Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

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

Weaknesses:

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

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

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

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

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

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

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

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

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

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

Overall appraisal and context:

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

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Comments on revisions:

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

Reviewer #2 (Public review):

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

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

Strengths:

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

Weaknesses:

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

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

Comments on Revised Version (from BRE):

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

Reviewer #2 (Public review):

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

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

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

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

Reviewer #2 (Public review):

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

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

Reviewer #2 (Public review):

Summary:

This work explores the phenotypic developmental traits associated with Cu and Cd responses in teosinte parviglumis, a species evolutionary related to extant maize crops. Cu and Cd could serve as a proxy for heavy metals present in the soils. The manuscript explores potential genetic loci associated with heavy metal responses and domestication identified in previous studies. This includes heavy metal transporters, which are unregulated during stress. To study that, the authors compare the plant architecture of maize defective in ZmHMA1 and speculate on its association with domestication.

Strengths:

Very few studies covered the responses of teosintes to heavy metal stress. The physiological function of ZmHMA1 in maize also gives some novelty in this study. The idea and speculation section is interesting and well-implemented.

Weaknesses:

The authors explored Cu/Cd stress but not a more comprehensive panel of heavy metals, making the implications of this study quite narrow. Some techniques used, such as end-point RT-PCR and qPCR, are substandard for the field. The phenotypic changes explored are not clearly connected with the potential genetic mechanisms associated with them, with the exception of nodal roots. If teosintes in response to heavy metal have phenotypic similarity with modern landraces of maize, then heavy metal stress might have been a confounding factor in the selection of maize and not a potential driving factor. Similar to the positive selection of ZmHMA1 and its phenotypic traits. In that sense, there is no clear hypothesis of what the authors are looking for in this study, and it is hard to make conclusions based on the provided results to understand its importance. The authors do not provide any clear data on the potential influence of heavy metals in the field during the domestication of maize. The potential role of Tb-1 is not very clear either.

Reviewer #2 (Public review):

The revised version of the paper clarifies the authors' discoveries regarding daily changes in metabolite concentrations in the gut of adult female Drosophila melanogaster. The authors have addressed all the questions and made the necessary changes, thereby strengthening the value of the article. They demonstrate that various factors influence metabolite oscillations: circadian clock genotype, dietary regime and composition, and gut microbiota.<br /> The notable strengths of this research article remain unchanged: the originality of the experimental design with multiple conditions tested, the variety of detected metabolites, and the clarity in data presentation.

Among the weaknesses, one may consider the following:<br /> Limitations of potential reproducibility: It is unclear whether another research team would identify the same set of cycling metabolites, although similar conclusions appear robust.<br /> Limitations of generalisation: While the conclusions regarding the influence of microbiota, circadian genotype, and dietary regime may be valid, the specific metabolic pathways affected might differ, whereas specific mechanistic explanations remain elusive.<br /> Accuracy of data interpretation: Addressed in comments to the authors. This point corresponds to interpretations discussed by the authors in the text of the manuscript, including beneficial effects of cycling metabolites and phenomenon of oscillation as a whole, its physiological relevance and lack of proofs for existence of any compensative effects, their relevance to metabolism in the gut.<br /> Nevertheless, the authors have clearly and thoroughly addressed all the reviewers' concerns, enabling a better interpretation of the entire study.

Reviewer #2 (Public review):

Summary:

This is a meta-analysis of the relative contributions of spring forcing temperature, winter chilling, photoperiod and environmental variables in explaining plant flowering and leafing phenology. The authors develop a new summary variable called phenology lag to describe why species might have different responses than predicted by spring temperature.

Strengths:

The summary statistic is used to make a variety of comparisons, such as between observational studies and experimental studies.

Weaknesses:

By combining winter chilling effects, photoperiod effects, and environmental stresses that might affect phenology, the authors create a new variable that is hard to interpret. The authors do not provide information in the abstract about new insights that this variable provides.

Comments:

It would be useful to have a map showing the sites of the studies.

The authors should provide a section in which the strengths and weaknesses of the approach are discussed. Is it possible that mixing different types of data, studies, sample sizes, number of years, experimental set-ups, and growth habits results in artifacts that influence the results?

Now that the authors have created this new variable, phenological lag, which of the components that contribute to it has the most influence on it? Or which components are most influential in which circumstances? For example, what are some examples where photoperiod causes a phenological lag?

Reviewer #2 (Public review):

The authors present an interesting study on calibrating and validating a biventricular cardiac electromechanical model. This is an important contribution, but some questions remain about the quantitative validation and verification aspects of the study.

Major comments:

(1) The title and paper stress the importance of validation on several occasions. However, the actual validation performed is limited to the section in lines 427-439. Furthermore, it is entirely qualitative, making assessing the model's quality difficult. Most of the paper is focused on sensitivity analysis, which is also interesting but unrelated to validation. Can you include a quantitative comparison with deformation biomarkers? E.g., spatially quantify strain differences between simulation and in vivo data, or overlay the current configuration of the geometry with MRI in various views, and calculate a displacement error norm.

(2) You mention the ASME V&V40 standards throughout your paper. Yet, you only address the "second V" validation, ignoring the "first V" verification. How did you ensure that your computational models are implemented correctly?

(3) All parameters discussed in this publication are physical parameters. What is the sensitivity of your model outputs concerning computational parameters?

Reviewer #2 (Public review):

Summary:

In this important study, the authors examine the role of two zinc uptake transporters, Zip6 and Zip10, which are important during the maturation of oocytes, and are critical for both successful fertilization and early embryogenesis.

Strengths:

The authors report that oocytes from Zip10 knockout mice exhibit lower labile zinc content during oocyte maturation, decreased amounts of zinc exocytosis during fertilization, and affect the rate of blastocyst generation in fertilized eggs relative to a control strain. They do not observe these changes in their Zip6 knockout animals. The authors present clear and well-documented results from a broad range of experimental modalities in support of their conclusions.

Weaknesses:

(1) The authors' statement that Zip10 is not expressed in the oocyte nuclei (line 252). Furthermore, in that study, ZIP10 was detected in the nuclear/nucleolar positions of oocytes of all follicular stages (Chen et al., 2023), which we did not observe. This is not supported by Figure 1, where some Zip10 signal is apparent in the primordial, primary, and secondary follicle oocytes. This statement should be corrected.

(2) Based on the FluoZin-3AM data, there appears to be less labile zinc in the Zip10d/d oocyte, eggs, and embryos; however, FluoZin-3AM has a number of well-known artifacts and does not accurately capture the localization of labile zinc pools. The patterns do not correspond to the well-documented zinc-containing cortical vesicles. Another zinc probe, such as ZinPyr-4 or ZincBY-1 should be used to visualize the zinc vesicles and confirm that there is less labile zinc in these locations as well.

(3) Line 268 The results indicate that ZIP10 is mostly responsible for the uptake of zinc ions in mouse oocytes. The situation seems a bit more complicated given that the differences in labile zinc content between oocytes from the WT and Zip10d/d animals are small (only 20-30 %) and that the zinc spark is diminished but still apparent at a low level in the Zip10d/d oocytes. Clearly, other factors are involved in zinc uptake at these stages. A variety of studies have suggested that Zip6 and Zip10 work together, perhaps even functioning as a heterodimer in some systems. The double KO would address this more clearly, but if it is not available, it might be more prudent to state that Zip10 plays some role in uptake of zinc in mouse oocytes while the role of Zip6 remains uncertain.

(4) Zip6d/d oocytes did not have changes in labile zinc, nor did the lack of Zip6 have an impact on the zinc spark. However, Figure S1 does show a small amount of detectable Zip6 in the western blot. It is possible that this small amount could compensate for the complete lack of Zip6. Can ZIP6 be found in immunofluorescence of GV oocytes or MII eggs from the Zip6d/d animals? Additionally, it is possible that Zip6's role is only supplementary to that of Zip10. The authors should discuss this possibility. It would also be interesting to see if the Zip6/Zip10 double knockout displays greater defects compared to the Zip10 knockout when considering previous studies.

Reviewer #2 (Public Review):

This study is inspired by the scanning movements observed in bees when performing visual recognition tasks. It uses a multilayered network, representing stages of processing in the visual lobes (lamina, medulla, lobula), and uses the lobula output as input to a model of associative learning in the mushroom body (MB). The network is first trained with short "scanning" sequences of natural images, in a non-associative adaptation process, and then several experimental paradigms where images are rewarded or punished are simulated, with the output of the MB able to provide the appropriate discriminative decisions (in some but not all cases). The lobula receptive fields formed by the initial adaptation process show spatiotemporal tuning to edges moving at particular orientations and speeds that are comparable to recorded responses of such neurons in the insect brain.

There are two main limitations to the study in my view. First, although described (caption fig 1) as a model "inspired by the micromorphology" of the insect brain, implying a significant degree of accuracy and detail, there are many arbitrary features (unsupported by current connectomics). For example, the strongly constrained delay line structure from medulla to­ lobula neurons, and the use of a single MB0N that has input synapses that undergo facilitation and decay according to different neuromodulators. Second, while it is reasonable to explore some arbitrary architectural features, given that not everything is yet known about these pathways, the presented work does not sufficiently assess the necessity and sufficiency of the different components, given the repeated claims that this is the "minimal circuit" required for the visual tasks explored.

Regarding the mushroom body (MB) learning model, it is strange that no reference is made to recent models closely tied to connectomic and other data in fruit flies, which suggests separate MBONS encode positive vs. negative value; that learning is not dependent on MB0N activity (so is not STDP); that feedback from MBONs to dopaminergic signalling plays an important role, etc. Possibly the MB of the bee operates in a completely different way to the fly, but the presented model relies on relatively old data about MB function, mostly from insects other than bees (e.g. locust) so its relationship to the increasingly comprehensive understanding emerging for the fly MB needs to be clarified. It is implied that the complex interaction of the differential effects of dopamine and octopamine, as modelled here, are required to learn the more complex visual paradigms, but it is not actually tested if simpler rules might suffice. Also, given previous work on models of view recognition in the MB, inspired by bees and ants, it seems plausible that simply using static 25×25 medulla activity as input to produce sparse activity in the KCs would be sufficient for MB0N output to discriminate the patterns used in training, including the face stimulus. Thus it is not clear whether the spatiotemporal input and the lobula encoding are necessary to solve these tasks.

It is also difficult to interpret the range of results in fig 3. The network sometimes learns well, sometimes just adequately (perhaps comparable to bees), and sometimes fails. The presentation of these results does not seem to identify any coherent pattern underlying success or failure, other than that the ability to generalise seems limited. That is, recognition (in most cases) requires the presentation of exactly the same stimulus in exactly the same way (same scanning pattern, distance and speed). In particular, it is hard to know what to conclude when the network appears able to learn some "complex patterns" (spirals, faces) but fails to learn the apparently simple plus vs. multiplication symbol discrimination if it is trained and tested with a scan passing across the whole pattern instead of just the lower half.

In summary, although it is certainly interesting to explore how active vision (scanning a visual pattern) might affect the encoding of stimuli and the ability to learn to discriminate rewarding stimuli, some claims in the paper need to be tempered or better supported by the demonstration that alternative, equally plausible, models of the visual and mushroom body circuits are not sufficient to solve the given tasks.

Reviewer #3 (Public review):

Summary:

The manuscript uses live imaging to study the role of microtubules in the movement of ribeye aggregates in neuromast hair cells in zebrafish. The main findings are that

(1) Ribeye aggregates, assumed to be ribbon precursors, move in a directed motion toward the active zone;<br /> (2) Disruption of microtubules and kif1aa increases the number of ribeye aggregates and decreases the number of mature synapses.

The evidence for point 2 is compelling, while the evidence for point 1 is less convincing. In particular, the directed motion conclusion is dependent upon fitting of mean squared displacement that can be prone to error and variance to do stochasticity, which is not accounted for in the analysis. Only a small subset of the aggregates meet this criteria and one wonders whether the focus on this subset misses the bigger picture of what is happening with the majority of spots.

Strengths:

(1) The effects of Kif1aa removal and nocodozole on ribbon precursor number and size is convincing and novel.<br /> (2) The live imaging of Ribeye aggregate dynamics provides interesting insight into ribbon formation. The movies showing fusion of ribeye spots are convincing and the demonstrated effects of nocodozole and kif1aa removal on the frequency of these events is novel.<br /> (3) The effect of nocodozole and kif1aa removal on precursor fusion is novel and interesting.<br /> (4) The quality of the data is extremely high and the results are interesting.

Weaknesses:

(1) To image ribeye aggregates, the investigators overexpressed Ribeye-a TAGRFP under control of a MyoVI promoter. While it is understandable why they chose to do the experiments this way, expression is not under the same transcriptional regulation as the native protein and some caution is warranted in drawing some conclusions. For example, the reduction in the number of puncta with maturity may partially reflect regulation of the MyoVI promoter with hair cell maturity. Similarly, it is unknown whether overexpression has the potential to saturate binding sites (for example to motors), which could influence mobility. In the revised manuscript, the authors provide evidence to suggest that overexpression is not at unreasonably high levels, which is reasonable. However, I think it remains important to think of these caveats while reading the paper--especially keeping in mind that expression timing is undoubtedly influenced by the transcriptional control of the exogenous promoter .<br /> (2) The examples of punctae colocalizing with microtubules look clear (fig 1 F-G), but the presentation is anecdotal. It would be better and more informative, if quantified.<br /> (3) It appears that any directed transport may be rare. Simply having an alpha >1 is not sufficient to declare movement to be directed (motor driven transport typically has an alpha approaching 2). Due to randomness of a random walk and errors in fits in imperfect data will yield some spread in movement driven by Brownian motion. Many of the tracks in figure 3H look as thought they might be reasonably fit by a straight line (i.e. alpha = 1).<br /> (4) The "directed motion" shown here does not really resemble motor driven transport observed in other systems (axonal transport, for example) even in the subset that have been picked out as examples here. While the role for microtubules and kif1aa in synapse maturation is strong, it seems likely that this role may be something non-canonical (which would be interesting). In the revision, the authors do an excellent job of considering the issues brought up in point 3 and 4. While perhaps no longer a weakness, I am leaving the critiques here for context for the readers to consider. The added taxol results may not completely settle the issue, but are interesting and provide important information.

Reviewer #2 (Public review):

Summary:

The manuscript by Chuah et al. reports the experimental results that suggest the occupancy of the HbYX pockets suffices for proteasome gate opening. The authors conducted cryo-EM reconstructions of two mutant archaeal proteasomes. The work is technically sound and may be of special interest in the field of structural biology of the proteasomes.

Strengths:

Overall, the work incrementally deepens our understanding of the proteasome activation and expands the structural foundation for therapeutic intervention of proteasome function. The evidence presented appears to be well aligned with the existing literature, which adds confidence in the presentation.

Weaknesses:

The paper may benefit from some minor revision by making improvements on the figures and necessary quantitative comparative studies.

Reviewer #2 (Public review):

Summary:

The manuscript offers an important contribution to the field of virology, especially concerning NNV entry mechanisms. The major strength of the study lies in the identification of MmMYL3 as a functional receptor for RGNNV and its role in macropinocytosis, mediated by the IGF1R-Rac1/Cdc42 signaling axis. This represents a significant advance in understanding NNV entry mechanisms beyond previously known receptors such as HSP90ab1 and HSC70. The data, supported by comprehensive in vitro and in vivo experiments, strongly justify the authors' claims about MYL3's role in NNV infection in marine medaka.

Strengths:

(1) The identification of MmMYL3 as a functional receptor for RGNNV is a significant contribution to the field. The study fills a crucial gap in understanding the molecular mechanisms governing NNV entry into host cells.

(2) The work highlights the involvement of IGF1R in macropinocytosis-mediated NNV entry and downstream Rac1/Cdc42 activation, thus providing a thorough mechanistic understanding of NNV internalization process. This could pave the way for further exploration of antiviral targets.

Comments on revisions:

The authors have addressed the concerns from reviewers. This manuscript can be published in the current form.

Reviewer #3 (Public review):

Summary

Tanaka and colleagues addressed the role of the C-C chemokine receptor 4 (CCR4) in early atherosclerotic plaque development using ApoE-deficient mice on a standard chow diet as a model. Because several CD4+ T cell subsets express CCR4, they examined whether CCR4-deficiency alters the immune response mediated by CD4+ T cells. By histological analysis of aortic lesions, they demonstrated that the absence of CCR4 promoted the development of early atherosclerosis, with heightened inflammation linked to increased macrophages and pro-inflammatory CD4+ T cells, along with reduced collagen content. Flow cytometry and mRNA expression analysis for identifying CD4+ T cell subsets showed that CCR4 deficiency promoted higher proliferation of pro-inflammatory effector CD4+ T cells in peripheral lymphoid tissues and accumulation of Th1 cells in the atherosclerotic lesions. Interestingly, the increased pro-inflammatory CD4+ T cell response occurred despite the expansion of T CD4+ Foxp3+ regulatory cells (Tregs), found in higher numbers in lymphoid tissues of CCR4-deficient mice, suggesting that CCR4 deficiency interfered with Treg's regulatory actions. The findings contrast with earlier studies in a murine model of advanced atherosclerosis, where CCR4 deficiency did not alter the development of the aortic lesions. The authors included a thoughtful discussion about hypothetical mechanisms explaining these contrasting results, including putative differences in the role played by the CCL17/CCL22-CCR4 axis along the stages of atherosclerosis development in this murine model.

Major strengths

• Demonstration of CCR4 deficiency's impact on early atherosclerosis. CCR4 deficiency effects on the early atherosclerosis development in the Apoe-/-mice model were demonstrated by a quantitative analysis of the lesion area, inflammatory cell content and the expression profile of several pro- and anti-inflammatory markers.<br /> • Analysis of the T CD4+ response in various lymphoid tissues (peripheral and para-aortic lymph nodes and spleen) and the atherosclerotic aorta during the early phase of atherosclerosis in the Apoe-/-mice model. This analysis, combining flow cytometry and mRNA expression, showed that CCR4 deficiency enhanced T CD4+ cell activation, favouring the amplification of the typical biased Th1-mediated inflammatory response observed in the lymphoid tissues of hypercholesterolemic mice.<br /> • Treg transference experiments. Transference of Treg from Apoe-/- or Ccr4-/- Apoe-/- mice to Apoe-/- mice under a standard chow diet was useful for addressing the relevance of CCR4 expression on Tregs for the atheroprotective effect of this regulatory T cell subset during early atherosclerosis.

Major weaknesses

• Methodological Limitations: The controls used in the flow cytometry analysis were suboptimal, as neither cell viability nor doublets were assessed. This may have introduced artifacts, particularly when measuring less-represented cell populations within complex samples, such as in assays evaluating Treg migration to the aorta in atherosclerotic mice.<br /> • Incomplete understanding of CCR4-Mediated Mechanisms: The mechanisms by which CCR4 regulates early inflammation and the development of atherosclerosis were not fully clarified.

