phenotype. The Cx3Cr1 CreER tamoxifen inducible system is currently the only macrophage Cre strategy that will avoid KC recombination. The authors need to rethink their results with the understanding that Chil1 is deleted from KCs in the LysM-Cre experiment. In addition, it appears that only one experiment was performed, with only 5 mice in each group for both the Clec4f and LysM-Cre data. This is generally not enough to make a firm conclusion for MASH diet experiments.

(2) The mouse weight gain is missing from Figure 2 and Supplementary Figure 4. This data is critical to interpret the changes in liver pathology, especially since they have worse insulin resistance.

(3) Figure 4 suggests that KC death is increased with KO of Chil1. However, this data cannot be concluded from the plots shown. In Supplementary Figure 6 the authors provide a more appropriate gating scheme to quantify resident KCs that includes TIM4. The TIM4 data needs to be shown and quantified in Figure 4. As shown in Supplementary Figure 6, the F4/80 hi population is predominantly KCs at baseline; however, this is not true with MASH diets. Most of the recruited MoMFs also reside in the F4/80 hi gate where they can be identified by their lower expression of TIM4. The MoMF gate shown in this figure is incorrect. The CD11b hi population is predominantly PMNs, monocytes, and cDC,2 not MoMFs (PMID:33997821). In addition, the authors should stain the tissue for TIM4, which would also be expected to reveal a decrease in the number of resident KCs.

(4) While the Clec4F Cre is specific to KCs, there is also less data about the impact of the Cre system on KC biology. Therefore, when looking at cell death, the authors need to include some mice that express Clec4F cre without the floxed allele to rule out any effects of the Cre itself. In addition, if the cell death phenotype is real, it should also be present in LysM Cre system for the reasons described above. Therefore, the authors should quantify the KC number and dying KCs in this mouse line as well.

(5) I am somewhat concerned about the conclusion that Chil1 is highly expressed in liver macrophages. Looking at our own data and those from the Liver Atlas it appears that this gene is primarily expressed in neutrophils. At a minimum, the authors should address the expression of Chil1 in macrophage populations from other publicly available datasets in mouse MASH to validate their findings (several options include - PMID: 33440159, 32888418, 32362324). If expression of Chil1 is not present in these other data sets, perhaps an environmental/microbiome difference may account for the distinct expression pattern observed. Either way, it is important to address this issue.

Joint Public Review:

This study presents a valuable contribution to our understanding of ion channel complex assembly by investigating whether BK and CaV1.3 channels begin to form functional associations early in the biosynthetic pathway, prior to reaching the plasma membrane. Using a combination of proximity ligation assays, single-molecule RNA imaging, and super-resolution microscopy, the authors provide convincing evidence that these channels co-localize intracellularly within the ER and Golgi, in both overexpression systems and a relevant endogenous cell model. The study addresses an important and underexplored aspect of membrane protein trafficking and organization, with broader implications for how ion channel signaling complexes are assembled and regulated. The experimental approaches are generally appropriate and the imaging data are clearly presented, with a commendable number of control experiments included. However, several limitations temper the interpretation of the results. The mechanisms underlying mRNA co-localization, and the role of co-translation in complex formation, remain insufficiently defined. Similarly, while intracellular colocalization is convincingly demonstrated, the study does not establish whether such early assembly is the predominant pathway for generating functional complexes at the plasma membrane. More rigorous quantification of channel co-association across compartments, and clarification of key terminology and image analysis methods, would strengthen the overall conclusions. Some of the language in the manuscript would also benefit from a more measured tone to avoid overstating the novelty of the findings. Despite these limitations, the study offers meaningful insights into intracellular ion channel organization and will be of interest to researchers in cell biology, membrane trafficking, and neurophysiology. With focused revisions addressing the outlined points, the manuscript has the potential to make a solid contribution to the field.

Reviewer #1 (Public review):

Summary:

The article by Zdraljevic et al. reports the discovery of a third toxin-antidote (TA) element in C. elegans, composed of the genes mll-1 (toxin) and smll-1 (antidote). Unlike previously characterized TA systems in C. elegans, this element induces larval arrest rather than embryonic lethality. The study identifies three distinct haplotypes at the TA locus, including a hyper-divergent version in the standard laboratory strain N2, which retains a functional toxin but lacks a functional antidote. The authors propose that small RNA-mediated silencing mechanisms, dependent on MUT-16 and PRG-1, suppress the toxicity of the divergent toxin allele. This work provides insights into the evolutionary dynamics of TA elements and their regulation through RNA interference (RNAi).

Overall, there are many things to like about this paper and only a few small quibbles, which will not require more than a little rewriting or relatively minor analyses.

Strengths:

(1) The discovery of a maternally deposited TA element with delayed toxicity due to delayed mRNA translation of the maternally deposited toxin mRNA is a significant addition to the literature on selfish genetic elements in metazoans.

(2) Identifying three haplotypes at the TA locus provides a snapshot of potential evolutionary trajectories for these elements, which are often inferred but rarely demonstrated in naturally occurring strains. The genomic analysis of 550 wild isolates contextualizes the findings within natural populations, revealing geographic clustering and evolutionary pressures acting on the TA locus.

(3) The study employs various techniques, including CRISPR/Cas9 knockouts, FISH, long-read RNA sequencing, and population genomics. The use of inducible systems to confirm toxicity and antidote functionality is particularly robust. This multifaceted approach strengthens the validity of the findings.

(4) The authors provide compelling evidence that small RNA pathways suppress toxin activity in strains lacking a functional antidote. This highlights an alternative mechanism for neutralizing selfish genetic elements.

Weaknesses:

(1) The introduction focuses strongly (for good reason) on bacterial TA systems and then jumps to TA systems in C. elegans. It's unclear why TA systems in other eukaryotes are not discussed.

(2) Similarly, there is a missed opportunity to discuss an analogy between the suppressor mechanism discovered here and the hairpin RNA suppressors of meiotic drive identified by Eric Lai and colleagues. Discussing these will provide a fuller context of the present study's findings and will not affect their novelty.