I have previously addressed the study limitations and their global impact in my earlier reviews.

Reviewer #2 (Public review):

This study presents an important finding to the field interested in recurrent processing and the role of NMDA-receptors herein. The evidence for improved decoding under memantine is convincing, while some open questions remain to be followed up in future studies (the lack of a behavioural effect, why is decoding improved rather than decreased?). It is an excellent example of how an unexpected finding can generate novel research ideas to the mechanisms underlying recurrent processing, suggesting that the answer lies in the differences in the effects of ketamine and memantine, rather than their commonalities.

I would like to thank the authors for the great care they have taken in addressing my concerns. I think the revised manuscript is significantly easier to follow now that specific hypothesis have been formulated in the introduction, and the direction of the results is explicitly stated throughout the manuscript. I further appreciate the dampening of some of the claims that are not completely supported by the appropriate interactions.

I think the resulting manuscript is an incredibly exciting contribution to our understanding of NMDA-receptor function, and a great example of how an unexpected finding can raise questions that could potentially drive the field forward. It shows how NMDA's role in recurrent processing is much more complicate than previously assumed, and reveals that it is not the commonalities between memantine and ketamine that are important in understanding recurrent processing, but rather the differences. I look forward to future studies that will target these differences.

Overall great job.

Reviewer #2 (Public Review):

Cell cycle control during nitrogen-fixing symbiosis is an important topic, but our understanding of the process is poor and lacks resolution, as the nodule is a complex organ with many cell types that undergo profound changes. The authors aim to define the cell cycle state of individual plant cells in the emerging nodule primordium, as a transcellular infection thread passes through the meristem to reach cells deep in the incipient nodule and releases bacteria to form symbiosomes. The authors used a number of cell cycle reporters, such as different Histone 3 variants and cyclins, to follow cell cycle progress in exquisite detail. They showed that the host cells in the path of an infection thread exhibit a cell fate distinct from their immediate neighbors: after entering the S phase similar to their neighbors, these cells exit the cell cycle and enter a special differentiated state. This is likely an important shift that allows the proper passage of the infection thread. Although definitive proof needs more investigation, they showed that a pioneering transcription factor, NF-YA1, likely represses these endoreduplicated cells from completing the cell cycle.

Reviewer #2 (Public review):

Summary:

The manuscript by Arabanian and colleagues presents studies showing how inhibition of mitochondrial transcription and replication with a novel inhibitor of the mitochondrial polymerase, IMT, can promote AML cell death in combination with the Bcl2 inhibitor venetoclax. They further show that this combinatorial efficacy is evident in vivo in both the AML cell line MV411 and in a PDX model. Given the multiple studies showing the importance of Oxphos in maintaining AML cell survival, the current studies provide an additional strategy to inhibit Oxphos and thus improve the therapeutic management of AML.

Strengths:

A novel aspect of this work is that IMT is a new class of mitochondrial inhibitor that acts through inhibiting the mitochondrial polymerase. In addition, the demonstration of therapeutic efficacy both in vitro and in vivo (including with PDX), together with some data showing minimal toxicity, adds to the impact of this work. Their overall conclusion that IMT increases the potency of Vex in treating AMLs is supported.

Comments on revisions:

In all, the authors responded to most of the critiques, while two of the major critiques were not experimentally addressed. The work will still have potential impact, but will depend on further studies under more clinically relevant conditions and with a better understanding of drug effects.

Reviewer #2 (Public review):

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

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

Some of the major issues are described below.

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

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

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

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

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

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

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

Reviewer #2 (Public review):

Summary:

This work highlights a novel role for platelet-derived growth factor (PDGF) in mitigating cellular senescence associated with age-related and painful intervertebral disc degeneration. Prior literature has demonstrated the importance of accumulation of senescent cells in mediating many of the pathological effects associated with the degenerate disc joint, such as inflammation and tissue breakdown. In this study the authors treat clinically relevant human nucleus pulposus and annulus fibrosus cells from patients undergoing discectomy with recombinant PDGF-AB/BB for 5 days and then deep phenotyped the outcomes using bulk RNA sequencing. In addition they irradiated healthy human disc cells which they subsequently treated with PDGF-AB/BB examining the expression of SASP-related markers and also PDGFRA receptor gene expression. Overall PDGF was able to down-regulate many senescent associated pathways and the degenerate phenotype in IVD cells. Altered pathways were associated with neurogenesis, mechanical stimuli, metabolism, cell cycle, reactive oxygen species and mitochondrial dysfunction. Overall the authors achieved their aims and the results by and large support their conclusions although improvements could be made to enhance the rigor of the study and findings

Strengths:

A major strength of this study is the use of human cells from patients undergoing discectomy for disc herniation as well as access to healthy human cells. Investigating the role of PDGF regarding cellular senescence in the degenerate disc joint is novel and an underexplored area of research which is a significant contribution to the field of spine. This study highlights a potential target for addressing cellular senescence where most of the prior focus has been on senolytic drugs. Such studies have broad implications to other age-related diseases where senescence plays a major role. The use of transcriptomics and therefore an unbiased approach to investigating the role of PDGF is also considered a strength as is the follow-up studies involving irradiating healthy human disc cells and treating these cells with PDGF. The combined assessment of both nucleus pulposus and annulus fibrosus cells in the context of these studies adds to the impact.

Weaknesses:

A weakness of these studies relates to qualitative data presented for the B-galactosidase assay. Quantification of such data sets would greatly strengthen the studies and lend further support to the hypotheses. The study in its current form could be strengthened by the inclusion of mechanistic studies probing the downstream PDGF receptor associated pathways for example specifically targeting or modulating the activity of the PDGF receptor PDGFRA.

Reviewer #2 (Public review):

Summary:

In their manuscript, Zhao et al. describe a link between JAK-STAT pathway activation in nephrocytes upon a high-fat diet. Nephrocytes are the homologs to mammalian podocytes, and it has been previously shown that metabolic syndrome and obesity is associated with worse outcomes for chronic kidney disease. A study from 2021 (Lubojemska et al.) could already confirm a severe nephrocyte phenotype upon feeding Drosophila a high fat diet and also linking lipid overflow by expressing adipose triglyceride lipase in the fat body to nephrocyte dysfunction. In this study, the authors identified a second pathway and mechanism, how lipid dysregulation impact on nephrocyte function. In detail, they show an activation of JAK-STAT signaling in nephrocytes upon feeding a high-fat diet, which was induced by Upd2 expression (a leptin-like hormone) in the fat body, the adipose tissue in Drosophila. Further, they could show genetic and pharmacological interventions can reduce JAK-STAT activation and thereby prevent the nephrocyte phenotype in the high-fat diet model.

Strengths:

The strength of this study is the combination of genetic tools and pharmacological intervention to confirm a mechanistic link between the fat body/adipose tissue and nephrocytes. Inter-organ communication is crucial in the development of several diseases, but the underlying mechanisms are only poorly understood. Using Drosophila, it is possible to investigate several players of one pathway, here JAK-STAT. This was done, by investigating the functional role of Hop, Socs36E and Stat92E in nephrocytes and has also been combined with feeding a high-fat diet, to assess restoration of nephrocyte morphology and function by inhibiting JAK-STAT signaling. Adding a translational approach was done by inhibiting JAK-STAT signaling with methotrexate, which also resulted in attenuated nephrocyte dysfunction. Expression of the leptin-like hormone upd2 in the fat body is a good approach to study inter-organ communication and the impact of other organs/tissue on nephrocyte function and expands their findings from nephrocyte function towards whole animal physiology.

Weaknesses:

Although the general findings of this study are of great interest, the number of flies investigated for the majority of the experiments is very low (6 flies). Also it is not clear whether the 6 flies used are from independent experiments to exclude differences in food/diet.

Reviewer #2 (Public review):

Summary:

The authors combined time-lapse microscopy with biophysical modeling to study the mechanisms and timescales of gliding and reversals in filamentous cyanobacterium Fluctiforma draycotensis. They observed the highly coordinated behavior of protein complexes moving in a helical fashion on cells' surfaces and along individual filaments as well as their de-coordination, which induces buckling in long filaments.

Strengths:

The authors provided concrete experimental evidence of cellular coordination and de-coordination of motility between cells along individual filaments. The evidence is comprised of individual trajectories of filaments that glide and reverse on surfaces as well as the helical trajectories of membrane-bound protein complexes that move on individual filaments and are implicated in generating propulsive forces.

Limitations:

The biophysical model is one-dimensional and thus does not capture the buckling observed in long filaments. I expect that the buckling contains useful information since it reflects the competition between bending rigidity, the speed at which cell synchronization occurs, and the strength of the propulsion forces.

Future directions:

The study highlights the need to identify molecular and mechanical signaling pathways of cellular coordination. In analogy to the many works on the mechanisms and functions of multi-ciliary coordination, elucidating coordination in cyanobacteria may reveal a variety of dynamic strategies in different filamentous cyanobacteria.

Reviewer #2 (Public review):

Summary:

This study explores how signals from all sides of a developing limb, front/back and top/bottom, work together to guide the regrowth of a fully patterned limb in axolotls, a type of salamander known for its impressive ability to regenerate limbs. Using a model called the Accessory Limb Model (ALM), the researchers created early limb regenerates (called blastemas) with cells from different sides of the limb. They discovered that successful limb regrowth only happens when the blastema contains cells from both the top (dorsal) and bottom (ventral) of the limb. They also found that a key gene involved in front/back limb patterning, called Shh (Sonic hedgehog), is only turned on when cells from both the dorsal and ventral sides come into contact. The study identified two important molecules, Wnt10B and FGF2, that help activate Shh when dorsal and ventral cells interact. Finally, the authors propose a new model that explains how cells from all four sides of a limb, dorsal, ventral, anterior (front), and posterior (back), contribute at both the cellular and molecular level to rebuilding a properly structured limb during regeneration.

Strengths:

The techniques used in this study, like delicate surgeries, tissue grafting, and implanting tiny beads soaked with growth factors, are extremely difficult, and only a few research groups in the world can do them successfully. These methods are essential for answering important questions about how animals like axolotls regenerate limbs with the correct structure and orientation. To understand how cells from different sides of the limb communicate during regeneration, the researchers used a technique called in situ hybridization, which lets them see where specific genes are active in the developing limb. They clearly showed that the gene Shh, which helps pattern the front and back of the limb, only turns on when cells from both the top (dorsal) and bottom (ventral) sides are present and interacting. The team also took a broad, unbiased approach to figure out which signaling molecules are unique to dorsal and ventral limb cells. They tested these molecules individually and discovered which could substitute for actual dorsal and ventral cells, providing the same necessary signals for proper limb development. Overall, this study makes a major contribution to our understanding of how complex signals guide limb regeneration, showing how different regions of the limb work together at both the cellular and molecular levels to rebuild a fully patterned structure.

Weaknesses:

Because the expressional analyses are performed on thin sections of regenerating tissue, they provide only a limited view of the gene expression patterns in their experiments, opening the possibility that they could be missing some expression in other regions of the blastema. Additionally, the quantification method of the expressional phenotypes in most of the experiments does not appear to be based on a rigorous methodology. Therefore, performing alternate expressional analysis, using RNA-seq or qRT-PCR (for example) on the entire blastema would help validate that the authors are not missing something.

Overall, the number of replicates per sample group is quite low (sometimes as low as 3), which is especially risky with challenging techniques like the ones the authors employ. The authors don't appear to have performed a power analysis to calculate the number of animals used in each experiment that are sufficient to identify possible statistical differences between groups. Increasing the sample sizes would substantially increase the rigor of their experiments.

Likewise, the authors' use of an AI-generated algorithm to quantify symmetry on the dorsal/ventral axis, and this approach doesn't appear to account for possible biases due to tissue sectioning angles. They also appear to arbitrarily pick locations in each sample group to compare symmetry measurements. There are other methods, which include using specific muscle groups and nerve bundles as dorsal/ventral landmarks, that would more clearly show differences in symmetry.

Reviewer #2 (Public review):

Summary:

The work presented in the manuscript by Tran et al deals with bacterial evolution in the presence of bacteriophage. Here, authors have taken three methicillin-resistant S. aureus strains that are also resistant to beta-lactams. Eventually, upon being exposed to phage, these strains develop beta-lactam sensitivity. Besides this, the strains also show other changes in their phenotype such as reduced binding to fibrinogen and hemolysis.

Strengths:

The experiments carried out are convincing to suggest such in vitro development of sensitivity to the antibiotics. Authors were also able to "evolve" phage in similar fashion thus showing enhanced virulence against the bacterium. In the end, authors carry out DNA sequencing of both evolved bacteria and phage and show mutations occurring in various genes. Overall, the experiments that have been carried out are convincing.

Weaknesses:

None. In the current version of the manuscript, I find the study complete.

Reviewer #2 (Public review):

Summary:

The work presented in the manuscript by Tran et al deals with bacterial evolution in the presence of bacteriophage. Here, the authors have taken three methicillin-resistant S. aureus strains that are also resistant to beta-lactams. Eventually, upon being exposed to phage, these strains develop beta-lactam sensitivity. Besides this, the strains also show other changes in their phenotype such as reduced binding to fibrinogen and hemolysis.

Strengths:

The experiments carried out are convincing to suggest such in vitro development of sensitivity to the antibiotics. Authors were also able to "evolve" phage in a similar fashion thus showing enhanced virulence against the bacterium. In the end, authors carry out DNA sequencing of both evolved bacteria and phage and show mutations occurring in various genes. Overall, the experiments that have been carried out are convincing.

Weaknesses:

Although more experiments are not needed, additional experiments could add more information. For example, the phage gene showing the HTH motif could be reintroduced in the bacterial genome and such a strain can then be assayed with wildtype phage infection to see enhanced virulence as suggested. At least one such experiment proves the discoveries regarding the identification of mutations and their outcome. Secondly, I also feel that authors looked for beta-lactam sensitivity and they found it. I am sure that if they look for rifampicin resistance in these strains, they will find that too. In this case, I cannot say that the evolution was directed to beta-lactam sensitivity; this is perhaps just one trait that was observed. This is the only weakness I find in the work. Nevertheless, I find the experiments convincing enough; more experiments only add value to the work.

Reviewer #3 (Public review):

Summary

This work investigated the immune response in the murine retina after focal laser lesions. These lesions are made with close to 2 orders of magnitude lower laser power than the more prevalent choroidal neovascularization model of laser ablation. Histology and OCT together show that the laser insult is localized to the photoreceptors and spares the inner retina, the vasculature and the pigment epithelium. As early as 1-day after injury, a loss of cell bodies in the outer nuclear layer is observed. This is accompanied by strong microglial proliferation to the site of injury in the outer retina where microglia do not typically reside. The injury did not seem to result in the extravasation of neutrophils from the capillary network, constituting one of the main findings of the paper. The demonstrated paradigm of studying the immune response and potentially retinal remodeling in the future in vivo is valuable and would appeal to a broad audience in visual neuroscience.

Strengths

Adaptive optics imaging of murine retina is cutting edge and enables non-destructive visualization of fluorescently labeled cells in the milieu of retinal injury. As may be obvious, this in vivo approach is a benefit for studying fast and dynamic immune processes on a local time scale - minutes and hours, and also for the longer days-to-months follow-up of retinal remodeling as demonstrated in the article. In certain cases, the in vivo findings are corroborated with histology.

The analysis is sound and accompanied by stunning video and static imagery. A few different sets of mouse models are used: a) two different mouse lines, each with a fluorescent tag for neutrophils and microglia, b) two different models of inflammation - endotoxin-induced uveitis (EAU) and laser ablation are used to study differences in the immune interaction.

One of the major advances in this article is the development of the laser ablation model for 'mild' retinal damage as an alternative to the more severe neovascularization models. This model would potentially allow for controlling the size, depth and severity of the laser injury opening interesting avenues for future study.

The time-course, 2D and 3D spatial activation pattern of microglial activation are striking and provide an unprecedented view of the retinal response to mild injury.

Editor's note: The authors have addressed all the previous concerns raised by the reviewers.

Reviewer #2 (Public review):

This revised study is an investigation of galanin and galanin receptor signaling on whole-brain activity in the context of recurrent seizure activity or under homeostatic basal conditions. The authors primarily use calcium imaging to observe whole-brain neuronal activity accompanied by galanin qPCR to determine how manipulations of galanin or the galr1a receptor affect the activity of the whole-brain under non-ictal conditions or when seizure activity occurs. The authors use their eaat2a-/- model (introduced in their Glia 2022 paper, PMID 34716961) that shows recurrent seizure activity as well as suppression of neuronal activity and locomotion interictally. It is compared to the well-known pentylenetetrazole (PTZ) pharmacological model of seizures in zebrafish. Given the literature cited in their Introduction, the authors hypothesize that galanin will exert a net inhibitory effect on brain activity in models of seizures/epilepsy. They were surprised to find that this hypothesis was only moderately supported in their eaat2a-/- model. In contrast, after PTZ, fish with galanin overexpression showed increased seizure number and reduced duration while fish with galanin KO showed reduced seizure number and increased duration.

Previous concerns about sex or developmental biological variables were addressed, as their model's seizure phenotype emerges rapidly and long prior to the establishment of zebrafish sexual maturity. However, in the course of re-review, some additional concerns (below) were detected that, if addressed, could further improve the manuscript. These concerns relate to how seizures were defined from the measurement of fluorescent calcium imaging data. Overall, this study is important and convincing, and carries clear value for understanding the multifaceted functions that neuronal galanin can perform under homeostatic and disease conditions.

Additional Concerns:

- The authors have validated their ability to measure behavioral seizures quantitatively in their 2022 Glia paper but the information provided on defining behavioral seizures was limited. The definition of behavioral seizure activity is not expanded upon in this paper, but could provide detail about how the behavioral seizures relate to a seizure detected via calcium imaging.

- Related to the previous point, for the calcium imaging, the difference between an increase in fluorescence that the authors think reflects increased neuronal activity and the fluorescence that corresponds to seizures is not very clear. This detail is necessary because exactly when the term "seizure" describes a degree of increased activity can be difficult to distinguish objectively.

- The supplementary movies that were added were very useful, but raised some questions. For example, what brain regions were pulsating? What areas seemed to constantly exhibit strong fluorescence and was this an artifact? It seemed that sometimes there was background fluorescence in the body. Perhaps an anatomical diagram could be provided for the readers. In addition, there were some movies with much greater fluorescence changes - are these the seizures? These are some reasons for our request for clarified definitions of the term "seizure".

Reviewer #2 (Public review):

Summary:

Shengsheng Zhao et al. investigated the role of nucleolar and coiled-body phosphoprotein 1 (NOLC1) in relegating gastric cancer (GC) development and cisplatin-induced drug resistance in GC. They found a significant correlation between high NOLC1 expression and the poor prognosis of GC. Meanwhile, upregulation of NOLC1 was associated with cis-resistant GC. Experimentally, the authors demonstrate that knocking down NOLC1 increased GC sensitivity to Cis possibly by regulating ferroptosis. Mechanistically, they found NOLC1 suppressed ferroptosis by blocking the translocation of P53 from the cytoplasm to the nucleus and promoting its degradation. In addition, the authors also evaluated the effect of combinational treatment of anti-PD-1 and cisplatin in NOLC1 -knockdown tumor cells, revealing a potential role of NOLC1 in the targeted therapy for GC.

Strengths:

Chemoresistance is considered a major reason causing failure of tumor treatment and death of cancer patients. This paper explored the role of NOLC1 in the regulation of Cis-mediated resistance, which involves a regulated cell death named ferroptosis. These findings provide more evidence highlighting the study of regulated cell death to overcome drug resistance in cancer treatment, which could give us more potential strategies or targets for combating cancer.

Weaknesses:

More evidence supporting the regulation of ferroptosis induced by Cisplatin by NOLC1 should be added. Particularly, the role of ferroptosis in the cisplatin-resistance should be verified and whether NOLC1 regulates ferroptosis induced by additional FINs should be explored. Besides, the experiments to verify the regulation of ferroptosis sensitivity by NOLC1 are sort of superficial. The role of MDM2/p53 in ferroptosis or cisplatin resistance mediated by NOLC1 should be further studied by genetic manipulation of p53, which is the key evidence to confirm its contribution to NOLC1 regulation of GC and relative cell death.

Reviewer #2 (Public review):

Summary:

In this study, the authors identified and characterized a regulatory mechanism based on transcriptional anti-termination that connects the two gene clusters, capsid and run-off replication (ROR) locus, of the bipartite Bartonella gene transfer agent (GTA). Among genes essential for GTA functionality identified in a previous transposon sequencing project, they found a potential antiterminatior of phage origin within the ROR locus. They employed fluorescence reporter and gene transfer assays of overexpression and knockout strains in combination with ChiPSeq and promoter-fusions to convincingly show that this protein indeed acts as an antiterminator counteracting attenuation of the capsid gene cluster expression.

Impact on the field:

The results provide valuable insights into the evolution of the chimeric BaGTA, a unique example of phage co-domestication by bacteria. A similar system found in the other broadly studied Rhodobacterales/Caulobacterales GTA family suggests that antitermination could be a general mechanism for GTA control.

Strengths:

Results of the selected and carefully designed experiments support the main conclusions.

Weaknesses:

The question why overexpression of the antiterminator does not increase the gene tranfer frequency needs to be answered in further studies.

Comments on revisions:

The authors further improved the already strong manuscript. All my concerns have been addressed. The addition of a summry figure helps to understand the proposed mechanism.

Reviewer #2 (Public review):

First, I would like to thank the authors for the response. I acknowledge that the authors show in previous studies that Rab3A acts from the presynaptic side at the NMJ, and that is, as the authors indicate, their impetus for the current study. However, mechanisms observed at a completely different type of synapses cannot be used as an argument for conclusions here. The authors also acknowledge that they should restrict their conclusions to the data in the current study, and they are merely proposing interpretations. Then perhaps they should restrict these interpretations to the discussion rather than make this claim in the abstract (lines 44-47). Here the authors ask whether Rab3A is involved in the homeostatic increase of postsynaptic AMPARs, am I understanding it correctly that their conclusion for this question is "increase in AMPAR levels in WT cultures is more variable than those in mEPSCs so that it is impossible to determine if Rab3A is involved at all"? If so, then this question has not been answered and should not be regarded as one of the main conclusions with the data presented here. It also remains unclear to me how this piece of inconclusive data serves the main objective of the study.

The authors state at the end that the current study is just an extension of their previous work, and therefore their interpretations here further support the idea that Rab3A is acting presynaptically. I would argue that it is the conclusive data, rather than interpretations that lack concrete evidence, that support ideas and models. I think that we would all agree that immunostaining measurements can be very variable. However, if the authors are determined to use this approach to answer one of their major questions, then perhaps one way to significantly strengthen their conclusions is to find ways to somewhat overcome this technical limitation.

Finally, I thank the authors for addressing other minor concerns of mine.

Reviewer #2 (Public review):

Summary:

The manuscript employs serial block‐face electron microscopy (SBEM) and cryofixation to obtain high‐resolution, three‐dimensional reconstructions of Drosophila antennal sensilla containing olfactory receptor neurons (ORNs) that detect CO2. This method has been used previously by the same lab in Gonzales et. al, 2021. (https://elifesciences.org/articles/69896), which had provided an exemplary model by integrating high-resolution EM with electrophysiology and cell-type-specific labeling. The previous study ended up correlating morphology with activity for multiple olfactory sensillar types. Compared to the 2021 study, this current manuscript appears somewhat incomplete and lacks integration with activity.

In fact older studies have also reported two-dimensional TEM images of the putative CO2 neuron in Drosophila (Shanbhag et al., 1999) and in mosquitoes (McIver and Siemicki, 1975; Lu et al, 2007), and in these instances reported that the dendritic architecture of the CO2 neuron was somewhat different (circular and flattened, lamellated) from other olfactory neurons.

The authors claim that this approach offers an artifact‐minimized ultrastructural dataset compared to earlier. In this study, not only do they confirm this different morphology but also classify it into distinct subtypes (loosely curled, fully curled, split, and mixed). This detailed morphological categorization was not provided in prior studies (e.g., Shanbhag et al., 1999 ). The authors would benefit from providing quantitative thresholds or objective metrics to improve reproducibility and to clarify whether these structural distinctions correlate with distinct functional roles.

Strengths:

The study makes a convincing case that ab1C neurons exhibit a unique, flattened dendritic morphology unlike the cylindrical dendrites found in ab1D neurons. This observation extends previous qualitative TEM findings by not only confirming the presence of flattened lamellae in CO₂ neurons but also quantifying key morphometrics such as dendritic length, surface area, and volume, and calculating surface area-to-volume ratios. The enhanced ratios observed in the flattened segments are speculated to be linked to potential advantages in receptor distribution (e.g., Gr21a/Gr63a) and efficient signal propagation.

Weaknesses:

While the manuscript offers valuable ultrastructural insights and reveals previously unappreciated heterogeneity among CO₂-sensing neurons, several issues warrant further investigation in addition to the points made above.

(1) Although this quantitative approach is robust compared to earlier descriptive reports, its impact is somewhat limited by the absence of direct electrophysiological data to confirm that ultrastructural differences translate into altered neuronal function. A direct comparison or discussion of how the present findings align with the functional data obtained from electrophysiology would strengthen the overall argument.

(2) Clarifying the criteria for dendritic subtype classification with quantitative parameters would enhance reproducibility and interpretability. Moreover, incorporating electrophysiological recordings from ab1C neurons would provide compelling evidence linking structure and function, and mapping key receptor proteins through immunolabeling could directly correlate receptor distribution with the observed morphological diversity.

(3) Even though Cryofixation is claimed to be superior to chemical fixation for generating fewer artifacts, authors need to confirm independently the variation observed in the CO2 neuron morphologies across populations. All types of fixation in TEMs cause some artifacts, as does serial sectioning. Without understanding the error rates or without independent validation with another method, it is hard to have confidence in the conclusions drawn by the authors of the paper.

Addressing these concerns and integrating additional experiments would significantly bolster the manuscript's completeness and advancement.

Reviewer #2 (Public review):

Summary:

The author of this manuscript aimed to uncover the mechanisms behind miRNA retention within cells. They identified PCBP2 as a crucial factor in this process, revealing a novel role for RNA-binding proteins. Additionally, the study discovered that SYNCRIP is essential for PCBP2's function, demonstrating the cooperative interaction between these two proteins. This research not only sheds light on the intricate dynamics of miRNA retention but also emphasizes the importance of protein interactions in regulating miRNA behavior within cells.

Strengths:

This paper makes important progress in understanding how miRNAs are kept inside cells. It identifies PCBP2 as a key player in this process, showing a new role for proteins that bind RNA. The study also finds that SYNCRIP is needed for PCBP2 to work, highlighting how these proteins work together. These discoveries not only improve our knowledge of miRNA behavior but also suggest new ways to develop treatments by controlling miRNA locations to influence cell communication in diseases. The use of liver cell models and thorough experiments ensures the results are reliable and show their potential for RNA-based therapies.

Reviewer #2 (Public review):

Summary:

The authors performed a genetic screen using deficiency lines and identified Uev1a as a factor that protects nurse cells from RasG12V-induced cell death. According to a previous study from the same lab, this cell death is caused by aberrant mitotic stress due to CycA upregulation (Zhang et al.). This paper further reveals that Uev1a forms a complex with APC/C to promote proteasome-mediated degradation of CycA.

In addition to polyploid nurse cells, the authors also examined the effect of RasG12V-overexpression in diploid germline cells, where RasG12V-overexpression triggers active proliferation, not cell death. Uev1a was found to suppress its overgrowth as well.

Finally, the authors show that the overexpression of the human homologs, UBE2V1 and UBE2V2, suppresses tumor growth in human colorectal cancer xenografts and cell lines. Notably, the expression of these genes correlates with the survival of colorectal cancer patients carrying the Ras mutation.

Strength:

This paper presents a significant finding that UBE2V1/2 may serve as a potential therapy for cancers harboring Ras mutations. The authors propose a fascinating mechanism in which Uev1a forms a complex with APC/C to inhibit aberrant cell cycle progression.

Weakness:

The quantification of some crucial experiments lacks sufficient clarity.

Reviewer #2 (Public review):

Summary:

In this paper, Drs. Kercmar, Murko, and Bombek make a series of observations related to the role of AVP in pancreatic islets. They use the pancreatic slice preparation that their group is well known for. The observations on the slide physiology are technically impressive. However, I am not convinced by the conclusions of this manuscript for a number of reasons. At the core of my concern is perhaps that this manuscript appears to be motivated to resolve 'controversies' surrounding the actions of AVP on insulin and glucagon secretion. This manuscript adds more observations, but these do not move the field forward in improving or solidifying our mechanistic understanding of AVP actions on islets. A major claim in this manuscript is the beta cell expression of the V1b Receptor for AVP, but the evidence presented in this paper falls short of supporting this claim. Observations on the activation of calcium in alpha cells via V1b receptor align with prior observations of this effect.

I have focused my main concerns below. I hope the authors will consider these suggestions carefully - please be assured that they were made with the intent to support the authors and increase the impact of this work.

Strengths:

The main strength of this paper is the technical sophistication of the approach and the analysis and representation of the calcium traces from alpha and beta cells.

Weaknesses:

(1) The introduction is long and summarizes a substantive body of literature on AVP actions on insulin secretion in vivo. There are a number of possible explanations for these observations that do not directly target islet cells. If the goal is to resolve the mechanistic basis of AVP action on alpha and beta cells, the more limited number of papers that describe direct islet effects is more helpful. There are excellent data that indicate that the actions of AVP are mediated via V1bR on alpha cells and that V1bR is a) not expressed by beta cells and b) does not activate beta cell calcium at all at 10 nM - which is the same concentration used in this paper (Figure 4G) for peak alpha cell Ca2+ activation (see https://doi.org/10.1016/j.cmet.2017.03.017; cited as ref 30 in the current manuscript).

(2) We know from bulk RNAseq data on purified alpha, beta, and delta cells from both the Huising and Gribble groups that there is no expression of V2a. I will point you to the data from the Huising lab website published almost a decade ago (http://dx.doi.org/10.1016/j.molmet.2016.04.007) - which is publicly available and can be used to generate figures (https://huisinglab.com/data-ghrelin-ucsc/index.html). They indicate the absence of expression of not only AVP2 receptors anywhere in the islet, but also the lack of expression of V1bra, V1brb, and Oxtr in beta cells. Instead of the detailed list of expression of these 4 receptors elsewhere in the body, it would be more directly relevant to set up their pancreatic slice experiments to summarize the known expression in pancreatic islets that is publicly available. It would also have helped ground the efforts that involved the generation of the V1aR agonist and V2R antagonist, which confirm these known AVP/OXT receptor expression patterns.

(3) Importantly, the lack of V1br from beta cells does not invalidate observations that AVP affects calcium in beta cells, but it does indicate that these effects are mediated a) indirectly, downstream of alpha cell V1br or b) via an unknown off-target mechanism (less likely). The different peak efficacies in Figure 4G would also suggest that they are not mediated by the same receptor.

(4) The rationale for the use of forskolin across almost all traces is unclear. It is motivated by a desire to 'study the AVP dependence of both alpha and beta cells at the same time'. As best as I can determine, the design choice to conduct all studies under sustained forskolin stimulation is related to the permissive actions of AVP on hormone secretion in response to cAMP-generating stimuli. The permissive actions by AVP that are cited are on hormone secretion, which in many cell types requires activation of both calcium and cAMP signaling. Whether the activation of V1br and subsequent calcium response is permitted by cAMP is unclear. I believe the argument the authors are making here is that the activation of beta cell calcium by AVP is permitted by forskolin. i.e., the cAMP stimulated by it in beta cells. However, the design does not account for the elevation of cAMP in alpha cells and subsequent release of glucagon, particularly upon co-stimulation with AVP, which permits glucagon release by activating a calcium response in alpha cells. This glucagon could then activate beta cells. If resolving the mechanism of action is the goal, often less is more. The activation of Gaq-mediated calcium is not cAMP dependent (although the downstream hormone secretion clearly often is). As was shown, AVP does not activate calcium in beta cells in the absence of cAMP. The experiments in Figures 1, 2, and 4 should have been completed in the absence of cAMP first.

(5) It is unexpected that epinephrine in Figure 2 does not activate the alpha cell calcium? A recent paper from the same group (Sluga et al) shows robust calcium activation in alpha cells in a similar prep by 1 nM epinephrine, which is similar to the dose used here.

(6) Figure 8 suggests a pharmacological activation of beta cell V1bR in the low pM range. How do the authors reconcile this comparison with the apparent absence of an effect of AVP stimulation at low pM to low nM doses in beta cells (Figure 4A)? I note that there are changes over time with sustained beta cell stimulation with 8 mM glucose, but these changes are relatively subtle, gradual, and quite likely represent the progression of calcium behaviors that would have occurred under sustained glucose, irrespective of these very low AVP concentrations. I will note that the Kd of the V1bR for AVP is around 1 nM, with tracer displacement starting around 100 pM according to the data in figure 5B, which is hard to reconcile with changes in beta cell calcium by AVP doses that start 10-100-fold lower than this dose at 1 and 10 pM (Figure 8).

Reviewer #2 (Public review):

Summary:

Mohanty et al. present a new deep learning method to identify intracellular allosteric modulators of GPCRs. This is an interesting field for e.g. the design of novel small molecule inhibitors of GPCR signalling. A key limitation, as mentioned by the authors, is the limited availability of data. The method presented, Gcoupler, aims to overcome these limitations, as shown by experimental validation of sterols in the inhibition of Ste2p, which has been shown to be relevant molecules in human and rat cardiac hypertrophy models.<br /> They have made their code available for download and installation, which can easily be followed to set up software on a local machine.

Strengths:

- Clear GitHub repository

- Extensive data on yeast systems

Weaknesses:

- No assay to directly determine the affinity of the compounds to the protein of interest.

In conclusion, the authors present an interesting new method to identify allosteric inhibitors of GPCRs, which can easily be employed by research labs. Whilst their efforts to characterize the compounds in yeast cells, in order to confirm their findings, it would be beneficial if the authors show their compounds are active in a simple binding assay.

Reviewer #2 (Public review):

Tittelmeier et al. investigated the role of sphingolipid (SL) metabolism in the maintenance of endolysosomal vesicle integrity. They find that both impaired SL biosynthesis and degradation in C. elegans, decrease the fluidity of endolysosomal membranes and promote their rupture, while it has little effect on plasma membrane fluidity. Endolysosomal membrane fluidity is also negatively affected in human cells upon knockdown (KD) of a gene (SPHK2) involved in the SL degradation pathway. Aggregated forms of tau in both models (C. elegans and human cells) can also cause rigidification of the endolysosomal membrane, with SL homeostasis disruption having an additive effect, exacerbating endolysosomal rupture. Notably, KD of SPHK2 also increased the formation of tau foci, suggesting that compromised endolysosomal integrity may promote tau aggregation. These data provide a clearer understanding of how genetic manipulation of SL metabolism affects endolysosomal membranes and their rigidification in the context of tau aggregation. Supplementation of polyunsaturated fatty acids (PUFAs), which has a beneficial effect on Alzheimer's patients, improved membrane fluidity and reduced tau propagation in human cells and tau-associated neurotoxicity in C. elegans, suggesting a possible mechanism of action.

Overall, the conclusions of this paper are supported by the data, with a few aspects requiring further clarification and elaboration.

(1) A reference to Figure S2E-G, which shows that KD of SL biosynthesis genes do not affect the plasma membrane, is missing from the main text.

(2) In Figure 3C, lipofectamine alone shows that it increases membrane rigidity (increased GP values), not membrane fluidity.

(3) In Figure 3F, the EV cntl condition expressing F3:mCh tau should have increased LGALS3 foci compared to the mCh EV cntl according to Ref (20) and its Figure 2G (at least for Day 5 animals), which would be indicative of the tau spreading in hypodermal tissue. What C. elegans age was examined in Figure 3F? Can the authors provide evidence of the transmission of the F3:mCh tau from the touch receptor neurons to the hypodermis in the EV [similar to Figure 2C & D from Ref (20)] and compare it to the KDs? Otherwise, it seems that KD of SL genes impacts not only endolysosomal rupture but significantly affects tau accumulation/spreading as well (e.g., shown later in HEK cells, where SPHK2 KD increases the formation of tau-Venus foci).

(4) Sphingolipids are essential membrane components and signaling molecules. Does KD of SL genes in C. elegans and the subsequent endolysosomal rupture cause any major, intermediate, or minor defects/phenotypes (in non-aggregation prone models, w/t..)?

Reviewer #2 (Public review):

The manuscript entitled "Structure of an oxygen-induced tubular nanocompartment in Pyrococcus furiosus" by Wenfei Song et al. employs whole-cell mass spectrometry and cryo-EM (including tomography, helical reconstruction, and single-particle analysis) to investigate the structure and function of the oxidoreductase Rubrerythrin (Rbr) from Pyrococcus furiosus. The study reports that under oxidative stress, Rbr forms a tubular structure, in contrast to its behaviour under anaerobic conditions. Authors characterized oxidoreductase Rubrerythrin (Rbr) from Pyrococcus furiosus under anaerobic conditions and formed a tubular structure when induced with oxidative stress. This study is well-designed. However, I have several questions related to the experimental design and the results obtained from those experiments, which are listed below.