(3) While the evidence for RNAi-mediated suppression is strong, the claim that positive selection drove diversification at piRNA binding sites requires further discussion and clarification. The elevated dN and dS are unusual (how unusual relative to other genes in vicinity? What is hyper-divergent statistically speaking?), but there is no a priori reason that there would be selection on piRNA binding sites within the mll-1 transcript to facilitate its recognition by endogenous RNAi machinery; what is the selective pressure for mll-1 to do so? Most TA systems would like to avoid being suppressed by the host. One cannot make the argument that this was motivated by the loss of the antidote because the loss of the antidote would be instantly suicidal, so the cadence of events described requiring hypermutation of the mll-1 transcript does not work.

Reviewer #1 (Public review):

Summary:

This work aims to improve our understanding of the factors that influence female-on-female aggressive interactions in gorilla social hierarchies, using 25 years of behavioural data from five wild groups of two gorilla species. Researchers analysed aggressive interactions between 31 adult females, using behavioural observations and dominance hierarchies inferred through Elo-rating methods. Aggression intensity (mild, moderate, severe) and direction (measured as the rank difference between aggressor and recipient) were used as key variables. A linear mixed-effects model was applied to evaluate how aggression direction varied with reproductive state (cycling, trimester-specific pregnancy, or lactation) and sex composition of the group. This study highlights the direction of aggressive interactions between females, with most interactions being directed from higher- to lower-ranking adult females close in social rank. However, the results show that 42% of these interactions are directed from lower- to higher-ranking females. Particularly, lactating and pregnant females targeted higher-ranking individuals, which the authors suggest might be due to higher energetic needs, which increase risk-taking in lactating and pregnant females. Sex composition within the group also influenced which individuals were targeted. The authors suggest that male presence buffers female-on-female aggression, allowing females to target higher-ranking females than themselves. In contrast, females targeted lower-ranking females than themselves in groups with a larger ratio of females, which supposes a lower risk for the females since the pool of competitors is larger. The findings provide an important insight into aggression heuristics in primate social systems and the social and individual factors that influence these interactions, providing a deeper understanding of the evolutionary pressures that shape risk-taking, dominance maintenance, and the flexibility of social strategies in group-living species.

The authors achieved their aim by demonstrating that aggression direction in female gorillas is influenced by factors such as reproductive condition and social context, and their results support the broader claim that aggression heuristics are flexible. However, some specific interpretations require further support. Despite this, the study makes a valuable contribution to the field of behavioural ecology by reframing how we think about intra-sexual competition and social rank maintenance in primates.

Strengths:

One of the study's major strengths is the use of an extensive dataset that compiles 25 years of behavioural data and 6871 aggressive interactions between 31 adult females in five social groups, which allows for a robust statistical analysis. This study uses a novel approach to the study of aggression in social groups by including factors such as the direction and intensity of aggressive interactions, which offers a comprehensive understanding of these complex social dynamics. In addition, this study incorporates ecological and physiological factors such as the reproductive state of the females and the sex composition of the group, which allows an integrative perspective on aggression within the broader context of body condition and social environment. The authors successfully integrate their results into broader evolutionary and ecological frameworks, enriching discussions around social hierarchies and risk sensitivity in primates and other animals.

Weaknesses:

Although the paper has a novel approach by studying the effect of reproductive state and social environment on female-female aggression, the use of observational data without experimental manipulation limits the ability to establish causation. The authors suggest that the difference observed in female aggression direction between groups with different sex composition might be indicative of male presence buffering aggression, which seems speculative, as no direct evidence of male intervention or support was reported. Similarly, the use of reproductive state as a proxy for energetic need is an indirect measure and does not account for actual energy expenditure or caloric intake, which weakens the authors' claims that female energetic need induces risk-taking. Overall, this paper would benefit from stronger justification and empirical support to strengthen the conclusions of the study about the mechanisms driving female aggression in gorillas.

Reviewer #2 (Public review):

Summary:

The authors observed gene ontologies associated with upregulated KLF2 target genes in HIV-1 RNA+ CD4 T Cells using scRNA-seq and scATAC-seq datasets from the PBMCs of early HIV-1-infected patients, showing immune responses contributing to HIV pathogenesis and novel targets for viral elimination.

Strengths:

The authors carried out detailed transcriptomics profiling with scRNA-seq and scATAC-seq datasets to conclude upregulated KLF2 target genes in HIV-1 RNA+ CD4 T Cells.

Comments on revisions:

The authors justified my comments.

Joint Public Review:

Summary:

Malita and colleagues investigated the mechanism by which infections increase sleep in Drosophila. Their work is important because it further supports the idea that the blood brain barrier is involved in brain-body communication, and because it advances the field of sleep research. Using knock-down and knock-out of cytokines and cytokine receptors specifically in the endocrine cells of the gut (cytokines) as well as in the glia forming the blood-brain barrier (BBB) (cytokines receptors), the authors show that cytokines, upd2 and upd3, secreted by entero-endocrine cells in response to infections increase sleep through the Dome receptor in the BBB. They also show that gut-derived Allatostatin (Alst) A promotes wakefulness by inhibiting the Alst A signaling that is mediated by Alst receptors expressed in BBB glia. Their results suggest there may be additional mechanisms that promote elevated sleep during gut inflammation. The evidence supporting most of their claims is compelling. Nevertheless, the activation of the sleep-promoting pathway by infection should be accomplished through bacterial infection of the gut.

Strengths:

The work is, in general, supported by well-designed and well-performed experiments, especially those that show that the endocrine cells from the gut are the sources of the Upd cytokines, the effects of these cytokines on daytime sleep, and that the glial cells of the BBB are the target cell for the Upds action. In addition, the evidence associating the downregulation of Alst receptors in the BBB by Upd and Jak/Stat pathways is compelling.

Weaknesses:

(1) The model of gut inflammation that is used is based on the increase in reactive oxygen species (ROS) that is caused by adding 1% H2O2 to the food. The use of the model is supported rather weakly by two papers (ref. 26 and 27 ). The paper by Jiang et al. (26) shows that the infection by Pseudomonas entomophila induces cytokine responses Upd2 and 3, which are also induced by the Jnk pathway; there is no mention of ROS. Buchon et al. (27) is a review that refers to results that indicate that as part of the immune response to pathogens in the gut, there is production of ROS by the NADPH oxidase DUOX. Thus, there is no strong support for the use of this model.

(2) There is no support for the use of ROS in the food instead a direct infection by pathogenic bacteria. It is known that ROS causes damage in the gut epithelium, which in turn induces the expression of the cytokines studied, which might be independent of infection and confound the results.

Reviewer #1 (Public review):

Summary:

The authors report a study on how stimulation of receptive-field surround of V1 and LGN neurons affects their firing-rates. Specifically, they examine stimuli in which a grey patch covers the classical RF of the cell and a stimulus appears in the surround. Using a number of different stimulus paradigms they find a long latency response in V1 (but not the LGN) which does not depend strongly on the characteristics of the surround grating (drifting vs static, continuous vs discontinuous, predictable grating vs unpredictable pink noise). They find that population responses to simple achromatic stimuli have a different structure that does not distinguish so clearly between the grey patch and other conditions and the latency of the response was similar regardless of whether the center or surround was stimulated by the achromatic surface. Taken together they propose that the surround-response is related to the representation of the grey surface itself. They relate their findings to previous studies which have put forward the concept of an 'inverse RF' based on strong responses to small grey patches on a full-screen grating. They also discuss their results in the context of studies that suggest that surround responses are related to predictions of the RF content or figure-ground segregation.

Strengths:

I find the study to be an interesting extension of the work on surround stimulation and the addition of the LGN data is useful showing that the surround-induced responses are not present in the feed-forward path. The conclusions appear solid, being based on large numbers of neurons obtained through Neuropixels recordings. The use of many different stimulus combinations provides a rich view of the nature of the surround-induced responses.

Weaknesses:

The LGN data comes from a small number of animals (n=2). Statistics are generally pooled across all recording sessions/animals without taking into account the higher covariance of neurons recorded in the same session. This is not a problem for paired comparisons, but for some statistics in the paper a hierarchical approach would have been more appropriate. The authors do present individual session data and the effects appear to be consistent across sessions.

Reviewer #1 (Public review):

The paper is well written and the figures well laid out. The methods are easy to follow, and the rational and logic for each experiment easy to follow. The introduction sets the scene well, and the discussion is appropriate. The summary sentences throughout the text help the reader.

The authors have done a lot of work addressing my previous concerns and those of the other Reviewers.

Reviewer #1 (Public review):

Summary:

This research group has consistently performed cutting-edge research aiming to understand the role of hormones in the control of social behaviors, specifically by utilizing the genetically-tractable teleost fish, medaka, and the current work is no exception. The overall claim they make, that estrogens modulate social behaviors in males and females is supported, with important caveats. For one, there is no evidence these estrogens are generated by "neurons" as would be assumed by their main claim that it is NEUROestrogens that drive this effect. While indeed the aromatase they have investigated is expressed solely in the brain, in most teleosts, brain aromatase is only present in glial cells (astrocytes, radial glia). The authors should change this description so as not to mislead the reader. Below I detail more specific strengths and weaknesses of this manuscript.

Strengths:

• Excellent use of the medaka model to disentangle the control of social behavior by sex steroid hormones

• The findings are strong for the most part because deficits in the mutants are restored by the molecule (estrogens) that was no longer present due to the mutation

• Presentation of the approach and findings are clear, allowing the reader to make their own inferences and compare them with the authors'

• Includes multiple follow-up experiments, which leads to tests of internal replication and an impactful mechanistic proposal

• Findings are provocative not just for teleost researchers, but for other species since, as the authors point out, the data suggest mechanisms of estrogenic control of social behaviors may be evolutionary ancient

Weaknesses:

• As stated in the summary, the authors are attributing the estrogen source to neurons and there isn't evidence this is the case. The impact of the findings doesn't rest on this either

• The d4 versus d8 esr2a mutants showed different results for aggression. The meaning and implications of this finding are not discussed, leaving the reader wondering

• Lack of attribution of previous published work from other research groups that would provide the proper context of the present study

• There are a surprising number of citations not included; some of the ones not included argue against the authors' claims that their findings were "contrary to expectation"

• The experimental design for studying aggression in males has flaws. A standard test like a resident-intruder test should be used.

• While they investigate males and females, there are fewer experiments and explanations for the female results, making it feel like a small addition or an aside

• The statistics comparing "experimental to experimental" and "control to experimental" isn't appropriate

Reviewer #1 (Public review):

Summary:

This work investigated whether cytoplasmic poroelastic properties play an important role in cellular mechanical response over length scales and time scales relevant to cell physiology. Overall, the manuscript concludes that intracellular cytosolic flows and pressure gradients are important for cell physiology and that they act of time- and length-scales relevant to mechanotransduction and cell migration.

Strengths:

Their approach integrates both computational and experimental methods. The AFM deformation experiments combined with measuring z-position of beads is a challenging yet compelling method to determine poroelastic contributions to mechanical realization.

The work is quite interesting and will be of high value to the field of cell mechanics and mechanotransduction.

Weaknesses:

However, there are several issues related to the lack of description of theoretical equations, experimental details, and data transparency that should be addressed, including the following:

(1) Some details are not described for experimental procedures. For example, what were the pharmacological drugs dissolved in, and what vehicle control was used in experiments? How long were pharmacological drugs added to cells?

(2) Details are missing from the Methods section and Figure captions about the number of biological and technical replicates performed for experiments. Figure 1C states the data are from 12 beads on 7 cells. Are those same 12 beads used in Figure 2C? If so, that information is missing from the Figure 2C caption. Similarly, this information should be provided in every figure caption so the reader can assess the rigor of the experiments. Furthermore, how heterogenous would the bead displacements be across different cells? The low number of beads and cells assessed makes this information difficult to determine.

(3) The full equation for displacement vs. time for a poroelastic material is not provided. Scaling laws are shown, but the full equation derived from the stress response of an elastic solid and viscous fluid is not shown or described.

Reviewer #1 (Public review):

Summary:

Selberg et al. present a small but apparently very relevant modification to the existing BUSTED model. The new model allows for a fraction of codons to be assigned to an error class characterized by a very high dN/dS value. This "omega_e" category is constrained to represent no more than 1% of the alignment. The analyses convincingly show that the method performs well and represents a real improvement for genome-wide scans of positive selection. Alignment and sequencing errors are a major concern in molecular evolution. This new method, which shows strong performance, is therefore a very welcome contribution.

Strengths:

By thoroughly reanalyzing four datasets, the manuscript convincingly demonstrates that omega_e effectively identifies genuine alignment errors. Next, the authors evaluate the reduction in power to detect true selection through simulations. This new model is simple, efficient, and computationally fast. It is already implemented and available in HYPHY software.