(1) The authors have mentioned that "Under aerobic conditions, Rbr levels are 3 to 13 times higher compared to anaerobic conditions (Figures 1a-d)." Also, they performed whole-cell mass spec to measure the overexpression of the Rbr enzyme under anaerobic conditions. Thus, from the above statement, I consider the authors' claim that P. furiosus cells were cultured under anaerobic conditions and then exposed to oxidative stress. While cell growth under anaerobic conditions appears perfectly fine, the authors conducted the rest of the experiment under aerobic conditions during mass spectrometry and cryo-EM sample preparation. As a baseline, the author first grew the cells in their preferred anaerobic environment and also imaged the same cells that were exposed to air (aerobic) after anaerobic growth. The cell growth in anaerobic conditions is perfectly fine. But how did authors make sure that during anaerobic conditions, the Rbr enzyme is not expressed or not formed? As a control experiment, authors should demonstrate that during mass spec and cryo-EM sample preparations, cells are not exposed to air or maintained in an anaerobic environment. From anaerobic conditions, whenever cells were selected for spec and cryo-EM, cells were exposed to O2, and definitely controlled cells were not in anaerobic conditions anymore.

The authors collected P. furiosus wild-type or Rbr knockout cells in an anaerobic hood, but after that, they centrifuged the cells and plunged them using a Vitrobot. Are the instrument, centrifuge, and Vitrobot kept in an anaerobic environment? Recently, a few studies (anaerobic plunge-freezing in cryo-electron microscopy, Cook et al. (2024), Hands-Portman and Bakker (2022) DOI: 10.1039/D2FD00060A ) have mentioned the anaerobic plunge freeze setup for protein sample or cell freezing. I guess the authors did not use that setup. In these circumstances, the cell is already exposed to O2 during centrifugation and Vitrobot freezing. How were the control experiments properly performed in anaerobic conditions? A similar argument is true for Lamella grid preparation, where the enzyme was already exposed to O2, and single-particle grid preparation, where the purified enzyme is already exposed to O2. How were the control experiments properly performed in anaerobic conditions?

(2) It is important to provide evidence that the overexpressed protein is actually in an anaerobic condition and is later induced with more O2. Also, authors should confirm biochemically that the overexpressed protein in their desired protein "oxidoreductase Rubrerythrin (Rbr)". No biochemical data were provided in this manuscript. During single-particle analysis, the authors had to purify the protein sample and confirm that these were their desired protein samples. No biochemical or biophysical experiments were performed to confirm that the overexpressed protein is the desired protein.

(3) Figure 3, the atomic model looks different in all four tetramers. However, I have fitted the atomic model into the cryo-EM map, which looks reasonable. However, it will be easier for the reader to evaluate the model if the authors show different orientations of the atomic model, as well as if the authors could show that the atomic model fits the cryo-EM map.

(4) How did the authors select initial particle sets like 24 lakhs when forming helices and not forming isolated particles?

(5) The authors proposed a model for electron transfer upon oxidative stress. However, the data is not convincing that VLP is surrounded by Rbr and forms a tube-like structure. Generally, VLP is a sphere-like structure, and Rbr can form a tube-like structure when it interacts with spherical VLP. Rbr will surround VLP, and it will form a Rbr-decorated sphere-like structure.

(6) It will also be important to comment on the diameter of Oxidative stress-induced tubules (OSITs) and 3D reconstruction and/or helical reconstruction of purified protein samples. The spherical cyan densities within the tube are not very clear. If VLP is surrounded by Rbr (Figure 4), extra Rbr densities will be observed on VLP in the tomogram (in Figure 1). However, in the tomogram, VLP is inside Oxidative stress-induced tubules (OSITs). Figure 1 is a contradicting Figure 4. The authors should explain it properly.

(7) The authors performed helical reconstruction. Where is the Layer line calculation in helical reconstruction, and how do authors identify helical parameters for reconstruction?

(8) The authors used an extremely confusing methodology, which was very difficult to follow. The authors performed tomography, helical reconstruction, and single-particle analysis. Why did the authors need 3 different image processing methods to resolve structures that are not clear to me? The authors should also show the proper fitting between the map and the model. In Supplemental Figure 6c, the overall fitting of the subdomain looks ok. However, many peptide chains and side chains are not fitted properly in the EM density map. It will be helpful to show proper side chain fitting. In Supplementary Fig. 6a, the authors binned the data (Bin 8 or Bin 2) but did not mention when they unbinned the data for data processing. Also, the authors implemented C2 symmetry during local refinement. Why do authors suddenly use C2 symmetry expansion?

Minor Comments:

(1) The authors should properly show a schematic diagram of the enzyme subdomains. It will help to understand interactions or tetrameric assembly.

(2) The introduction is poorly written. It will really be helpful for the reader if the authors provide a proper introduction.

(3) The atomic model did not fit into the cryo-EM, so it was hard to determine the overall fitting.

(4) 17.1A pixel size? It's surprising.

(5) It will be better to calculate local resolution and show the map's angular distribution. It is obvious that resolution at the peripheral region will be poorer than core region. Therefore, it will be better to calculate local resolution. Additionally, authors should show the map to model fitting.

Reviewer #2 (Public review):

Summary:

This manuscript addresses the gap in knowledge related to the cardiac function of the S-denitrosylase SNO-CoA Reductase 2 (SCoR2; product of the Akr1a1 gene). Genetic variants in SCoR2 have been linked to cardiovascular disease, yet their exact role in the heart remains unclear. This paper demonstrates that mice deficient in SCoR2 show significant protection in a myocardial infarction (MI) model. SCoR2 also affected ketolytic energy production, antioxidant levels, and polyol balance through the S-nitrosylation of crucial metabolic regulators.

Strengths:

(1) Addresses a well-defined gap in knowledge related to the cardiac role of SNO-CoA Reductase 2. Besides the in-depth case for this specific player, the manuscript sheds more light on the links between S-nitrosylation and metabolic reprogramming in the heart.

(2) Rigorous proof of requirement through the combination of gene knockout and in vivo myocardial ischemia/reperfusion.

(3) Identification of precise Cys residue for SNO-modification of BDH1 as SCoR2 target in cardiac ketolysis

Weaknesses:

(1) The experiments with BDH1 stability were performed in mutant 293 cells. Was there a difference in BDH1 stability in myocardial tissue or primary cardiomyocytes from SCoR2-null vs -WT mice? The same question extends to PKM2.

(2) In the absence of tracing experiments, the cross-sectional changes in ketolysis, glycolysis, or polyol intermediates presented in Figures 4 and 5 are suggestive at best. This needs to be stressed while describing and interpreting these results.

(3) The findings from human samples with ischemic and non-ischemic cardiomyopathy do not seem immediately or linearly in line with each other and with the model proposed from the KO mice. While the correlation holds up in the non-ischemic cardiomyopathy (increased SNO-BDH1, SNO-PKM2 with decreased SCoR2 expression), how do the authors explain the decreased SNO-BDH1 with preserved SCoR2 expression in ischemic cardiomyopathy? This seems counterintuitive as activation of ketolysis is a quite established myocardial response to ischemic stress. It may help the overall message clarity to focus the human data part on only NICM patients.

(4) This issue is partially linked to point #(3). Currently, important evidence that is lacking is the demonstration of sufficiency for SCoR2 in S-nytrosylation of targets and cardiac remodeling. Does SCoR2 overexpression in the heart or isolated cardiomyocytes reduce S-nitrosylation of BDH1 and other targets, thus affecting heart function at baseline or under stress?

DOI: 10.1038/s41598-025-03429-2

Resource: (RRID:CVCL_AX39)

Curator: @dhovakimyan1

SciCrunch record: RRID:CVCL_AX39

What is this?

Reviewer #2 (Public review):

This is an excellent paper. The ability to measure the immune response to multiple viruses in parallel is a major advancement for the field, which will be relevant across pathogens (assuming the assay can be appropriately adapted). I only have a few comments, focused on maximising the information provided by the sera.

Firstly, one of the major findings is that there is wide heterogeneity in responses across individuals. However, we could expect that individuals' responses should be at least correlated across the viruses considered, especially when individuals are of a similar age. It would be interesting to quantify the correlation in responses as a function of the difference in ages between pairs of individuals. I am also left wondering what the potential drivers of the differences in responses are, with age being presumably key. It would be interesting to explore individual factors associated with responses to specific viruses (beyond simply comparing adults versus children).

Relatedly, is the phylogenetic distance between pairs of viruses associated with similarity in responses?

Figure 5C is also a really interesting result. To be able to predict growth rates based on titers in the sera is fascinating. As touched upon in the discussion, I suspect it is really dependent on the representativeness of the sera of the population (so, e.g., if only elderly individuals provided sera, it would be a different result than if only children provided samples). It may be interesting to compare different hypotheses - so e.g., see if a population-weighted titer is even better correlated with fitness - so the contribution from each individual's titer is linked to a number of individuals of that age in the population. Alternatively, maybe only the titers in younger individuals are most relevant to fitness, etc.

In Figure 6, the authors lump together individuals within 10-year age categories - however, this is potentially throwing away the nuances of what is happening at individual ages, especially for the children, where the measured viruses cross different groups. I realise the numbers are small and the viruses only come from a small numbers of years, however, it may be preferable to order all the individuals by age (y-axis) and the viral responses in ascending order (x-axis) and plot the response as a heatmap. As currently plotted, it is difficult to compare across panels

Reviewer #2 (Public review):

Uphoff and colleagues present the results of a study focused on characterizing the binding of SVEP1 to TIE1 along with Angiopoietin-2. Starting with computational prediction of SVEP1 binding to TIE1, the authors identify the region of SVEP1 that serves as a high-affinity ligand for TIE1. Advanced studies identify a weak secondary binding site within SVEP1 that appears to be sufficient but not necessary for its interaction with TIE1 based on in vivo rescue experiments. The most novel contribution of the manuscript seems to be the identification of angiopoietin-1 and -2 as co-factors that seem to enhance the binding of SVEP1 with TIE1 and impact downstream AKT signaling. They propose a complex in which SVEP1 binds to TIE1 and ANG2.

Although the first set of results is essentially confirmatory, the identification of ANG-2 as a "co-factor" enhancing the binding of SVEP1 to TIE1 and associated downstream signaling (i.e., Figures 3 and 4) is novel and is of interest. However, the manuscript and its conclusions would greatly benefit from some clarifying details and additional experiments to ensure rigor and support specific claims.

Reviewer #2 (Public review):

In the present study the authors have investigated the effects of mutations on Rep protein ability to package DNA within the gene therapy vector, AAV. A detailed investigation of Rep mutants selected from a library has been probed for their ability to produce active virions. While the concept is interesting the outcome effects are very limited.

The major issue is the lack of immediate applicability and relevance in the vector production pipeline for AAV. The authors have found that with the synthetic GFP transgene cargo, mutations of the p19 promoter did not lead to enhanced AAV vector packing. Thus the data is quite preliminary and a complete characterization may be necessary to further enhance the translational potential of the approach.

Reviewer #2 (Public review):

Summary:

This manuscript studies the prey capture by archer fish, which observe the initial values of motion of aerial prey they made fall by spitting on them, and then rapidly turn to reach the ballistic landing point on the water surface. The question raised by the article is whether this incredibly fast decision-making process is hardwired and thus unmodifiable or can be adjusted by experience to follow a new rule, namely that the landing point is deflected from a certain amount from the expected ballistic landing point. The results show that the fish learn the new rule and use it afterwards in a variety of novel situations that include height, side and speed of the prey, and which preserve the speed of the fish's decision. Moreover, a remarkable finding presented in this work is the fact that fish that have learned to use the new rule can relearn to use the ballistic landing point for an object based on its shape (a triangle) while keeping simultaneously the 'deflected rule' for an object differing in shape (a disc); in other words, fish can master simultaneously two decision-making rules based on the different shape of objects.

Strengths:

The manuscript relies on a sophisticated and clever experimental design that allows changing the apparent landing point of a virtual prey using a virtual reality system. Several robust controls are provided to demonstrate the reliability and usefulness of the experimental setup.

Overall, I like very much the idea conveyed by the authors that even stimuli triggering apparently hardwired responses can be relearned in order to be associated to a different response, thus showing the impressive flexibility of circuits that are sometimes considered as mediating pure reflexive responses. This is the case - as an additional example - of the main component of the Nasanov pheromone of bees (geraniol), which triggers immediate reflexive attraction and appetitive responses, and which can, nevertheless, be learned by bees in association with an electric shock so that bees end up exhibiting avoidance and the aversive response of sting extension to this odorant(1), which is a fully unnatural situation, and which shows that associative aversive learning is strong enough to override preprogrammed responding, thus reflecting an impressive behavioral flexibility.

Weaknesses:

As a general remark, there is some information that I missed and that are mandatory in the analysis of behavioral changes: one is the variability in the performances displayed. The authors mentioned that the results reported come from 6 fish (which is a low sample size). How were the individual performances in terms of consistency? Were all fish equally good in adjusting/learning the new rule? How did errors vary according to individual identity? It seems to me that this kind of information should be available as the authors reported that individual fish could be recognized and tracked (see lines 620-635) and is essential for appreciating the flexibility of the system under study.

The other information that I could not find explained in a proper way is referred to the speed of the learning process. Admittedly, fish learn in an impressive way the new rule and even two rules simultaneously; yet, how long did they need to achieve this? In the article, Fig 2 mention that at least 6 training stages (each defined as a block of 60 evaluated turn decisions, which actually shows that the standard term 'Training Block' would be more appropriate) were required for the fish to learn the 'deflected rule'. While this means 360 trials (turning starts), I was left with the question of how long did this process last? How many hours, days, weeks were needed for the fish to learn? And as mentioned above, were al fish equally fast in learning? I would appreciate explaining this very important point because learning dynamics is relevant to understanding the flexibility of the system.

Comments After Revision:

There was consensus among reviewers that the authors addressed the initial critiques adequately and that the manuscript improved accordingly. The revision clarified several methodological aspects, and the addition of the new Fig. 2 was particularly helpful. It elucidates the experimental approach used in the study and offers essential context for understanding points that may have been unclear in the previous version.

Reviewer #2 (Public review):

This study advances our understanding of information encoding in the DLPFC and PMD brain regions. The conclusions are supported with convincing and robust analyses conducted on monkey datasets and trained RNN models. However, there are some concerns regarding the interpretation of findings related to the information bottleneck theory and the mapping of brain areas in the RNN simulations.

The authors' justification regarding mapping between model areas and anatomical areas remains insufficient, in my opinion. However, I recognize that my initial critique may not have been fully clear. The issue I see is this: whichever area is mapped to the first RNN module will trivially exhibit stimulus information, and downstream regions will naturally show a gradual loss of that information if one simply reads out their responses.

Thus, the observed stimulus loss in later modules could be an inevitable consequence of the model's structure, rather than a meaningful analog to the PFC-PMd transition. This point requires more careful justification or a reevaluation of the proposed mapping.

Reviewer #2 (Public review):

Summary:

The authors have previously shown that the mouse neonatal cerebellum can regenerate damage to granule cell progenitors in the external granular layer, through reprogramming of gliogenic nestin-expressing progenitors (NEPs). The mechanisms of this reprogramming remain largely unknown. Here the authors used scRNAseq and ATACseq of purified neonatal NEPs from P1-P5 and showed that ROS signatures were transiently upregulated in gliogenic NEPs ve neurogenic NEPs 24 hours post injury (P2). To assess the role of ROS, mice transgenic for global catalase activity were assessed to reduce ROS. Inhibition of ROS significantly decreased gliogenic NEP reprogramming and diminished cerebellar growth post-injury. Further, inhibition of microglia across this same time period prevented one of the first steps of repair - the migration of NEPs into the external granule layer. This work is the first demonstration that the tissue microenvironment of the damaged neonatal cerebellum is a major regulator of neonatal cerebellar regeneration. Increased ROS is seen in other CNS damage models, including adults, thus there may be some shared mechanisms across age and regions, although interestingly neonatal cerebellar astrocytes do not upregulate GFAP as seen in adult CNS damage models. Another intriguing finding is that global inhibition of ROS did not alter normal cerebellar development.

Strengths:

This paper presents a beautiful example of using single cell data to generate biologically relevant, testable hypotheses of mechanisms driving important biological processes. The scRNAseq and ATACseq analyses are rigorously conducted and conclusive. Data is very clearly presented and easily interpreted supporting the hypothesis next tested by reduce ROS in irradiated brains.

Analysis of whole tissue and FAC sorted NEPS in transgenic mice where human catalase was globally expressed in mitochondria were rigorously controlled and conclusively show that ROS upregulation was indeed decreased post injury and very clearly the regenerative response was inhibited. The authors are to be commended on the very careful analyses which are very well presented and again, easy to follow with all appropriate data shown to support their conclusions.

Weaknesses:

The authors also present data to show that microglia are required for an early step of mobilizing gliogenic NEPs into the damaged EGL. While the data that PLX5622 administration from P0-P5 or even P0-P8 clearly shows that there is an immediate reduction of NEPs mobilized to the damaged EGL, there is no subsequent reduction of cerebellar growth such that by P30, the treated and untreated irradiated cerebella are equivalent in size. There is speculation in the discussion about why this might be the case. Additional experiments and tools are required to assess mechanisms. Regardless, the data still implicate microglia in the neonatal regenerative response, and this finding remains an important advance.

Reviewer #3 (Public review):

Summary:

In the present work, Zhang et al investigate the involvement of the bacterial DNA damage repair SOS response in the evolution of beta-lactam drug resistance in Escherichia coli. Using a combination of microbiological, bacterial genetics, laboratory evolution, next-generation, and live-cell imaging approaches, the authors propose short-term (transient) drug resistance evolution can take place in RecA-deficient cells in an SOS response-independent manner. They propose the evolvability of drug resistance is alternatively driven by the oxidative stress imposed by accumulation of reactive oxygen species and compromised DNA repair. Overall, this is a nice study that addresses a growing and fundamental global health challenge (antimicrobial resistance).

Strengths:

The authors introduce new concepts to antimicrobial resistance evolution mechanisms. They show short-term exposure to beta-lactams can induce durably fixed antimicrobial resistance mutations. They propose this is due to compromised DNA repair and oxidative stress. Antibiotic resistance evolution under transient stress is poorly studied, so the authors' work is a nice mechanistic contribution to this field.

Weaknesses:

The authors revisions have significantly addressed weaknesses previously identified earlier in the review process.