As a side note, I found it particularly interesting how the authors tested the statistical support for the new method compared to the simpler version without the error class. In many cases, the simpler model could not be statistically rejected in favor of the more complex model, despite producing biologically incorrect results in terms of parameter inference. This highlights a broader issue in molecular evolution and phylogenomics, where model selection often relies too heavily on statistical tests, potentially at the expense of biological realism. The analyses also reveal a trade-off between statistical power and the false positive rate. As with other methods, BUSTED-E cannot distinguish between alignment/sequencing errors and episodes of very strong positive selection. The authors are transparent about this limitation in the discussion.

Weaknesses:

Regarding the structure of the manuscript, the text could be clearer and more precise. Clear, practical recommendations for users could also be provided in the Results section. Additionally, the simulation analyses could be further developed to include scenarios with both alignment errors and positive selection, in order to better assess the method's performance. Finally, the model is evaluated only in the context of site models, whereas the widely used branch-site model is mentioned as possible but not assessed.

Reviewer #1 (Public review):

Summary:

The authors provide a simple yet elegant approach to identifying therapeutic targets that synergize to prevent therapeutic resistance using cell lines, data-independent acquisition proteomics, and bioinformatic analysis. The authors identify several combinations of pharmaceuticals that were able to overcome or prevent therapeutic resistance in culture models of ovarian cancer, a disease with an unmet diagnostic and therapeutic need.

Strengths:

The manuscript utilizes state-of-the-art proteomic analysis, entailing data-independent acquisition methods, an approach that maximizes the robustness of identified proteins across cell lines. The authors focus their analysis on several drugs under development for the treatment of ovarian cancer and utilize straightforward thresholds for identifying proteomic adaptations across several drugs on the OVSAHO cell line. The authors utilized three independent and complementary approaches to predicting drug synergy (NetBox, GSEA, and Manual Curation). The drug combination with the most robust synergy across multiple cell lines was the inhibition of MEK and CDK4/6 using PD-0325901+Palbociclib, respectively. Additional combinations, including PARPi (rucaparib) and the fatty acid synthase inhibitor (TVB-2640). Collectively, this study provides important insight and exemplifies a solid approach to identifying drug synergy without large drug library screens.

Weaknesses:

The manuscript supports their findings by describing the biological function(s) of targets using referenced literature. While this is valuable, the number of downstream targets for each initial target is extensive, thus, the current work does not attempt to elucidate the mechanism of their drug synergy. Responses to drugs are quantified 72 hours after treatment and exclusively focused on cell viability and protein expression levels. The discovery phase of experimentation was solely performed on the OVSAHO cell line. An additional cell line(s) would increase the impact of how the authors went about identifying synergistic targets using bioinformatics. Ovarian cancer is elusive to treatment as primary cancer will form spheroids within ascites/peritoneal fluids in a state of pseudo-senescence to overcome environmental stress. The current manuscript is executed in 2D culture, which has been demonstrated to deviate from 3D, PDX, and primary tumours in terms of therapeutic resistance (DOI: 10.3390/cancers13164208). Collectively, the manuscript is insufficient in providing additional mechanistic insight beyond the literature, and its interpretation of data is limited to 2D culture until further validated.

Reviewer #1 (Public review):

Van Arsdale and colleagues evaluated whether human-HPV DNA junctions could be detected in serum, cell-free DNA from 16 patients with cervical cancer by hybrid capture and Illumina sequencing. Junctions were identified in seven patients, and these junctions were concordant with junctions identified in tumor DNA except for one patient, suggesting that, in most cases, the cfDNA is originating from a clone of the primary tumor. Junction detection at 6 months was found to be statistically significant prognostic for recurrence. The study further validates that type-specific E7 DNA, which is essential for tumorigenesis, was detectable by PCR for most patient sera, but had no association with recurrence. Furthermore, the study provides additional evidence that tumors harboring non-alpha-9 clade HPVs had shorter recurrence-free survival and overall worse outcome from the study's patients, as well as reanalysis of TCGA data. However, these findings need to be more extensively discussed in the context of previous publications. One identified limitation of this approach is the detection of non-tumor HPVs, but this was only seen in one patient. The major shortcoming of this study is the limited number of patients that were evaluated, but for a retrospective study, this is a reasonable number of patients evaluated, and the findings are appropriately not overstated. The design, execution, and detailed analysis of the sequencing data are a major strength. This study provides important foundational evidence for further evaluating the clinical utility of HPV DNA detection from cfDNA and specifically assessing for integration junctions.

Reviewer #1 (Public review):

Summary:

Y-family polymerases, such as polymerases eta, iota, and kappa, have low fidelity relative to other polymerases involved in DNA replication and repair. This is believed to be due to their active sites being less constrained than those of other polymerases. Paradoxically, work by this lab and others shows that in vivo, these Y-family polymerases are more error-free (less error-prone) during DNA damage bypass than would be expected given their low fidelity. For this reason, the authors have been focusing on other cellular factors that may increase the fidelity of Y-family polymerases. The current paper focuses on two such factors: WRN, which possesses exonuclease and helicase activities, and WRNIP1, which possesses a DNA-dependent ATPase.

Previously, this group showed that defects in the exonuclease function of WRN lead to a loss in the fidelity of polymerases eta and iota during DNA damage bypass, presumably by removing nucleotide misinsertions. The current paper extends this work by considering the ATPase activities of WRN and WRNIP1. The authors looked at the impact of various amino acid substitutions in these proteins on the fidelity of DNA damage bypass by Y-family polymerases. They did this by both measuring the mutation frequencies of these cell lines as well as the mutation spectra observed in them. They showed that the ATPase activities of both WRN and WRNIP1, as well as the exonuclease activities of WRN, are necessary high fidelity of Y-family polymerases in cells. They specifically examined the bypass of cyclobutene pyrimidine dimers by polymerase eta, the bypass of 6-4 photoproducts by polymerases eta and iota, and the bypass of ethenoadenine by polymerase iota. Moreover, they showed that WRNIP1 ATPase defects impair the WRN exonuclease from removing misinsertions by polymerase iota at thymine glycol lesions. These defects generally do not affect the efficiency of the bypass, only its fidelity.

Strengths:

The manuscript by Yoon et al is the latest in a series of important and impactful papers by this research group examining the cellular factors that enhance the fidelity of translesion synthesis by Y-family polymerases in human cell lines. Overall, the study is well designed, the data are clearly presented, and the conclusions are well supported and convincing. The authors also discuss a reasonable possibility that complex formation between the WRN and WRNIP1 proteins and Y-family polymerases could tighten the active sites of these polymerases to improve fidelity. Further studies are required to demonstrate this model, but it is a very exciting model that is well supported by the current data.