Reviewer #2 (Public review):

Hypothalamic neural circuits that control body weight develop during the lactation period in rodents. Exposure to maternal high-fat diet during this period (MHFD-L) program has lasting effects on their neuroanatomical organization and function. Microglia sense environmental signals and can sculpt developing circuits by promoting or pruning synaptic connections. Here, the authors examine the contribution of microglia to the effects of MHFD-L to reduce projections from AgRP neurons in the ARH to the PVH, a critical node in circuits regulating energy balance. Using detailed histomorphometric analyses of Iba-1+ cells in the three brain regions (ARH, PVH, and BNST) at two time points (P16 and P30), the authors show that microglial volume and complexity increase, while cell numbers decrease across this period. Exposure to MHFD-L is associated with a transient increase in microglial complexity/volume at P16 in the PVH but not in the other brain regions or time points assessed. Depleting microglia using a pharmacological approach reversed effects of MHD-L on AgRP outgrowth and body weight.

Strengths:

(1) The Introduction is well-written and provides a good overview of what is known about the roles of microglia in sculpting developing circuits in the hippocampus and cortex. This provides a strong rationale for the current investigations in the hypothalamus.

(2) High-quality imaging and detailed 3-D reconstructions of Iba-1 staining in microglia are used to perform unbiased analyses of microglial complexity and to quantify the spatial relationship between microglial processes and AgRP terminals.

Weaknesses:

(1) The central claim of the manuscript is that microglia in the PVH sculpt the density of AgRP inputs to the PVH in a temporally and spatially restricted manner. While the findings of the microglial ablation experiment are consistent with this hypothesis, they do not prove causality, since their manipulations were not limited to the PVH. Further studies are needed to exclude the possibility that increased outgrowth from AgRP neurons results from direct actions in the ARH or indirect consequences of changes in growth rates.

(2) Impacts of microglial depletion were only assessed in adulthood. Given the hypothesized importance of differences in microglia at P16 and not at P30, it would be helpful to demonstrate that PLX5622 does indeed affect microglia at P16, when the circuit is most sensitive to maternal influences.

Reviewer #2 (Public review):

Summary

This important study uncovers a novel mechanism for L-leucine uptake by M. tuberculosis and shows that targeting this pathway with 'Semapimod' interferes with bacterial metabolism and virulence. These results identify the leucine uptake pathway as a potential target to design new anti-tubercular therapy.

Strengths

The authors took numerous approaches to prove that L-leucine uptake of M. tuberculosis is an important physiological phenomenon and may be effectively targeted by 'Semapimod'. This study utilizes a series of experiments using a broad set of tools to justify how the leucine uptake pathway of M. tuberculosis may be targeted to design new anti-tubercular therapy.

Weaknesses

The study does not explain how L-leucine is taken up by M. tuberculosis, leaving the mechanism unclear. Even though 'Semapimod' binds to the PpsB protein, the relevant connection between changes in PDIM and amino acid transport remains incomplete. Also, the fact that the drug does not function on WT bacteria makes it a weak candidate to consider its usefulness for a therapeutic option.

Reviewer #2 (Public review):

Summary:

This study shows that type I interferon (IFN-I) signaling helps protect against mycobacterial infection. Using human gene expression data and a zebrafish model, the authors find that reduced IFN-I activity is linked to more severe disease. They also show that zebrafish lacking the IFN-I signaling gene stat2 are more vulnerable to infection due to poor macrophage migration. These results suggest a protective role for IFN-I in mycobacterial disease, challenging previous findings from other animal models.

Strengths:

Strengths of the manuscript include the use of human clinical samples to support relevance to disease, along with a genetically tractable zebrafish model that enables mechanistic insight.

Weaknesses:

(1) The manuscript presents intriguing human data showing an inverse correlation between IFN-I gene signatures and TB disease, but the findings remain correlative and may be cohort-specific. Given that the skin is not a primary site of TB and is relatively immunotolerant, the biological relevance of downregulated IFN-I-related genes in this tissue to systemic or pulmonary TB is unclear.

(2) The reliance on stat2 CRISPants in zebrafish offers a limited view of IFN-I signaling. Including additional crispant lines targeting other key regulators (e.g., ifnar1, tyk2, irf3, irf7) would strengthen the interpretation and clarify whether the observed effects reflect broader IFN-I pathway disruption.

(3) The conclusion that IFN-I is protective contrasts with established findings from murine and non-human primate models, where IFN-I is often detrimental. While the authors highlight species differences, the lack of functional human data and reliance on M. marinum in zebrafish limit the translational relevance. A more balanced discussion addressing these discrepancies would improve the manuscript.

(4) Quantification of bacterial burden using fluorescence intensity alone may not accurately reflect bacterial viability. Complementary methods, such as qPCR for bacterial DNA, would provide a more robust assessment of antimicrobial activity.

(5) Finally, the authors should clarify whether impaired macrophage recruitment in stat2 crispants results from defects in chemotaxis, differentiation, or survival, and address discrepancies between their human blood findings and prior studies.

Reviewer #2 (Public review):

Summary:

The majority of CD8+ T cell responses rely on the proper presentation of antigens through stable MHC-I (but not requiring a stable immunological synapse). This work highlights a new approach to build an array of stable peptide MHC-I using temperature exchange, which can be used to identify antigen-specific CD8+ T cells.

Strengths:

In this work, the authors have proposed an alternative method to reload the peptide MHC-I molecule. Their temperature-exchange approach is distinct from current reloadable peptide MHC technologies involving photolabile peptide, empty MHC-I (Nat Commun 11, 1314 (2020). https://doi.org/10.1038/s41467-020-14862-4), tapasin/TAPBPR chaperone-assisted (eLife 7:e40126.), enzyme exchangeable (WO2020226570) and small alcohol (Curr Res Immunol. 2022 Aug 18;3:167-174. doi: 10.1016/j.crimmu.2022.08.002) approaches.

Weaknesses:

However, the proposed temperature-exchange approach does not substantially improve the quality of antigen-specific T cells that can be identified using the photolabile peptide MHC-I molecules.

The time saved using the temperature-exchange protocol may not be a pull factor as the photolabile peptide MHC-I approach is not unreasonably laborious.

Reviewer #2 (Public review):

Summary:

In the manuscript by Mahen et al., entitled "Gut Microbe-Derived Trimethylamine Shapes Circadian Rhythms Through the Host Receptor TAAR5," the authors investigate the interplay between a host G protein-coupled receptor (TAAR5), the gut microbiota-derived metabolite trimethylamine (TMA), and the host circadian system. Using a combination of genetically engineered mouse and bacterial models, the study demonstrates a link between microbial signaling and circadian regulation, particularly through effects observed in the olfactory system. Overall, this manuscript presents a novel and valuable contribution to our understanding of host-microbe interactions and circadian biology. However, several sections would benefit from improved clarity, organization, and mechanistic depth to fully support the authors' conclusions.

Strengths:

(1) The manuscript addresses an important and timely topic in host-microbe communication and circadian biology.

(2) The studies employ multiple complementary models, e.g., Taar5 knockout mice, microbial mutants, which enhance the depth of the investigation.

(3) The integration of behavioral, hormonal, microbial, and transcript-level data provides a multifaceted view of the observed phenotype.

(4) The identification of olfactory-linked circadian changes in the context of gut microbes adds a novel perspective to the field.

Weaknesses:

While the manuscript presents compelling data, several weaknesses limit the clarity and strength of the conclusions.

(1) The presentation of hormonal, cytokine, behavioral, and microbiome data would benefit from clearer organization, more detailed descriptions, and functional grouping to aid interpretation.

(2) Some transitions-particularly from behavioral to microbiome data-are abrupt and would benefit from better contextual framing.

(3) The microbial rhythmicity analyses lack detail on methods and visualization, and the sequencing metadata (e.g., sample type, sex, method) are not clearly stated.

(4) Several figures are difficult to interpret due to dense layouts or vague legends, and key metabolites and gene expression comparisons are either underexplained or not consistently assessed across models.

(5) Finally, while the authors suggest a causal role for TAAR5 and its ligand in circadian regulation, the current data remain correlative; mechanistic experiments or stronger disclaimers are needed to support these claims.

Reviewer #2 (Public review):

Summary:

The authors meticulously characterized EC-specific Tgfbr1, Tgfbr2, or double knockout in the retina, demonstrating through convincing immunostaining data that loss of TGF-β signaling disrupts retinal angiogenesis and choroidal neovascularization. Compared to other genetic models (Fzd4 KO, Ndp KO, VEGF KO), the Tgfbr1/2 KO retina exhibits the most severe immune cell infiltration. The authors proposed that TGF-β signaling loss triggers vascular inflammation, attracting immune cells - a phenotype specific to CNS vasculature, as non-CNS organs remain unaffected.

Strengths:

The immunostaining results presented are clear and robust. The authors performed well-controlled analyses against relevant mouse models. snRNA-seq corroborates immune cell leakage in the retina and vascular inflammation in the brain.

Weaknesses:

The causal link between TGF-β loss, vascular inflammation, and immune infiltration remains unresolved. The authors' model posits that EC-specific TGF-β loss directly causes inflammation, which recruits immune cells. However, an alternative explanation is plausible: Tgfbr1/2 KO-induced developmental defects (e.g., leaky vessels) permit immune extravasation, subsequently triggering inflammation. The observations that vein-specific upregulation of ICAM1 staining and the lack of immune infiltration phenotypes in the non-CNS tissues support the alternative model. Late-stage induction of Tgfbr1/2 KO (avoiding developmental confounders) could clarify TGF-β's role in retinal angiogenesis versus anti-inflammation.

Reviewer #2 (Public review):

Summary:

The current study seeks to understand the neural mechanisms underlying geometric reasoning. Using fMRI with both children and adults, the authors found that contrasting simple geometric shapes with naturalistic images (faces, tools, houses) led to responses in the dorsal visual stream, rather than ventral regions that are generally thought to represent shape properties. The authors followed up on this result using computational modeling and MEG to show that geometric properties explain distinct variance in the neural response beyond what is captured by a CNN.

Strengths:

These findings contribute much-needed neural and developmental data to the ongoing debate regarding shape processing in the brain and offer additional insights into why CNNs may have difficulty with shape processing. The motivation and discussion for the study are appropriately measured, and I appreciate the authors' use of multiple populations, neuroimaging modalities, and computational models to explore this question.

Weaknesses:

Given that the primary take away from this study is that geometric shape information is found in the dorsal stream, rather than the ventral stream there is very little there is very little discussion of prior work in this area (for reviews, see Freud et al., 2016; Orban, 2011; Xu, 2018). Indeed, there is extensive evidence of shape processing in the dorsal pathway in human adults (Freud, Culham, et al., 2017; Konen & Kastner, 2008; Romei et al., 2011), children (Freud et al., 2019), patients (Freud, Ganel, et al., 2017), and monkeys (Janssen et al., 2008; Sereno & Maunsell, 1998; Van Dromme et al., 2016), as well as the similarity between models and dorsal shape representations (Ayzenberg & Behrmann, 2022; Han & Sereno, 2022).

The presence of activation in aIPS led the authors to interpret their results to mean that geometric reasoning draws on the same processes as mathematical thinking. However, there is not enough evidence in the current study to support this claim.

Reviewer #2 (Public review):

Summary:

Lipid transfer between membranes is essential for lipid biosynthesis across different organelle membranes. Ups1-Mdm35 is one of the best-characterized lipid transfer proteins, responsible for transferring phosphatidic acid (PA) between the mitochondrial outer membrane (OM) and inner membrane (IM), a process critical for cardiolipin (CL) synthesis in the IM. Upon dissociation from Mdm35, Ups1 binds to the intermembrane space (IMS) surface of the OM, extracts a PA molecule, re-associates with Mdm35, and moves through the aqueous IMS to deliver PA to the IM. Here, the authors analyzed the early steps of this PA transfer - membrane binding and PA extraction - using a combination of in vitro biochemical assays with lipid liposomes and purified Ups1-Mdm35 to measure liposome binding, lipid transfer between liposomes, and lipid extraction from liposomes. The authors found that membrane curvature, a previously overlooked property of the membrane, significantly affects PA extraction but not PA insertion into liposomes. These findings were further supported by MD simulations.

Strengths:

The experiments are well-designed, and the data are logically interpreted. The present study provides an important basis for understanding the mechanism of lipid transfer between membranes.　

Weaknesses:

The physiological relevance of membrane curvature in lipid extraction and transfer still remains open.

Reviewer #2 (Public review):

Summary:

This study describes the key observation that SATB1 binds directly to so-called BUR elements. This is in contrast to several other reports describing SATB1 binding to promoters and enhancers. This discrepancy is explained by the authors to depend on the features of the ChIP technique being used. Urea-ChIP, innovated by the authors, strips off protein-protein interactions that compound conventional ChIP methods. The authors convincingly make the case that SATB1 and a key genome organiser, CTCF, largely bind different sites, as particularly evident in Figure 2A. In contrast, standard ChIP shows considerable overlap between their sites (Figure 2-figure supplement 1). The report documents convincingly that SATB1 partitions the genome independent of so-called TADs to influence expression patterns. SATB1 controls long-range interactions in thymocytes, and knock down of SATB1 does not affect the TAD patterns.

Strengths:

A new and innovative adaptation of ChIP-seq (urea ChIP-seq) has enabled the authors to successfully question existing data on the patterns of SATB1 binding to the genome. The authors provide a wealth of data to reinforce their claims. This report thus rectifies misconceptions about SATB1 function, which is particularly important given its role in metastasising cancer cells.

Weaknesses:

None

Reviewer #2 (Public review):

Summary:

The authors proposed a neural network model to explore the spatial representations of the hippocampal CA1 and entorhinal cortex (EC) and the remapping of these representations when multiple environments are learned. The model consists of a recurrent network and output units (a decoder) mimicking the EC and CA1, respectively. The major results of this study are: the EC network generates cells with their receptive fields tuned to a border of the arena; the decoder develops neuron clusters arranged in a hexagonal lattice. Thus, the model accounts for entrohinal border cells and CA1 place cells. It suggests that the remapping of place cells occurs between different environments through state transitions corresponding to unstable dynamical modes in the recurrent network.

Strengths:

The authors found a spatial arrangement of receptive fields similar to their model's prediction in experimental data recorded from CA1. Thus, the model proposes plausible mechanisms to generate hippocampal spatial representations without relying on grid cells. The model also suggests an interesting possibility that path integration is not the speciality of grid cells.

Weaknesses:

The role of grid cells in the proposed view, i.e., the boundary-to-place-to-grid model, remains elusive. The model can generate place cells without generating entorhinal grid cells. Moreover, the model can generate hexagonal grid patterns of place cells in a large arena. Whether and how the proposed model is integrated into the entire picture of the hippocampal-entorhinal meｍory processing remains elusive.

Reviewer #2 (Public review):

This revised paper describes an investigation of galanin and galanin receptor signaling on whole-brain activity in the context of recurrent seizure activity or under homeostatic basal conditions. The authors primarily use calcium imaging to observe whole-brain neuronal activity accompanied by galanin qPCR to determine how manipulations of galanin or the galr1a receptor affect the activity of the whole-brain under non-ictal conditions or when seizure activity occurs. The authors use their eaat2a-/- model (introduced in their Glia 2022 paper, PMID 34716961) that shows recurrent seizure activity as well as suppression of neuronal activity and locomotion interictally. It is compared to the well-known pentylenetetrazole (PTZ) pharmacological model of seizures in zebrafish. Given the literature cited in their Introduction, the authors hypothesize that galanin will exert a net inhibitory effect on brain activity in models of seizures/epilepsy. They were surprised to find that this hypothesis was only moderately supported in their eaat2a-/- model. In contrast, after PTZ, fish with galanin overexpression showed increased seizure number and reduced duration while fish with galanin KO showed reduced seizure number and increased duration.

Previous concerns about sex or developmental biological variables were addressed, as their model's seizure phenotype emerges rapidly and long prior to the establishment of zebrafish sexual maturity. However, it remains unclear whether all seizures detected via calcium imaging alone are also seizures that are detectable at the level of animal behavior. To confirm this, a validation of the threshold used for calcium imaging of "neuronal seizures" would be required to determine if this threshold detects only "neuronal seizures" that co-occur with behavioral seizures. Overall, this study is important and convincing, and carries clear value for understanding the multifaceted functions that neuronal galanin can perform under homeostatic and disease conditions.

Additional Concerns:

- The authors have validated their ability to measure behavioral seizures quantitatively in their 2022 Glia paper but the information provided on defining behavioral seizures as they map onto seizures detected via imaging alone was limited. The definition of behavioral seizure activity as it relates to calcium fluctuations is not expanded upon in this paper, but could provide detail about how the behavioral seizures relate to a seizure detected via calcium imaging alone.

Reviewer #2 (Public review):

In their manuscript with the title "Integrated transcriptomic analysis of human induced pluripotent stem cell (iPSC)-derived osteogenic differentiation reveals a regulatory role of KLF16", Ru et al. have analyzed the gene expression changes during the osteogenic differentiation of iPSC-derived mesenchymal stem/stromal cells into preosteoblasts and osteoblasts. As part of the computational analyses, they have investigated the transcription factor regulatory network mediating this differentiation process, which has also led to the identification of the transcription factor KLF16. Overexpression experiments in vitro and the analysis of heterozygous KLF16 knockout mice in vivo indicate that KLF16 is an inhibitor of osteogenic differentiation.

The integrated analysis of iPSC bulk transcriptomic data is a major strength of the study, and it is also great that the authors provide deeper functional characterization of the transcription factor KLF16, one of the newly identified candidate regulators of osteogenic differentiation.

However, characterization of KLF16 expression in the mouse and validation of the knockout model are currently lacking. Alternative explanations for the mutant phenotype should be considered to improve the strength of the conclusions.

If all issues can be addressed, the study would provide an important resource for the field that would facilitate future research on the regulation of osteogenesis in vitro and in vivo, with potential implications for preclinical and clinical research as well as bioengineering.

Reviewer #2 (Public review):

Summary:

In this manuscript, Krishnan et al devised three paradigms to perform contextual fear conditioning in head-fixed mice. Each of the paradigms relied on head-fixed mice running on a treadmill through virtual reality arenas. The authors tested the validity of three versions of the paradigms by using various parameters. The authors have addressed some of my initial concerns in their revised manuscript.

Strengths:

The authors have devised three new contextual fear conditioning paradigms in head-fixed mice. The authors tested a number of parameters towards optimization of this approach.

Weaknesses:

While some experimental parameters were tested in the manuscript, it appears that a large amount of additional testing and optimization will be required before reliable behavioral responses can be acquired and ultimately for the paradigm(s) to be useful for answering biological questions. One major factor will be optimizing parameters such that head-fixed mice in this paradigm can (largely) recapitulate what is observed in freely behaving mice. This may be challenging however, as they have previously published one of the three paradigms and the extensive additional testing they did in this current manuscript did not greatly improve the experimental setup. This may indicate limited immediate usefulness for the community as significant work likely remains for optimization.