Weaknesses:

No weaknesses were identified by this reviewer.

Reviewer #3 (Public review):

Summary: In this paper the authors examined the effects of strip cropping, a relatively new agricultural technique of alternating crops in small strips of several meters wide, on ground beetle diversity. The results show an increase in species diversity (i.e. abundance and species richness) of the ground beetle communities compared to monocultures.

Strengths: The article is well written; it has an easily readable tone of voice without too much jargon or overly complicated sentence structure. Moreover, as far as reviewing the models in depth without raw data and R scripts allows, the statistical work done by the authors looks good. They have well thought out how to handle heterogenous, unbalanced and taxonomically unspecific yet spatially and temporarily correlated field data. The models applied and the model checks performed are appropriate for the data at hand. Combining RDA and PCA axes together is a nice touch. Moreover, after the first round of reviews, the authors have done a great job at rewriting the paper to make it less overstated, more relevant to the data at hand and more solid in the findings. Many of the weaknesses noted in the first review have been dealt with. The overall structure of the paper is good, with a clear introduction, hypotheses, results section and discussion.

Weaknesses: The weaknesses that remain are mainly due to a difficult dataset and choices that could have stressed certain aspects more, like the relationship between strip cropping and intercropping. The mechanistic understanding of strip cropping is what is at stake here. Does strip cropping behave similar to intercropping, a technique which has been proven to be beneficial to biodiversity because of added effects due to increased resource efficiency and greater plant species richness.

Unfortunately, the authors do not go into this in the introduction or otherwise and simply state that they consider strip cropping a form of intercropping.

I also do not like the exclusive focus on percentages, as these are dimensionless. I think more could have been done to show underlying structure in the data, even after rarefaction.

A further weakness is a limited embedding into the larger scientific discourses other than providing references. But this may be a matter of style and/or taste

Reviewer #1 (Public review):

Summary:

Recent work has demonstrated that the hummingbird hawkmoth, Macroglossum stellatarum, like many other flying insects, use ventrolateral optic flow cues for flight control. However, unlike other flying insects, the same stimulus presented in the dorsal visual field, elicits a directional response. Bigge et al., use behavioral flight experiments to set these two pathways in conflict in order to understand whether these two pathways (ventrolateral and dorsal) work together to direct flight and if so, how. The authors characterize the visual environment (the amount of contrast and translational optic flow) of the hawkmoth and find that different regions of the visual field are matched to relevant visual cues in their natural environment and that the integration of the two pathways reflects a prioritization for generating behavior that supports hawkmoth safety rather than the prevalence for a particular visual cue that is more prevalent in the environment.

Strengths:

This study creatively utilizes previous findings that the hawkmoth partitions their visual field as a way to examine parallel processing. The behavioral assay is well-established and the authors take the extra steps to characterize the visual ecology of the hawkmoth habitat to draw exciting conclusions about the hierarchy of each pathway as it contributes to flight control.

Reviewer #1 (Public review):

Summary:

The aim of the experiment reported in this paper is to examine the nature of the representation of a template of an upcoming target. To this end, participants were presented with compound gratings (consisting of tilted to the right and tilted to the left lines) and were cued to a particular orientation - red left tilt or blue right tilt (counterbalanced across participants). There are two directly compared conditions: (i) no ping: where there was a cue, that was followed by a 5.5-7.5s delay, then followed by a target grating in which the cued orientation deviated from the standard 45 degrees; and (ii) ping condition in which all aspects were the same with the only difference that a ping (visual impulse presented for 100ms) was presented after the 2.5 seconds following the cue. There was also a perception task in which only the 45 degrees to the right or to the left lines were presented. It was observed that during the delay, only in the ping condition, were the authors able to decode the orientation of the to-be-reported target using the cross-task generalization. Attention decoding, on the other hand, was decoded in both ping and non-ping conditions. It is concluded that the visual system has two different functional states associated with a template during preparation: a predominantly non-sensory representation for guidance and a latent sensory-like for prospective stimulus processing.

Strengths:

There is so much to be impressed with in this report. The writing of the manuscript is incredibly clear. The experimental design is clever and innovative. The analysis is sophisticated and also innovative - the cross-task decoding, the use of Mahalanobis distance as a function of representational similarity, the fact that the question is theoretically interesting, and the excellent figures.

Weaknesses:

While I think that this is an interesting study that addresses an important theoretical question, I have several concerns about the experimental paradigm and the subsequent conclusions that can be drawn.

(1) Why was V1 separated from the rest of the visual cortex, and why the rest of the areas were simply lumped into an EVC ROI? It would be helpful to understand the separation into ROIs.

(2) It would have been helpful to have a behavioral measure of the "attended" orientation to show that participants in fact attended to a particular orientation and were faster in the cued condition. The cue here was 100% valid, so no such behavioral measure of attention is available here.

(3) As I was reading the manuscript I kept thinking that the word attention in this manuscript can be easily replaced with visual working memory. Have the authors considered what it is about their task or cognitive demand that makes this investigation about attention or working memory?

(4) If I understand correctly, the only ROI that showed a significant difference for the cross-task generalization is V1. Was it predicted that only V1 would have two functional states? It should also be made clear that the only difference where the two states differ is V1.

(5) My primary concern about the interpretation of the finding is that the result, differences in cross-task decoding within V1 between the ping and no-ping condition might simply be explained by the fact that the ping condition refocuses attention during the long delay thus "resharpening" the template. In the no-ping condition during the 5.5 to 7.5 seconds long delay, attention for orientation might start getting less "crisp." In the ping condition, however, the ping itself might simply serve to refocus attention. So, the result is not showing the difference between the latent and non-latent stages, rather it is the difference between a decaying template representation and a representation during the refocused attentional state. It is important to address this point. Would a simple tone during the delay do the same? If so, the interpretation of the results will be different.

(6) The neural pattern distances measured using Mahalanobis values are really great! Have the authors tried to use all of the data, rather than the high AMI and low AMI to possibly show a linear relationship between response times and AMI?

(7) After reading the whole manuscript I still don't understand what the authors think the ping is actually doing, mechanistically. I would have liked a more thorough discussion, rather than referencing previous papers (all by the co-author).

Comments on revisions:

I am impressed with the thoroughness with which the authors addressed my concerns. I don't have any further concerns and think that this paper makes an interesting and significant contribution to our understanding of VWM. I would only suggest adding citations to the newly added paragraph where the authors state "It could be argued that preparatory attention relies on the same mechanisms as working memory maintenance." They could cite work by Bettencourt and Xu, 2016; and Sheremata, Somers, and Shomstein (2018).

Reviewer #1 (Public review):

This study is part of an ongoing effort to clarify the effects of cochlear neural degeneration (CND) on auditory processing in listeners with normal audiograms. This effort is important because ~10% of people who seek help for hearing difficulties have normal audiograms and current hearing healthcare has nothing to offer them.

The authors identify two shortcomings in previous work that they intend to fix. The first is a lack of cross-species studies that make direct comparisons between animal models in which CND can be confirmed and humans for which CND must be inferred indirectly. The second is the low sensitivity of purely perceptual measures to subtle changes in auditory processing. To fix these shortcomings, the authors measure envelope following responses (EFRs) in gerbils and humans using the same sounds, while also performing histological analysis of the gerbil cochleae, and testing speech perception while measuring pupil size in the humans.

The study begins with a comprehensive assessment of the hearing status of the human listeners. The only differences found between the young adult (YA) and middle aged (MA) groups are in thresholds at frequencies > 10 kHz and DPOAE amplitudes at frequencies > 5 kHz. The authors then present the EFR results, first for the humans and then for the gerbils, showing that amplitudes decrease more rapidly with increasing envelope frequency for MA than for YA in both species. The histological analysis of the gerbil cochleae shows that there were, on average, 20% fewer IHC-AN synapses at the 3 kHz place in MA relative to YA, and the number of synapses per IHC was correlated with the EFR amplitude at 1024 Hz.

The study then returns to the humans to report the results of the speech perception tests and pupillometry. The correct understanding of keywords decreased more rapidly with decreasing SNR in MA than in YA, with a noticeable difference at 0 dB, while pupillary slope (a proxy for listening effort) increased more rapidly with decreasing SNR for MA than for YA, with the largest differences at SNRs between 5 and 15 dB. Finally, the authors report that a linear combination of audiometric threshold, EFR amplitude at 1024 Hz, and a few measures of pupillary slope is predictive of speech perception at 0 dB SNR.

I only have two questions/concerns about the specific methodologies used:

(1) Synapse counts were made only at the 3 kHz place on the cochlea. But the EFR sounds were presented at 85 dB SPL, which means that a rather large section of the cochlea will actually be excited. Do we know how much of the EFR actually reflects AN fibers coming from the 3 kHz place? And are we sure that this is the same for gerbils and humans given the differences in cochlear geometry, head size, etc.?

[Note added after revision: the authors have added new data, references, and discussion that have answered my initial questions].

(2) Unless I misunderstood, the predictive power of the final model was not tested on held out data. The standard way to fit and test such model would be to split the data into two segments, one for training and hyperparameter optimization, and one for testing. But it seems that the only spilt was for training and hyperparameter optimization.

[Note added after revision: the authors now make it clear in their response that the modeling tells us how much of the current data can be explained but not necessary about generalization to other datasets.]

While I find the study to be generally well executed, I am left wondering what to make of it all. The purpose of the study with respect to fixing previous methodological shortcomings was clear, but exactly how fixings these shortcomings has allowed us to advance is not. I think we can be more confident than before that EFR amplitude is sensitive to CND, and we now know that measures of listening effort may also be sensitive to CND. But where is this leading us?

I think what this line of work is eventually aiming for is to develop a clinical tool that can be used to infer someone's CND profile. That seems like a worthwhile goal but getting there will require going beyond exploratory association studies. I think we're ready to start being explicit about what properties a CND inference tool would need to be practically useful. I have no idea whether the associations reported in this study are encouraging or not because I have no idea what level of inferential power is ultimately required.

[Note added after revision: the authors have added to the Discussion to put their work into a broader perspective.]

That brings me to my final comment: there is an inappropriate emphasis on statistical significance. The sample size was chosen arbitrarily. What if the sample had been half the size? Then few, if any, of the observed effects would have been significant. What if the sample had been twice the size? Then many more of the observed effects would have been significant (particularly for the pupillometry). I hope that future studies will follow a more principled approach in which relevant effect sizes are pre-specified (ideally as the strength of association that would be practically useful) and sample sizes are determined accordingly.

[Note added after revision: my intention with this comment was not to make a philosophical or nitty-gritty point about statistics. It was more of a follow on to the previous point. Because I don't know what sort of effect size is big enough to matter (for whatever purpose), I don't find the statistical significance (or lack thereof) of the effect size observed to be informative. But I don't think there is anything more that the authors can or should do in this regard.]

So, in summary, I think this study is a valuable but limited advance. The results increase my confidence that non-invasive measures can be used to infer underlying CND, but I am unsure how much closer we are to anything that is practically useful.

Reviewer #1 (Public review):

Wang et al. investigated how sexual failure influences sweet taste perception in male Drosophila. The study revealed that courtship failure leads to decreased sweet sensitivity and feeding behavior via dopaminergic signaling. Specifically, the authors identified a group of dopaminergic neurons projecting to the subesophageal zone that interact with sweet-sensing Gr5a+ neurons. These dopaminergic neurons positively regulate the sweet sensitivity of Gr5a+ neurons via DopR1 and Dop2R receptors. Sexual failure diminishes the activity of these dopaminergic neurons, leading to reduced sweet taste sensitivity and sugar feeding behavior in the male flies. These findings highlight the role of dopaminergic neurons in integrating reproductive experiences to modulate appetitive sensory responses.

Previous studies have explored the dopaminergic-to-Gr5a+ neuronal pathways in regulating sugar feeding under hunger conditions. Starvation has been shown to increase dopamine release from a subset of TH-GAL4 labeled neurons, known as TH-VUM, in the subesophageal zone. This enhanced dopamine release activates dopamine receptors in Gr5a+ neurons, heightening their sensitivity to sugar and promoting sucrose acceptance in flies. Since the function of the dopaminergic-to-Gr5a+ circuit motif has been well established, the primary contribution of Wang et al. is to show that mating failure in male flies can also engage this circuit to modulate sugar feeding behavior. This contribution is valuable because it highlights the role of dopaminergic neurons in integrating diverse internal state signals to inform behavioral decisions.