Achievement of Aims:

The authors have put a significant amount of work in testing the paradigms, and as a result, progress has been made towards their usefulness in the field. However, a significant amount of optimization likely exists.

Impact on the field:

The development of a reliable paradigm for studying contextual fear in head-fixed animals would be a strong contribution to the field as it would enable sophisticated cell and circuit imaging analyses. This study is a good start towards this goal, but significant optimization is required for the paradigm(s) to fully benefit the field - especially to allow those who may have less experience in these approaches to use it in their own research.

Reviewer #2 (Public review):

I appreciate the author's responses to my original review. This is a comprehensive analysis of CAPE on C. difficile activity. It seems like this compound effects all aspects of C. difficile, which could make it effective during infection but also make it difficult to understand the mechanism. Even considering the authors responses, I think it is critical for the authors to work on the conclusions regarding the infection model. There is some protection from disease by CAPE but some parameters are not substantially changed. For instance, weight loss is not significantly different in the C. difficile only group versus the C. difficile + CAPE group. Histology analysis still shows a substantial amount of pathology in the C. difficile + CAPE group. This should be discussed more thoroughly using precise language.

The authors did a good job addressing my concerns regarding the infection model by providing a more accurate descriptions in the Results section for histology. However, the weight loss improvement by CAPE does not look like a significant effect, although it is trending towards improvement. This should be more accurately described.

Another minor concern is that the current Abstract is overstating the amount of disease attenuation. I would replace "remarkably reduces the pathology" with "reduces some of the pathology"

Reviewer #2 (Public review):

Summary:

The authors build a gene expression model based on histone post-translational modifications, and find that H3K27ac is correlated with gene expression. They compare to other gene prediction methods such as DeepChrome. They proceed to perturb H3K27ac at 13 gene promoters in two cell types, and measure gene expression changes to test their model.

Strengths:

The combination of multiple methods to model expression, along with utilizing 6 histone datasets in 13 cell types allowed the authors to build a model that correlates between 0.7-0.79 with gene expression.<br /> They compare three cells types to other prediction models, and this figure should be included in the main figures.<br /> They use dCas9-p300 fusions to perturb H3K27ac and monitor gene expression to test their model. Ranked correlations of the HEK293 data showed some support for the predictions after perturbation of H3K27ac.

Weaknesses:

The authors state in the latest submission that the primary use case of this work is related to predicting epigenome editing outcomes, not predicting gene expression from chromatin. However the first four figures all relate to gene expression prediction. The only main figure that shows epigenome editing prediction is panel 6E. If this authors wish to highlight the use case of this work they should redo figures, including moving panels from current supplemental figures to show this.

The perturbation of 5 genes in K562 with perturb-seq data shows a modest correlation of ~0.5 and is still only shown in supplemental figures, which is odd as this is the true test case of their model in my opinion. The authors are then left to speculate the reasons why the outcome of epigenome editing doesn't fit their predictions, which highlights the limited value in the current version of this method.<br /> As mentioned before, testing genes that were not expressed being most activated by dCas9-p300 weaken the correlations vs. looking at a broad range of different gene expression as the original model was trained on.

If the authors want this method to be used to predict outcomes of epigenome editing, expanding to dCas9-KRAB and other CRISPRa methods (SAM and VPR) would be useful. Those datasets are published and could be analyzed for this manuscript and show how the model holds up across cell types and epigenome editing methods.

The utility of this method as described here, to predict gRNA outcomes seems modest and limited. It is fairly trivial to test 10 or more gRNAs for a single gene to find the best one, and the authors show limited prediction and occasionally no benefit. For example, with CHD8 and CD79 the gRNA with the highest prediction had the lowest actual impact on gene expression of the gRNAs tested. For many other genes the gRNA's prediction and gene expression outcome show no correlation.

Reviewer #2 (Public review):

Summary:

Authors in this study previously reported that BYL719, an inhibitor of PI3Kα, suppressed heterotopic ossification in mice model of a human genetic disease, fibrodysplasia ossificans progressive, which is caused by the activation of mutant ACVR1/R206H by Activin A. The aim of this study is to identify the mechanism of BYL719 for the inhibition of heterotopic ossification. They found that BYL719 suppressed heterotopic ossification in two ways: one is to inhibit the specification of precursor cells for chondrogenic and osteogenic differentiation and the other is to suppress the activation of inflammatory cells.

Strengths:

This study is based on authors' previous reports and the experimental procedures including the animal model are established. In addition, to confirm the role of PI3Kα, authors used the conditional knock-out mice of the subunit of PI3Kα. They clearly demonstrated the evidence indicating that the targets of PI3Kα is not members of TGFBR by a newly established experimental method.

Weaknesses:

Overall, the presented data were closely related to those previously published by authors' group or others and there were very few new findings. The molecular mechanisms through which BYL719 inhibits HO remain unclear, even in the revised manuscript.<br /> Heterotopic ossification in mice model was not stable and inappropriate for the scientific evaluation.<br /> The method for chondrogenic differentiation was not appropriate, and the scientific evidence of successful differentiation was lacking.<br /> The design of gene expression profile comparison was not appropriate and failed to obtain the data for the main aim of this study.<br /> The experiments of inflammatory cells were performed cell lines without ACVR1/R206H mutation, and therefore the obtained data were not precisely related to the inflammation in FOP.

Comments on revisions:

In the R2 version, the authors performed additional experiments using mice with inducible human R206H ACVR1A. BM-MSCs isolated from these mice were used to investigate the effect of Activin-A. The results again suggested that BYL79 inhibited the chondrogenic differentiation of BM-MSCs. However, there are still no data demonstrating the effect of BYL79 on cell growth in these in vitro experiments. In Figures 7A-D, 10 μM BYL79 strongly inhibited the proliferation of inflammatory cells, suggesting that growth inhibition may have contributed to the results shown in Figure 5.

The main point of discussion concerns the significance of the comparisons made. The fundamental disagreement arises from the role of Activin-A in R206H cells and its effect on chondrogenic differentiation. The authors' rebuttal regarding my comments on the RNA-seq analyses should be reconsidered. The core issue lies in the interpretation of Activin-A's role in R206H cells and the distinction between chondrogenic differentiation and ossification.

A key feature of R206H mutant cells is that they respond to Activin-A by activating Smad1/5 signaling-comparable in quality to the signaling induced by BMP6 in WT cells. Another important point, as also acknowledged by the authors, is that Activin-A can transduce Smad2/3 signaling via its canonical receptor, ACVR1B. These dual signaling pathways synergistically contribute to chondrogenic differentiation in precursor cells such as FAPs. Several reports have demonstrated that the combined activation of TGF-β and BMP signaling promotes chondrogenesis more strongly than either pathway alone.

Since the PI3Kα inhibition effect on HO is already known, a critical question in this study is whether BYL79 also inhibits the Smad2/3 pathway. A straightforward experiment would be to compare WT cells treated with Activin-A alone versus Activin-A plus BYL79, and to perform GO term enrichment analyses related specifically to chondrogenic differentiation, not ossification. Additionally, comparing R206H cells treated with Activin-A/BYL79 and WT cells treated with BMP6/BYL79 could help identify gene sets inhibited by BYL79 via Smad2/3 signaling. If these comparisons reveal no specific effect on genes related to chondrogenesis, the effect of BYL79 may be limited to suppression of BMP-mediated osteogenesis. Unfortunately, the authors appear to show little interest in addressing this issue.

Regarding Figure 7, the authors' rebuttal should also be reconsidered. Since the R2 version employed FOP model mice, it would have been possible to evaluate the effects of BYL79 on inflammatory cells harboring the R206H mutation. This could have enabled a more precise assessment of BYL79's influence on inflammatory signaling. While the authors repeatedly claim that BYL79's effect is not specific to any particular ligand or the presence of the FOP mutation, the role of TGF-β signaling in the development of endochondral heterotopic ossification is well recognized. Therefore, the mechanism of BYL79 should be clarified before considering its therapeutic application

Reviewer #2 (Public review):

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

Reviewer #3 (Public review):

Summary:

In this manuscript, "Neocortical Layer-5 tLTD Relies on Non-Ionotropic Presynaptic NMDA Receptor Signaling", Thomazeau et al. seek to determine the role of presynaptic NMDA receptors and the mechanism by which they mediate expression of frequency-independent timing-dependent long-term depression (tLTD) between layer-5 (L5) pyramidal cells (PCs) in the developing mouse visual cortex. By utilizing sophisticated methods, including sparse Cre-dependent deletion of GluN1 subunit via neonatal iCre-encoding viral injection, in vitro quadruple patch clamp recordings, and pharmacological interventions, the authors elegantly show that L5 PC->PC tLTD is 1) dependent on presynaptic NMDA receptors, 2) mediated by non-ionotropic NMDA receptor signaling, and 3) is reliant on JNK2/Syntaxin-1a (STX1a) interaction (but not RIM1αβ) in the presynaptic neuron. The study elegantly and pointedly addresses a long-standing conundrum regarding the lack of frequency dependence of tLTD.

Strengths:

The authors did a commendable job presenting a very polished piece of work with high-quality data that this Reviewer feels enthusiastic about. The manuscript has several notable strengths. Firstly, the methodological approach used in the study is highly sophisticated and technically challenging, and successfully produced high-quality data that were easily accessible to a broader audience. Secondly, the pharmacological interventions used in the study targeted specific players and their mechanistic roles, unveiling the mechanism in question step-by-step. Lastly, the manuscript is written in a well-organized manner that is easy to follow. Overall, the study provides a series of compelling evidence that leads to a clear illustration of mechanistic understanding.

Weakness:

No major weaknesses were noted.

Reviewer #2 (Public review):

Summary:

This study investigates the developmental and lifelong consequences of reduced foxf2 dosage in zebrafish, a gene associated with human stroke risk and cerebral small vessel disease (CSVD). The authors show that a ~50% reduction in foxf2 function through homozygous loss of foxf2a leads to a significant decrease in brain pericyte number, along with striking abnormalities in pericyte morphology-including enlarged soma and extended processes-during larval stages. These defects are not corrected over time but instead persist and worsen with age, ultimately affecting the surrounding endothelium. The study also makes an important contribution by characterizing pericyte behavior in wild-type zebrafish using a clever pericyte-specific Brainbow approach, revealing novel interactions such as pericyte process overlap not previously reported in mammals.

Strengths:

This work provides mechanistic insight into how subtle, developmental changes in mural cell biology and coverage of the vasculature can drive long-term vascular pathology. The authors make strong use of zebrafish imaging tools, including longitudinal analysis in transgenic lines to follow pericyte number and morphology over larval development, and then applied tissue clearing and whole brain imaging at 3 and 11 months to further dissect the longitudinal effects of foxf2a loss. The ability to track individual pericytes in vivo reveals cell-intrinsic defects and process degeneration with high spatiotemporal resolution. Their use of a pericyte-specific Zebrabow line also allows, for the first time, detailed visualization of pericyte-pericyte interactions in the developing brain, highlighting structural features and behaviors that challenge existing models based on mouse studies. Together, these findings make the zebrafish a valuable model for studying the cellular dynamics of CSVD.

Weaknesses:

While the findings are compelling, several aspects could be strengthened. First, quantifying pericyte coverage across distinct brain regions (forebrain, midbrain, hindbrain) would clarify whether foxf2a loss differentially impacts specific pericyte lineages, given known regional differences in developmental origin, with forebrain pericytes being neural crest-derived and hindbrain pericytes being mesoderm-derived. Second, measuring foxf2b expression in foxf2a mutants would better support the interpretation that total FOXF2 dosage is reduced in a graded fashion in heterozygote and homozygote foxf2a mutants. Finally, quantifying vascular density in adult mutants would help determine whether observed endothelial changes are a downstream consequence of prolonged pericyte loss. Correlating these vascular changes with local pericyte depletion would also help clarify causality.

Reviewer #2 (Public review):

Summary:

This study investigates the role of GMCL1 in regulating the mitotic surveillance pathway (MSP), a protective mechanism that activates p53 following prolonged mitosis. The authors identify a physical interaction between 53BP1 and GMCL1, but not with GMCL2. They propose that the ubiquitin ligase complex CRL3-GMCL1 targets 53BP1 for degradation during mitosis, thereby preventing the formation of the "mitotic stopwatch" complex (53BP1-USP28-p53) and subsequent p53 activation. The authors show that high GMCL1 expression correlates with resistance to paclitaxel in cancer cell lines that express wild-type p53. Importantly, loss of GMCL1 restores paclitaxel sensitivity in these cells, but not in p53-deficient lines. They propose that GMCL1 overexpression enables cancer cells to bypass MSP-mediated p53 activation, promoting survival despite mitotic stress. Targeting GMCL1 may thus represent a therapeutic strategy to re-sensitize resistant tumors to taxane-based chemotherapy.

Strengths:

This manuscript presents potentially interesting observations. The major strength of this article is the identification of GMCL1 as a 53BP1 interaction partner. The authors identified relevant domains and showed that GMCL1 controls 53BP1 stability. The authors further show a potentially interesting link between GMCL1 status and sensitivity to Taxol.

Weaknesses:

However, the manuscript is significantly weakened by unsubstantiated mechanistic claims, overreliance on a non-functional model system (U2OS), and overinterpretation of correlative data. To support the conclusions of the manuscript, the authors must show that the GMCL1-dependent sensitivity to Taxol depends on the mitotic surveillance pathway.

Reviewer #2 (Public review):

The yeast double-stranded RNA endonuclease Rnt1, a homolog of bacterial RNAse III, mediates the processing of pre-rRNA, pre-snRNA, and pre-snoRNA molecules. Cells lacking Rnt1 exhibit pronounced growth defects, particularly at lower temperatures. In this manuscript, Notice-Sarpaning examines whether these growth defects can be attributed at least in part to a function of Rnt1 in mRNA degradation. To test this, the authors apply parallel analysis of RNA ends (PARE), which they developed in previous work, to identify polyA+ fragments with 5' monophosphates in RNT1 yeast that are absent in rnt1Δ cells. Because such RNAs are substrates for 5' to 3' exonucleolytic decay by Rat1 in the nucleus or Xrn1 in the cytoplasm, these analyses were performed in a rat1-ts xrn1Δ background. The data recapitulate known Rtn1 cleavage sites in rRNA, snRNAs, and snoRNAs, and identify 122 putative novel substrates, approximately half of which are mRNAs. Of these, two-thirds are predicted to contain double-stranded stem loop structures with A/UGNN tetraloops, which serve as a major determinant of Rnt1 substrate recognition. Rtn1 resides in the nucleus, and it likely cleaves mRNAs there, but cleavage products seem to be degraded after export to the cytoplasm, as analysis of published PARE data shows that some of them accumulate in xrn1Δ cells. The authors then leverage the slow growth of rnt1Δ cells for experimental evolution. Sequencing analysis of thirteen faster-growing strains identifies mutations predominantly mapping to genes encoding nuclear exosome co-factors. Some of the strains have mutations in genes encoding a larat-debranching enzyme, a ribosomal protein nuclear import factor, poly(A) polymerase 1, and the RNA-binding protein Puf4. In one of the puf4 mutant strains, a second mutation is also present in YDR514C, which the authors identify as an mRNA substrate cleaved by Rnt1. Deletion of either puf4 or ydr514C marginally improves the growth of rnt1Δ cells, which the authors interpret as evidence that mRNA cleavage by Rnt1 plays a role in maintaining cellular homeostasis by controlling mRNA turnover.

While the PARE data and their subsequent in vitro validation convincingly demonstrate Rnt1-mediated cleavage of a small subset of yeast mRNAs, the data supporting the biological significance of these cleavage events is substantially less compelling. This makes it difficult to establish whether Rnt1-mediated mRNA cleavage is biologically meaningful or simply "collateral damage" due to a coincidental presence of its target motif in these transcripts.

(1) A major argument in support of the claim that "several mRNAs rely heavily on Rnt1 for turnover" comes from comparing number of PARE reads at the transcript start site (as a proxy for fraction of decapped transcripts) and at the Rnt1 cleavage site (as a proxy for fraction of Rnt1-cleaved transcripts). The argument for this is that "the major mRNA degradation pathway is through decapping". However, polyA tail shortening usually precedes decapping, and transcripts with short polyA tails would be strongly underrepresented in PARE sequencing libraries, which were constructed after two rounds of polyA+ RNA selection. This will likely underestimate the fraction of decapped transcripts for each mRNA. There is a wide range of well-established methods that can be used to directly measure differences in the half-life of Rnt1 mRNA targets in RNT1 vs rnt1Δ cells. Because the PARE data rely on the presence of a 5' phosphate to generate sequencing reads, they also cannot be used to estimate what fraction of a given mRNA transcript is actually cleaved by Rnt1.

(2) Rnt1 is almost exclusively nuclear, and the authors make a compelling case that its concentration in the cytoplasm would likely be too low to result in mRNA cleavage. The model for Rnt1-mediated mRNA turnover would therefore require mRNAs to be cleaved prior to their nuclear export in a manner that would be difficult to control. Alternatively, the Rnt1 targets would need to re-enter prior to cleavage, followed by export of the cleaved fragments for cytoplasmic decay. These processes would need to be able to compete with canonical 5' to 3' and 3' to 5' exonucleolytic decay to influence mRNA fate in a biologically meaningful way.

(3) The experimental evolution clearly demonstrates that mutations in nuclear exosome factors are the most frequent suppressors of the growth defects caused by Rnt1 loss. This can be rationalized by stabilization of nuclear exosome substrates such as misprocessed snRNAs or snoRNAs, which are the major targets of Rnt1. The rescue mutations in other pathways linked to ribosomal proteins (splicing, ribosomal protein import, ribosomal mRNA binding) support this interpretation. By contrast, the potential suppressor mutation in YDR514C does not occur on its own but only in combination with a puf4 mutation; it is also unclear whether it is located within the Rnt1 cleavage motif or if it impacts Rnt1 cleavage at all. This can easily be tested by engineering the mutation into the endogenous YDR514C locus with CRISPR/Cas9 or expressing wild-type and mutant YDR514C from a plasmid, along with assaying for Rnt1 cleavage by northern blot. Notably, the growth defect complementation of YDR514C deletion in rnt1Δ cells is substantially less pronounced than the growth advantage afforded by nuclear exosome mutations (Figure S9, evolved strains 1 to 5). These data rather argue for a primary role of Rnt1 in promoting cell growth by ensuring efficient ribosome biogenesis through pre-snRNA/pre-snoRNA processing.