An intriguing discrepancy between Wang et al. and earlier studies lies in the involvement of dopamine receptors in Gr5a+ neurons. Prior research has shown that Dop2R and DopEcR, but not DopR1, mediate starvation-induced enhancement of sugar sensitivity in Gr5a+ neurons. In contrast, Wang et al. report that DopR1 and Dop2R, but not DopEcR, are involved in the mating failure-induced suppression of sugar sensitivity in these neurons. Further investigation is needed to clarify how dopamine selectively engages different receptor types depending on internal state.

The data in this revised version are largely convincing and support the authors' conclusions. However, I remain concerned about the results shown in Figure 6E. The authors show that knocking down DopR1 or Dop2R in Gr5a+ neurons restores sucrose-evoked activity in Failed flies to levels seen in Naive and Satisfied animals. This appears to contradict the proposed model, in which these receptors positively modulate Gr5a+ activity through dopaminergic input. If dopamine signaling is reduced in Failed flies, further receptor knockdown should have no effect or further reduce activity-not restore it. I encourage the authors to clarify this apparent inconsistency and, if possible, provide a mechanistic explanation.

Reviewer #1 (Public review):

Summary:

Horizontal gene transfer is the transmission of genetic material between organisms through ways other than reproduction. Frequent in prokaryotes, this mode of genetic exchange is scarcer in eukaryotes, especially in multicellular eukaryotes. Furthermore, the mechanisms involved in eukaryotic HGT are unknown. This article by Banerjee et al. claims that HGT occurs massively between cells of multicellular organisms. According to this study, the cell free chromatin particles (cfChPs) that are massively released by dying cells are incorporated in the nucleus of neighboring cells. These cfChPs are frequently rearranged and amplified to form concatemers, they are made of open chromatin, expressed, and capable of producing proteins. Furthermore, the study also suggests that cfChPs transmit transposable elements (TEs) between cells on a regular basis, and that these TEs can transpose, multiply, and invade receiving cells. These conclusions are based on a series of experiments consisting in releasing cfChPs isolated from various human sera into the culture medium of mouse cells, and using FISH and immunofluorescence to monitor the state and fate of cfChPs after several passages of the mouse cell line.

Strengths:

The results presented in this study are interesting because they may reveal unsuspected properties of some cell types that may be able to internalize free-circulating chromatin, leading to its chromosomal incorporation, expression, and unleashing of TEs. The authors propose that this phenomenon may have profound impacts in terms of diseases and genome evolution. They even suggest that this could occur in germ cells, leading to within-organism HGT with long-term consequences.

Weaknesses:

The claims of massive HGT between cells through internalization of cfChPs are not well supported because they are only based on evidence from one type of methodological approach: immunofluorescence and fluorescent in situ hybridization (FISH) using protein antibodies and DNA probes. Yet, such strong claims require validation by at least one, but preferably multiple, additional orthogonal approaches. This includes, for example, whole genome sequencing (to validate concatemerization, integration in receiving cells, transposition in receiving cells), RNA-seq (to validate expression), ChiP-seq (to validate chromatin state).

Should HGT through internalization of circulating chromatin occur on a massive scale, as claimed in this study, and as illustrated by the many FISH foci observed on Fig 3 for example, one would expect that the level of somatic mosaicism may be so high that it would prevent assembling a contiguous genome for a given organism. Yet, telomere-to-telomere genomes have been produced for many eukaryote species, calling into question the conclusions of this study.

Reviewer #1 (Public review):

Summary:

Fallah and colleagues characterize the connectivity between two basal ganglia output nuclei, the SNr and GPe, and a the pedunculopontine nucleus, a brainstem nucleus that is part of the mesencephalic locomotor region. Through a series of systematic electrophysiological studies, they find that these regions target and inhibit different populations of neurons, with anatomical organization. Overall, SNr projects to PPN and inhibits all major cell types, while the GPe inhibits glutamatergic and GABAergic PPN neurons, and preferentially in the caudal part of the nucleus. Optogenetic manipulation of these inputs in the had opposing effects on behavior - SNr terminals in the PPN drove place aversion, while GPe terminals drove place preference.

Strengths:

This work is thorough and systematic characterization of a set of relatively understudied circuits. They build on the classic notions of basal ganglia connectivity and suggest a number of interesting future directions to dissect motor control and valence processing in brainstem systems.

Limitations:

All the cell type recording studies showing subtle differences in the degree of inhibition and anatomical organization of that inhibition suggest a complex effect of general optogenetic manipulation of SNr or GPe terminals in the PPN. It will be important to determine if SNr or GPe inputs onto a particular cell type in PPN are more or less critical for the how the locomotion and valence effects demonstrated here.

Reviewer #1 (Public review):

Summary:

This work studies representations in a network with one recurrent layer and one output layer that needs to path-integrate so that its position can be accurately decoded from its output. To formalise this problem, the authors define a cost function consisting of the decoding error and a regularisation term. They specify a decoding procedure that, at a given time, averages the output unit center locations, weighted by the activity of the unit at that time. The network is initialised without position information, and only receives a velocity signal (and a context signal to index the environment) at each timestep, so to achieve low decoding error it needs to infer its position and keep it updated with respect to its velocity by path integration.

The authors take the trained network and let it explore a series of environments with different geometries while collecting unit activities to probe learned representations. They find localised responses in the output units (resembling place fields) and border responses in the recurrent units. Across environments, the output units show global remapping and the recurrent units show rate remapping. Stretching the environment generally produces stretched responses in output and recurrent units. Ratemaps remain stable within environments and stabilise after noise injection. Low-dimensional projections of the recurrent population activity forms environment-specific clusters that reflect the environment's geometry, which suggests independent rather than generalised representations. Finally, the authors discover that the centers of the output unit ratemaps cluster together on a triangular lattice (like the receptive fields of a single grid cell), and find significant clustering of place cell centers in empirical data as well.

The model setup and simulations are clearly described, and are an interesting exploration of the consequences of a particular set of training requirements - here: path integration and decodability. But it is not obvious to what extent the modelling choices are a realistic reflection of how the brain solves navigation. Therefore, it is not clear whether the results generalize beyond the specifics of the setup here.