Reviewer #2 (Public review):

Summary:

This paper measures associations between RNA transcript levels and important reproductive traits in the model organism C. elegans. The authors go beyond determining which gene expression differences underlie reproductive traits, but also (1) build a model that predicts these traits based on gene expression and (2) perform experiments to confirm that some transcript levels indeed affect reproductive traits. The clever study design allows the authors to determine which transcript levels impact reproductive traits, and also which transcriptional differences are driven by stochastic vs environmental differences. In sum, this is a rather comprehensive study that highlights the power of gene expression as a driver of phenotype, and also teases apart the various factors that affect the expression levels of important genes.

Strengths:

Overall, this study has many strengths, is very clearly communicated, and has no substantial weaknesses that I can point to. One question that emerges for me is about the extent to which these findings apply broadly. In other words, I wonder whether gene expression levels are predictive of other phenotypes in other organisms. I think this question has largely been explored in microbes, where some studies (PMID: 17959824) but not others (PMID: 38895328) find that differences in gene expression are predictive of phenotypes like growth rate. Microbes are not the primary focus here, and instead, the discussion is mainly focused on using gene expression to predict health and disease phenotypes in humans. This feels a little complicated since humans have so many different tissues. Perhaps an area where this approach might be useful is in examining infectious single-cell populations (bacteria, tumors, fungi). But I suppose this idea might still work in humans, assuming the authors are thinking about targeting specific tissues for RNAseq.

In sum, this is a great paper that really got me thinking about the predictive power of gene expression and where/when it could inform about (health-related) phenotypes.

Reviewer #2 (Public review):

Summary:

In this paper, the authors used MEFs expressing the R1441G mutant of leucine-rich repeat kinase 2 (LRRK2), a mutant associated with the early onset of Parkinson's disease. They report that in these cells LAMP2 fluorescence is higher but BMP fluorescence is lower, MVE size is reduced, and that MVEs contain less ILVs. They also report that LAMP2-positive EVs are increased in mutant cells in a process sensitive to LRRK2 kinase inhibition but are further increased by glucocerebrosidase (GCase) inhibition, and that total di-22:6-BMP and total di-18:1-BMP are increased in mutant LRRK2 MEFs compared to WT cells by mass spectrometry. They also report that LRRK2 kinase inhibition partially restores cellular BMP levels, and that GCase inhibition further increases BMP levels, and that in EVs from the LRRK2 mutant, LRRK2 inhibition decreases BMP while GCase inhibition has the opposite effect. Moreover, they report that the BMP increase is not due to increased BMP synthesis, although the authors observe that CLN5 is increased in LRRK2 mutant cells. Finally, they report that GW4869 decreases EV release and exosomal BMP, while bafilomycin A1 increases EV release. They conclude that LRRK2 regulates BMP levels (in cells) and release (via EVs). They also conclude that the process is modulated by GCase in LRRK2 mutant cells, and that these studies may contribute to the use of BMP-positive EVs as a biomarker for Parkinson's disease and associated treatments.

Strengths:

This is an interesting paper, which provides novel insights into the biogenesis of exosomes with exciting biomedical potential. However, I have comments that authors need to address to clarify some aspects of their study.

Weaknesses:

(1) The intensity of LAMP2 staining is increased significantly in cells expressing the R1441G mutant of LRRK2 when compared to WT cells (Figure 1C). Yet mutant cells contain significantly smaller MVEs with fewer ILVs, and the MVE surface area is reduced (Figure 1D-F). This is quite surprising since LAMP2 is a major component of the limiting membrane of late endosomes. Are other proteins of endo-lysosomes (eg, LAMP1, CD63, RAB7) or markers (lysotracker) also decreased (see also below)?

(2) LRRK2 has been reported to interact with endolysosomal membranes. Does the R1441G mutant bind LAMP2- and/or BMP-positive membranes? Does the mutant affect endolysosomes?

(3) Immunofluorescence data indicate that BMP is decreased in mutant LRRK2-expressing cells compared to WT (Figure 1A-B), but mass spec data indicate that di-22:6-BMP and di-18:1-BMP are increased (Figure 3). Authors conclude that the BMP pool detected by mass spec in mutant cells is less antibody-accessible than that present in wt cells, or that the anti-BMP antibody is less specific and that it detects other analytes. This is an awkward conclusion, since the IF signal with the antibody is lower (not higher): why would the antibody be less specific? Could it be that the antibody does not see all BMP isoforms equally well? Moreover, the observations that mutant cells contain smaller MVEs (Figure 1D-F) with fewer ILVs are consistent with the IF data and reduced BMP amounts. This needs to be clarified.

Mass spectrometry data are only shown for two BMP species (di-22:6, di-18:1). What are the major BMP isoforms in WT cells? The authors should show the complete analysis for all BMP species if they wish to draw quantitative conclusions about the amounts of BMP in wt and mutant cells. Finally, BMP and PG are isobaric lipids. Fragmentation of BMPs or PGs results in characteristic fingerprints, but the presence of each daughter ion is not absolutely specific for either lipid. This should be clarified, e.g., were BMP and PG separated before mass spec analysis? Was PG affected? The authors should also compare the BMP data with mass spec data obtained with a control lipid, e.g., PC.

(4) It is quite surprising that the amounts of labeled BMP continue to increase for up to 24h after a short 25min pulse with heavy BMP precursors (Figure 4B).

(5) It is argued that upregulation of CLN5 may be due to an overall upregulation of lysosomal enzymes, as LAMP2 levels were also increased (Figure 2A, C, E). Again, this is not consistent with the observed decrease in MVE size and number (Figure 1D-F). As mentioned above, other independent markers of endo-lysosomes should be analyzed (eg, LAMP1, CD63, RAB7), and/or other lysosomal enzymes (e.g. cathepsin. D).

(6) The authors report that the increase in BMP is not due to an increase in BMP synthesis (Figure 4), although they observe a significant increase in CLN5 (Figure 5A) in LRRK2 mutant cells. Some clarification is needed.

(7) Authors observe that both LAMP2 and BMP are decreased in EVs by GW4869 and increased by bafilomycin (Figure 6). Given my comments above on Figure 1, it would also be nice to illustrate/quantify the effects of these compounds on cells by immunofluorescence.

Reviewer #3 (Public review):

Summary:

This study examines the metabolic regulation of progenitor proliferation and differentiation in the developing retina. The authors observe dynamic changes in glycolytic gene expression in retinal progenitors and use various strategies to test the role of glycolysis. They find that elevated glycolysis in Pten-cKO retinas results in alteration of RPC fate, while inhibition of glycolysis has converse effects. They specifically test the role of elevated glycolysis using dominant active cytoPFKB3, which demonstrates the selective effects of elevated glycolysis on progenitor proliferation and rod differentiation. They then show that elevated glycolysis modulates both pHi and Wnt signaling, and provide evidence that these pathways impact proliferation and differentiation of progenitors, particularly affecting rod photoreceptor differentiation.

Strengths:

This is a compelling and rigorous study that provides an important advance in our understanding of metabolic regulation of retina development, addressing a major gap in knowledge. A key strength is that the study utilizes multiple genetic and pharmacological approaches to address how both increased or decreased glycolytic flux affect retinal progenitor proliferation and differentiation. They discover elevated Wnt signaling pathway genes in Pten cKO retina, revealing a potential link between glycolysis and Wnt pathway activation. Altogether the study is comprehensive and adds to the growing body of evidence that regulation of glycolysis plays a key role in tissue development.

Weaknesses:

(1) Following expression of cytoPFKB3, which results in increased glycolytic flux, BrDU labeling was performed at e12.5 and increased labeled cells were detected in the outer nuclear layer. But whether these are cones or rods is not established. The rest of the analysis is focused on the precocious maturation of rhodopsin-labelled outer segments, and the major conclusions emphasize rod photoreceptor differentiation. Therefore it is unclear whether there is an effect on cone differentiation for either Pten cKO or cytoPFKB3 transgenic retina. It is also not established whether rods are born precociously. Presumably this would be best detected by BrDU labeling at later embryonic stages.

(2) The authors find that there is upregulation of multiple Wnt pathway components in Pten cKO retina. They further show that inhibiting Wnt signaling phenocopies the effects of reducing glycolysis. However, they do not test whether pharmacological inhibition of Wnt signaling reverses the effects of high glycolytic activity in Pten cKO retinas. Thus the argument that Wnt is a key downstream effector pathway regulating rod photoreceptor differentiation is weak.

(3) The use of sodium acetate to force protein acetylation is quite non-specific and will have effects beyond beta-catenin acetylation (which the authors acknowledge). Thus it is a stretch to state that "forced activation of beta-catenin acetylation" mimics the impact of Pten<br /> loss/high glycolytic activity in RPCs since the effects could be due to acetylation of other proteins.

Reviewer #2 (Public review):

Summary:

Homologous recombination (HR) is a critical pathway for repairing double-strand DNA breaks and ensuring genomic stability. At the core of HR is the RAD51-mediated strand-exchange process, in which the RAD51-ssDNA filament binds to homologous double-stranded DNA (dsDNA) to form a characteristic D-loop structure. While decades of biochemical, genetic, and single-molecule studies have elucidated many aspects of this mechanism, the atomic-level details of the strand-exchange process remained unresolved due to a lack of atomic-resolution structure of RAD51 D-loop complex.<br /> In this study, the authors achieved this by reconstituting a RAD51 mini-filament, allowing them to solve the RAD51 D-loop complex at 2.64 Å resolution using a single particle approach. The atomic resolution structure reveals how specific residues of RAD51 facilitate the strand exchange reaction. Ultimately, this work provides unprecedented structural insight into the eukaryotic HR process and deepens the understanding of RAD51 function at the atomic level, advancing the broader knowledge of DNA repair mechanisms.

Strengths:

The authors overcame the challenge of RAD51's helical symmetry by designing a minifilament system suitable for single-particle cryo-EM, enabling them to resolve the RAD51 D-loop structure at 2.64 Å without imposed symmetry. This high resolution revealed precise roles of key residues, including F279 in Loop 2, which facilitates strand separation, and basic residues on site II that capture the displaced strand. Their findings were supported by mutagenesis, strand exchange assays, and single-molecule analysis, providing strong validation of the structural insights.

Weaknesses:

Despite the detailed structural data, some structure-based mutagenesis data interpretation lacks clarity. Additionally, the proposed 3′-to-5′ polarity of strand exchange relies on assumptions from static structural features, such as stronger binding of the 5′-arm-which are not directly supported by other experiments. This makes the directional model compelling but contradicts several well-established biochemical studies that support a 5'-to-3' polarity relative to the complementary strand (e.g., Cell 1995, PMID: 7634335; JBC 1996, PMID: 8910403; Nature 2008, PMID: 18256600).

Overall:

The 2.6 Å resolution cryoEM structure of the RAD51 D-loop complex provides remarkably detailed insights into the residues involved in D-loop formation. The high-quality cryoEM density enables precise placement of each nucleotide, which is essential for interpreting the molecular interactions between RAD51 and DNA. Particularly, the structural analysis highlights specific roles for key domains, such as the N-terminal domain (NTD), in engaging the donor DNA duplex.

This structural interpretation is further substantiated by single-molecule fluorescence experiments using the KK39,40AA NTD mutant. The data clearly show a significant reduction in D-loop formation by the mutant compared to wild-type, supporting the proposed functional role of the NTD observed in the cryoEM model.

However, the strand exchange activity interpretation presented in Figure 5B could benefit from a more rigorous experimental design. The current assay measures an increase in fluorescence intensity, which depends heavily on the formation of RAD51-ssDNA filaments. As shown in Figure S6A, several mutants exhibit reduced ability to form such filaments, which could confound the interpretation of strand exchange efficiency. To address this, the assay should either: (1) normalize for equivalent levels of RAD51-ssDNA filaments across samples, or (2) compare the initial rates of fluorescence increase (i.e., the slope of the reaction curve), rather than endpoint fluorescence, to better isolate the strand exchange activity itself.

Based on the structural features of the D-loop, the authors propose that strand pairing and exchange initiate at the 3'-end of the complementary strand in the donor DNA and proceed with a 3'-to-5' polarity. This conclusion, drawn from static structural observations, contrasts with several well-established biochemical studies that support a 5'-to-3' polarity relative to the complementary strand (e.g., Cell 1995, PMID: 7634335; JBC 1996, PMID: 8910403; Nature 2008, PMID: 18256600). While the structural model is compelling and methodologically robust, this discrepancy underscores the need for further experiments.

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

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

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

Weaknesses:

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

I have a few minor concerns regarding new data that was added during the revision:

- The statement ´ Moreover, we found that the fluorescence of low accumulators decreased over time when bacteria were treated with 20 μg mL´ is, in my opinion, not supported by the data shown in Figure S4C. That figure shows that the abundance of ´low accumulator´ cells decreases over time. Following the rationale that protease K treatment may cleave surface-associated/extracellular tachyplesin-NDB, this should lead to a shift of ´low accumulator´population to the left, indicating reduced fluorescence intensity per cell. This is not so case, but the population just disappears. However, after 120 min of treatment more cells appear in the ´high accumulator´ state. This result is somewhat puzzling.

- The authors used the metabolic dye resazurin to measure the metabolic activity of low vs. high accumulators. I am not entirely convinced that the lower fluorescence resorufin-fluorescence in tachyplesin-NBD accumulators really indicates lower metabolic activity, since a cell's fluorescence levels would also be affected by the cellular uptake and efflux. It appears plausible that the lower resorufin-fluorescence may result from reduced accumulation/increased efflux in the´low-tachyplesin NBD´ population.

Comment on revisions: All my previous comments have been satisfactorily addressed by the authors.

Reviewer #2 (Public review):

Summary:

The authors made a thorough revision of the manuscript, strengthening the message. They also considered all the comments made by the reviewers and provided appropriate and convincing arguments.

Strengths:

The revised manuscript clarifies all the major points raised by the reviewers, and the way the information is presented (in the text, figures and tables) is clear.

Weaknesses:

The authors provided an appropriate and convincing rebuttal regarding the potential weakness I pointed out in the first review of the manuscript. Therefore, I do not see any major issue in their work.

Reviewer #2 (Public review):

Summary:

The manuscript by Nosaka et al is a comprehensive study exploring the involvement of IL1beta signaling in a 2-hit model of lung injury + ventilation, with a focus on modulation by hypothermia.

Strengths:

The authors demonstrate quite convincingly that interleukin 1 beta plays a role in the development of ventilator-induced lung injury in this model, and that this role includes the regulation of neutrophil extracellular trap formation. The authors use a variety of in vivo animal-based and in vitro cell culture work, and interventions including global gene knockout, cell-targeted knockout and pharmacological inhibition, which greatly strengthen the ability to make clear biological interpretations.

Comments on revised version:

The authors have addressed my concerns/queries.

Reviewer #3 (Public review):

Summary:

In this study, Zhang et al., reported that CHMP5 restricts bone formation by controlling endolysosome-mitochondrion-mediated cell senescence. Zhang et al., report a novel role of CHMP5 on osteogenesis through affecting cell senescence. Overall, it is an interesting study and provides new insights in the field of cells senescence and bone.

Strengths:

Analyzed the bone phenotype OF CHMP5-periskeletal progenitor-CKO mouse model and found the novel role of senescent cells on osteogenesis and migration.

Weaknesses:

(1) The role and mechanism of CHMP5 gene deletion in enhancing osteogenesis via cellular senescence remain insufficiently elucidated.

(2) The use of the ADTC5 cell line as a skeletal precursor/progenitor model is suboptimal.

Overall, the results support their conclusions.

The impact of this work on the field is its proposal that cellular senescence may exert either inhibitory or promotive effects on osteogenic capacity, depending on cell type and context.

The revised manuscript has addressed most of the concerns raised during the initial review.

Reviewer #2 (Public review):

Summary:

In this study, "Forecasting protein evolution by integrating birth-death population models with structurally constrained substitution models", David Ferreiro and co-authors present a forward-in-time evolutionary simulation framework that integrates a birth-death population model with a fitness function based on protein folding stability. By incorporating structurally constrained substitution models and estimating fitness from ΔG values using homology-modeled structures, the authors aim to capture biophysically realistic evolutionary dynamics. The approach is implemented in a new version of their open-source software, ProteinEvolver2, and is applied to four viral proteins from HIV-1 and SARS-CoV-2.

Overall, the study presents a compelling rationale for using folding stability as a constraint in evolutionary simulations and offers a novel framework and software to explore such dynamics. While the results are promising, particularly for predicting biophysical properties, the current analysis provides only partial evidence for true evolutionary forecasting, especially at the sequence level. The work offers a meaningful conceptual advance and a useful simulation tool, and sets the stage for more extensive validation in future studies.

Strengths:

The results demonstrate that fitness constraints based on protein stability can prevent the emergence of unrealistic, destabilized variants - a limitation of traditional, neutral substitution models. In particular, the predicted folding stabilities of simulated protein variants closely match those observed in real variants, suggesting that the model captures relevant biophysical constraints.

Weaknesses:

The predictive scope of the method remains limited. While the model effectively preserves folding stability, its ability to forecast specific sequence content is not well supported. Only one dataset (HIV-1 MA) is evaluated for sequence-level divergence using KL divergence; this analysis is absent for the other proteins. The authors use a consensus Omicron sequence as a representative endpoint for SARS-CoV-2, which overlooks the rich longitudinal sequence data available from GISAID. The use of just one consensus from a single time point is not fully justified, given the extensive temporal and geographical sampling available. Extending the analysis to include multiple timepoints, particularly for SARS-CoV-2, would strengthen the predictive claims. Similarly, applying the model to other well-sampled viral proteins, such as those from influenza or RSV, would broaden its relevance and test its generalizability.

It would also be informative to include a retrospective analysis of the evolution of protein stability along known historical trajectories. This would allow the authors to assess whether folding stability is indeed preserved in real-world evolution, as assumed in their model.

Finally, a discussion on the impact of structural templates - and whether the fixed template remains valid across divergent sequences - would be valuable. Addressing the possibility of structural remodeling or template switching during evolution would improve confidence in the model's applicability to more divergent evolutionary scenarios.

Reviewer #2 (Public review):

This study presents an investigation of the visual and chemical properties and mating behaviour in Morpho butterflies, aimed at addressing the nature of divergence between closely related species in sympatry. The study species consists of three subspecies of Morpho helenor (bristowi, theodorus, and helenor), and the conspecific Morpho achilles achilles. The authors postulate that whereas the iridescent blue signals of all (sub)species should function as a predator reduction signal (similar to aposematism) and therefore exhibit convergence, the same signals should indicate divergence if used as a mating signal, particularly in sympatric populations. They also assess chemical profiles among the species to assess the potential utility of scent in mediating species/sex discrimination.

The authors first used reflectance spectrometry to calculate hue, brightness, and chroma, plus two measures of "iridescence" (perhaps better phrased as angular dependence) in each (sub)species. This indicated the ubiquitous presence of sexual dimorphism in brightness (males brighter), which also appears to be the case for iridescence (Figure 3A-B). Analysis of these data also indicated that whereas there is evidence for divergence among subspecies in allopatry, the same evidence is lacking for species in sympatry (P = 0.084). This was supported further by visual modelling, which showed that both conspecifics and birds should be (theoretically) capable of perceiving the colour difference among allopatric populations of M. helenor, whereas the same is not true for the sympatric species.