Strengths:

The authors introduce a very minimal set of model requirements, assumptions, and constraints. In that sense, the model can function as a useful 'baseline', that shows how spatial representations and remapping properties can emerge from the requirement of path integration and decodability alone. Moreover, the authors use the same formalism to relate their setup to existing spatial navigation models, which is informative.

The global remapping that the authors show is convincing and well-supported by their analyses. The geometric manipulations and the resulting stretching of place responses, without additional training, are interesting. They seem to suggest that the recurrent network may scale the velocity input by the environment dimensions so that the exact same path integrator-output mappings remain valid (but maybe there are other mechanisms too that achieve the same).

The simulations and analyses in the appendices serve as insightful controls for the main results.

The clustering of place cell peaks on a triangular lattice is intriguing, given there is no grid cell input. It could have something to do with the fact that a triangular lattice provides optimal coverage of 2d space? The included comparison with empirical data is valuable as a first exploration, showing a promising example, but doesn't robustly support the modelling results.

Reviewer #1 (Public review):

Summary:

In their comprehensive analysis Diallo et al. deorphanise the first olfactory receptor of a non-hymenopteran eusocial insect - a termite and identified the well established trail pheromone neocembrene as the receptor's best ligand. By using a large set of odorants the authors convincingly show that, as expected for a pheromone receptor, PsimOR14 is very narrowly tuned. While the authors first make use of an ectopic expression system, the empty neuron of Drosophila melanogaster, to characterise the receptor's responses, they next perform single sensillum recordings with different sensilla types on the termite antenna. By that they are able to identify a sensillum which houses three neurons, of which the B neuron exhibits the narrow responses described for PsimOR14. Hence the authors do not only identify the first pheromone receptor in a termite but can even localise its expression on the antenna. The authors in addition perform a structural analysis to explain the binding properties of the receptor and its major and minor ligands (as this is beyond my expertise, I cannot judge this part of the manuscript). Finally, they compare expression patterns of ORs in different castes and find that PsimOR14 is more strongly expressed in worker than in soldier termites, which corresponds well with stronger antennal responses in the worker caste.

Strengths:

The manuscript is well written and a pleasure to read.

Weaknesses:

Whenever it comes to the deorphanization of a receptor and its potential role in behaviour (in the case of the manuscript it would be trail following of the termite) one thinks immediately of knocking out the receptor to check whether it is necessary for the behaviour. However, I definitely do not want to ask for this (especially as the establishment of CRISPR Cas-9 in eusocial insects usually turns out to be a nightmare). I also do not know either, whether knock downs via RNAi have been established in termites, but maybe the authors could consider some speculation on this in the discussion.

Comments on revisions:

I appreciate how the authors have replied to my comments and I have the feeling that also the other reviewers' comments have been dealt with carefully. I therefore support the acceptance of this very nice and interesting manuscript.

Reviewer #1 (Public review):

The authors examine how probabilistic reversal learning is affected by dopamine by studying the effects of methamphetamine (MA) administration. Based on prior evidence that the effects of pharmacological manipulation depend on baseline neurotransmitter levels, they hypothesized that MA would improve learning in people with low baseline performance. They found this effect, and specifically found that MA administration improved learning in noisy blocks, by reducing learning from misleading performance, in participants with lower baseline performance. The authors then fit participants' behavior to a computational learning model and found that an eta parameter, responsible for scaling learning rate based on previously surprising outcomes, differed in participants with low baseline performance on and off MA.

Questions:

(1) It would be helpful to confirm that the observed effect of MA on the eta parameter is responsible for better performance in low baseline performers. If performance on the task is simulated for parameters estimated for high and low baseline performers on and off MA, does the simulated behavior capture the main behavioral differences shown in Figure 3?

(2) In Figure 4C, it appears that the main parameter difference between low and high baseline performance is inverse temperature, not eta. If MA is effective in people with lower baseline DA, why is the effect of MA on eta and not IT?

Also, this parameter is noted as temperature but appears to be inverse temperature as higher values are related to better performance. The exact model for the choice function is not described in the methods.

Comments on revisions:

Thanks to the authors for their thorough responses and revisions. One typo to note: in the Methods, the "drug effects" paragraph is repeated.

Reviewer #1 (Public review):

Summary:

In this study, Bonnifet et al. profile the presence of L1 ORF1p in the mouse and human brain and report that ORF1p is expressed in the human and mouse brain specifically in neurons at steady state and that there is an age-dependent increase in expression. This is a timely report as two recent papers have extensively documented the presence of full-length L1 transcripts in the mouse and human brain (PMID: 38773348 & PMID: 37910626). Thus, the finding that L1 ORF1p is consistently expressed in the brain is important to document and will be of value to the field.

Strengths:

Several parts of this manuscript appear to be well done and include the necessary controls. In particular, the documentation of neuron-specific expression of ORF1p in the mouse brain is an interesting finding with nice documentation. This will be very useful information for the field.

Weaknesses:

Several parts of the manuscript appear to be more preliminary and need further experiments to validate their claims. In particular, the data suggesting expression of L1 ORF1p in the human brain and the data suggesting increased expression in the aged brain need further validation. Detailed comments:

(1) The expression of ORF1p in the human brain shown in Fig. 1j is puzzling. Why are there two strong bands in the WB? How can the authors be sure that this signal represents ORF1p expression and not non-specific labelling? While the authors discuss that others have found double bands when examining human ORF1p, there are also several labs that report only one band. This discrepancy in the field should at least be discussed and the uncertainties with their findings should be acknowledged.

(2) The data showing a reduction in ORF1p expression in the aged mouse brain is an interesting observation, but the effect magnitude of effect is very limited and somewhat difficult to interpret. This finding should be supported by orthogonal methods to strengthen this conclusion. For example, by WB and by RNA-seq (to verify that the increase in protein is due to an increase in transcription).

(3) The transcriptomic data using human postmortem tissue presented in Figure 4 and Figure 5 are not convincing. Quantification of transposon expression on short read sequencing has important limitations. Longer reads and complementary approaches are needed to study the expression of evolutionarily young L1s (see PMID: 38773348 & PMID: 37910626 for examples of the current state of the art). As presented, the human RNA data is inconclusive due to the short read length and small sample size. The value of including an inconclusive analysis in the manuscript is difficult to understand. With this data set, the authors cannot investigate age-related changes in L1 expression in human neurons.

(4) In line with these comments, the title should be changed to better reflect the findings in the manuscript. A title that does not mention "L1 increase with aging" would be better.