The authors then conducted mate choice trials, first using live individuals and second using female dummies. The live experiments indicated the presence of assortative mating among the two subspecies of M. helenor (bristowi and theodorus). The dummy presentations indicated (a) bristowi males prefer conspecific wings, whereas theodorus have no preference, (b) bristowi males prefer the con(sub)specific colour pattern, (c) theodorus prefer the con(sub)specific iridescence when the pattern is manipulated to be similar among female dummies. A fourth experiment, using sympatric M. achilles and M. helenor, indicated no preference for conspecific female dummies. Finally, chemical analysis indicated substantial differences between these two species in putative pheromone compounds, and especially so in the males.

The authors conclude that the similarity of iridescence among species in sympatry is suggestive of convergence upon a common anti-predation signal. Despite some behavioural evidence in favour of colour (iridescence)-based mate discrimination, chemical differences between Achilles and Helenor are posed as more likely to function for species isolation than visual differences.

Overall, I enjoyed reading this manuscript, which presents a valiant attempt at studying visual, chemical and behavioural divergence in this iconic group of butterflies.

Major comments

My only major comment concerns the authors' favoured explanation for aposematism (or evasive mimicry) for convergence among species, which is based upon the you-can't-catch-me hypothesis first presented by Young 1971. Although there is supporting work showing that iridescent-like stimuli are more difficult to precisely localize by a range of viewers, most of the evidence as applied to the Morpho system is circumstantial, and I'm not certain that there is widespread acceptance of this hypothesis. Given that the present study deals with closely-related (sub)species, one alternative explanation - a "null" hypothesis of sorts - is for a lack of divergence (from a common starting point) as opposed to evolutionary convergence per se. in other words, two subspecies are likely to retain ancestral character states unless there is selection that causes them to diverge. I feel that the manuscript would benefit from a discussion of this alternative, if not others. Signalling to predators could very well be involved in constraining the extent of convergence, but this seems a little premature to state as an up-front conclusion of this work. There is also the result of a *dorsal* wing manipulation by Vieira-Silva et al. 2024 (https://doi.org/10.1111/eth.13517), which seems difficult to reconcile in light of this explanation. Whereas this paper is cited by the authors, a more nuanced discussion of their experimental results would seem appropriate here.

Reviewer #2 (Public review):

The authors extend their SPLASH framework with single-cell RNA-seq in mind, in two ways. First, they introduce "compactors", which are possible paths branching out from an anchor. Second, they introduce a workflow to classify compactors according to the type of biological sequence variation represented (splicing, SNV, etc). They focus on simulated data for fusion detection, and then focus on analyzing the Tabula sapiens Smart-seq2 data, showing extensive results on alternative splicing analysis, VDJ, and repeat elements.

This is strong work with an impressive array of biological investigations and results for a methods paper. I have various concerns about terminology and comparisons, as follows (in a somewhat arbitrary order, apologies).

(1) The discussion of the weaknesses of the consensus sequence approach of SPLASH is an odd way to motivate SPLASH+ in my opinion, in that SPLASH is not yet so widely used, so the baseline for SPLASH+ is really standard alignment-based approaches. It is fine to mention consensus sequence issues briefly, but it felt belabored.

(2) Regarding compactors reducing alignment cost: the comparison should really be between compactor construction and alignment vs read alignment (and maybe vs modern contig construction algorithms and alignment).

(3) The language around "compactors" is a bit confusing, where the authors sometimes refer to the tree of possibilities from an anchor as a "compactor", and sometimes a compactor is a single branch. Presumably, ideally, compactors should be DAGs, not trees, i.e., they can connect back together. Perhaps the authors could comment on whether this matters/would be a valuable extension.

(4) The main oddness of the splicing analysis to me is not using cell-type/state in any way in the statistical testing. This need not be discrete cell types: psiX, for example, tested whether exonic PSI was variable with reference to a continuous gene expression embedding. Intuitively, such transcriptome-wide signal should be valuable for a) improving power and b) distinguishing cell-type intrinsic/"noisy" from cell-type specific splicing variation. A straightforward way of doing this would be pseudobulking cell types. Possibly a more sophisticated hierarchical model could be constructed also.

(5) A secondary weakness is that some informative reads will not be used, for example, unspliced reads aligning to an alterantive exons. This relates to the broader weakness of SPLASH that it is blind to changes in coverage that are not linked to a specific anchor (which should be acknowledged somewhere, maybe in the Discussion). In the deeply sequenced SS2 data, this is likely not an issue, but might be more limiting in sparser data. A related issue is that coverage change indicative of, e.g., alternative TSS or TES (that do not also include a change in splice junction use) will not be detected. In fairness, all these weaknesses are shared by LeafCutter. It would be valuable to have a comparison to a more "traditional" splicing analysis approach (pick your favorite of rMATS, MISO, SUPPA).

(6) "We should note that there is no difference between gene fusions and other RNA variants (e.g., RNA splicing) from a sequence assembly viewpoint". Maybe this is true in an abstract sense, but I don't think it is in reality. AS can produce hundreds of isoforms from the same gene, and be variable across individual cells. Gene fusions are generally less numerous/varied and will be shared across clonal populations, so the complexity is lower. That simplicity is balanced against the challenge that any genes could, in principle, fuse.

(7) For the fusion detection assessment, SPLASH+ is given the correct anchor for detection. This feels like cheating since this information wouldn't usually be available. Can the authors motivate this? Are the other methods given comparable information? Also, TPM>100 seems like a very high expression threshold for the assessment.

(8) Why are only 3'UTRs considered and not 5'? Is this because the analysis is asymmetric, i.e., only considering upstream anchors and downstream variation? If so, that seems like a limitation: how much additional variation would you find if including the other direction?

(9) I don't find the theoretical results very meaningful. Assuming independent reads (equivalently binomial counts) has been repeatedly shown to be a poor assumption in sequencing data, likely due to various biases, including PCR. This has motivated the use of overdispersed distributions such as the negative Binomial and beta binomial. The theory would be valuable if it could say something at a specified level of overdispersion. If not, the caveat of assuming no overdispersion should be clearly stated.

Reviewer #2 (Public review):

Tanja Nielsen et al. present a novel strategy for the identification of candidate genes in Congenital Heart Disease (CHD). Their methodology, which is based on comprehensive experiments across cell models, Drosophila and zebrafish models, represents an innovative, refreshing and very useful set of tools for the identification of disease genes, in a field which are struggling with exactly this problem. The authors have applied their methodology to investigate the pathomechanisms of Hypoplastic Left Heart Syndrome (HLHS) - a severe and rare subphenotype in the large spectrum of CHD malformations. Their data convincingly implicates ribosomal proteins (RPs) in growth and proliferation defects of cardiomyocytes, a mechanism which is suspected to be associated with HLHS.

By whole genome sequencing analysis of a small cohort of trios (25 HLHS patients and their parents), the authors investigated a possible association between RP encoding genes and HLHS. Although the possible association between defective RPs and HLHS needs to be verified, the results suggest a novel disease mechanism in HLHS, which is a potentially substantial advance in our understanding of HLHS and CHD. The conclusions of the paper are based on solid experimental evidence from appropriate high- to medium-throughput models, while additional genetic results from an independent patient cohort are needed to verify an association between RP encoding genes and HLHS in patients.

Reviewer #2 (Public review):

Summary:

This study elucidated the impact of GATA4 on aging- and injury-induced cartilage degradation and osteoarthritis (OA) progression, based on the team's finding that GATA expression is positively correlated with aging in human chondrocytes. By integrating cell culture of human chondrocytes, gene manipulation tools (siRNA, lentivirus), biological/biochemical analyses and murine models of post-traumatic OA, the team found that increasing GATA4 levels reduced anabolism and increased catabolism of chondrocytes from young donors, likely through upregulation of the BMP pathway, and that this impact is not correlated with TGF-β stimulation. Conversely, silencing GATA4 by siRNA attenuated catabolism and elevated aggrecan/collagen II biosynthesis of chondrocytes from old donors. The physiological relevance of GATA4 was further validated by the accelerated OA progression observed in lentivirus-infected mice in the DMM model.

Strengths:

This is a highly significant and innovative study that provides new molecular insights into cartilage homeostasis and pathology in the context of aging and disease. The experiments were performed in a comprehensive and rigorous manner. The data were interpreted thoroughly in the context of the current literature.

Weaknesses:

(1) While it is convincing that GATA4 expression is elevated in elderly individuals, and that it has a detrimental impact on cartilage health, the authors might want to add further discussion on the variability among individual human donors, especially given the finding that the elevation of GATA4 was not observed in chondrocytes from donor O1 (Figure 1G).

(2) It might also be worth adding additional discussion on the interplay between senescent chondrocytes and the dysfunctional ECM during aging. As noted by the authors, aging is associated with decreased sGAG content and likely degenerative changes in the collagen II network, so the microniche of chondrocytes, and thus cell-matrix crosstalk through the pericellular matrix, is also altered or impaired.

Reviewer #1 (Public review):

Summary:

This foundational study builds on prior work from this group to reveal the complexities underlying ligand-dependent RXRγ-Nur77 heterodimer formation, offering a compelling re-evaluation of their earlier conclusions. The authors examine how a library of RXR ligands influences the biophysical, structural, and functional properties of Nur77. They find that although the Nur77-RXRγ heterodimer shares notable functional similarities with the Nurr1-RXRα complex, it also exhibits unique features, notably, both dimer dissociation and classical agonist-driven activities. This work advances our understanding of the nuanced behaviors of nuclear receptor heterodimers, which have important implications for health and disease.

Strengths:

(1) Builds on previous work by providing a comprehensive analysis that examines whether Nur77-RXRγ heterodimer formation parallels that of the Nurr1-RXRα complex.

(2) Systematic evaluation of a library of RXR ligands provides a broad survey of functional outputs.

(3) Careful reanalysis of previous work sheds new light on how NR4A heterodimers function.

Weaknesses:

(1) Some conclusions appear overstated or are not well substantiated by the work presented. It's unclear how the data support a non-classical mode of agonism, for example, based on the data shown.

(2) Some assays have relatively few replicates, with only two in some cases.

Reviewer #2 (Public review):

Summary:

In this manuscript, Pavri and colleagues examine in-depth how the local transcriptional landscape affects somatic hypermutation (SHM) of variable region genes. They use the human Burkitt lymphoma Ramos cell line as a model system to examine AID-induced SHM.

The authors delete Emu and demonstrate that the epigenetic marks at the Ig loci do not correlate with their mutability. They define algorithms to map the V gene promoters and their mutational load in Ramos cells overexpressing AID and failed to find a correlation between mutation frequency and nascent transcription or transcription strength or between mutation frequency and polII stalling. Additionally, the authors show that convergent transcription may not be a major player for SHM. The authors additionally knock-in two other human V genes into the endogenous Vh gene in Ramos cells, and again failed to observe any significant correlation between PolII stalling and SHM. The authors also observe a similar lack of correlation between SHM (at the B-18 gene) and nascent transcription features in germinal center B cells. Overall, the authors conclude that mutation patterns in V genes are not linked to transcriptional features but are rather hard-wired into the sequence. The authors propose that premature transcription termination might have a role in promoting AID recruitment and activity at Ig genes.

Strengths:

The mechanisms that allow AID recruitment to Ig genes during SHM are very poorly understood. Many mechanisms have been proposed, with most invoking transcriptional features, including stalling, convergent transcription, etc. This work, demonstrating the lack of correlation with the proposed models, is of much importance to the field. The experiments are well done, and even though the results are generally "negative", they are highly relevant to our current understanding of SHM.

Weaknesses:

The authors propose premature transcription termination as a possible mechanism to determine V gene mutability, but the study does not experimentally address such possibilities.

Comments:

(1) It would be important for the authors to compare their results in Figure S1 at the B1-8 locus with those reported several years ago by Schatz and colleagues (Odegard et al, Immunity, 2005) and discuss if the results are different from what the authors report here. This is important as the first two figures essentially corroborate previous results that the Emu enhancer is important for transcription through the V genes.

(2) The authors mention that AID recruitment is facilitated by Ig enhancers. Is endogenous AID recruited to the V genes in the absence of Emu in the Ramos cells?

(3) The authors should explain how their results are different from those reported by the Schatz lab in their recent study (Wu et al, Mol Cell, 2025), demonstrating that ELOF1-mediated transcriptional pausing might promote SHM.

Reviewer #2 (Public review):

Summary:

This paper aims at understanding the effects of plasticity in shaping dynamics and structure of cortical circuits, as well as on how that depends on aspects as network structure and dendritic processing.

Strengths:

The level of biological detail included is impressive, and the numerical simulations appear to be well executed. Additionally, they have done a commendable job in open-sourcing the model.

Weaknesses (after revision):

- As noted in my initial review, the observation that network activity remains stable without an explicit homeostatic mechanism-while acknowledged by the authors as consistent with previous findings (e.g., Higgins et al., 2014)-is not clearly framed as a replication or validation step in the current manuscript. For instance, the abstract states: "In our exploratory simulations, plasticity acted sparsely and specifically, firing rates and weight distributions remained stable without additional homeostatic mechanisms," without noting that this outcome has been previously reported, albeit in models with different levels of biological detail. Furthermore, in the general response to reviewers, the authors list this as the first item in their summary of phenomena accounted for by the model, which gives the impression that it is being presented as a primary result.<br /> If this finding is instead meant to serve as a necessary validation that prior results continue to hold under the authors' extended modeling framework-including multicompartmental neurons, stochastic synaptic transmission, and a modified calcium-based plasticity rule-this should be made more explicit in both the abstract and main text. Unless there were specific reasons to suspect that these model extensions might disrupt previously observed stability, the conceptual contribution of this validation step remains unclear.<br /> I would encourage the authors to revise the manuscript to clarify the role and novelty of this result in the context of existing literature and to briefly motivate why confirming this property in their model was an important step.

- While the revised manuscript includes improvements in the discussion of the generality and specificity of the findings, it still offers limited interpretability and mechanistic insight. As it stands, the simulations provide limited understanding of the underlying principles or mechanisms at play, which constrains the broader conclusions that can be drawn from the work.

- In my first review, I suggested that the comparison with the MICrONS dataset could be made more informative-specifically by showing the same quantification of Figure 7D (7B in the previous version) in a version of the model without plasticity and clarifying the interpretation of Figure 8B, where the data appears to align closely with the model before plasticity.<br /> In their response, the authors explain that several of these features remain largely unchanged before and after plasticity. For example, they note that total $g_{\text{AMPA}}$ increases with $k$-edge indegree even in the initial model configuration. I appreciate this clarification, but it highlights a conceptual point that should be more clearly addressed in the manuscript. If the aspects of the model that align with MICrONS data are already present before plasticity, then these similarities reflect properties of the initial network architecture or baseline dynamics, rather than outcomes shaped by the plasticity process itself.<br /> If this interpretation is correct, it represents an interesting and potentially important finding. However, it is not currently articulated in the text. The manuscript places strong emphasis on the role of plasticity in shaping network structure and dynamics, yet the comparisons with MICrONS data appear to reflect features that do not depend on plasticity. Clarifying this distinction would help readers better appreciate the implications of the model-data comparison and discern which conclusions are genuinely supported by the data.

Reviewer #2 (Public review):

Summary:

This study aims the describe the single-cell transcriptomes of H pylori-associated (Hp) gastric cancers and tumour microenvironment (TME), as a starting point to understand TME diversity stratified by Hp status.<br /> RNAseq was performed for gastric cancers with current Hp+ (from N=9 people), ex-Hp+ (N=6), non-Hp (N=6), and healthy gastric tissue (N=6).<br /> The study expands on previous single-cell transcriptomic studies of gastric cancers and was motivated by previous observations about the effect of H pylori status on therapeutic outcomes. The study includes a brief review of previous work and provides valuable context for this study.

Strengths:

The observations are supported by solid RNAseq study design and analysis. The authors describe correlations between Hp status and inferred molecular characteristics including cell lineages, enrichment for cell subclusters identifed as tumour-infiltrating lyphocyte cell types, tumour-infiltrating myeloid cells and cancer-associated fibroblasts.<br /> The observed correlations between Hp status and enrichment of cell subclusters were broadly corroborated using comparisons to deconvolved bulk RNAseq from publicly available gastric cancer data, providing a convincing starting point for understanding the diversity of tumour microenvironment by Hp-status.

Weaknesses:

The authors acknowledge several limitations of this study.<br /> The correlations with HP-status are based on a small number of participants per Hp category (N=9 with current Hp+; N=6 for ex-HP+ and non-HP), and would benefit from further validation to establish reproducibility in other cohorts.<br /> The ligand-receptor cross-talk analysis and the suggestion that suppressive T cells could interact with the malignant epithelium through TIGIT-NECTIN2/PVR pairs, are preliminary findings based on transcriptomic analysis and immunostaining and will require further validation.

Reviewer #2 (Public review):

Summary:

This revised manuscript investigates the role and the mechanism by which PDE1 impacts NSCLC progression, providing solid data to demonstrate that PDE1 binds to m6A reader YTHDF2, in turn, regulating STAT3 signaling pathway through its interaction, promoting metastasis and angiogenesis. The study provides a valuable information to lung cancer field.

Strength:

The study uncovers a novel PDE1A/YTHDF2/SOCS2/STAT3 pathway in NSCLC progression and the findings provide a potential treatment strategy for NSCLC patients with metastasis.

Weakness:

Given that physical interaction of PDE1A and YTHDF2 plays a critical role in PDE1A-mediated NSCLC metastasis, the in vivo data to show that YTHDF2 mimics the effect of PDE1A in metastasis will strength the manuscript although this point was mentioned in the revised manuscript.

Reviewer #2 (Public review):

The authors investigate the phosphotransfer capacity of Ser/Thr kinase IκB kinase (IKK), a mediator of cellular inflammation signaling. Canonically, IKK activity is promoted by activation loop phosphorylation at Ser177/Ser181. Active IKK can then unleash NF-κB signaling by phosphorylating repressor IκBα at residues Ser32/Ser26. Noting the reports of other IKK phosphorylation sites, the authors explore the extent of autophosphorylation.

Semi-phosphorylated IKK purified from Sf9 cells, exhibits the capacity for further autophosphorylation. Anti-phosphotyrosine immunoblotting indicated unexpected tyrosine phosphorylation. Contaminating kinase activity was tested by generating a kinase-dead K44M variant, supporting the notion that the unexpected phosphorylation was IKK-dependent. In addition, the observed phosphotyrosine signal required phosphorylated IKK activation loop serines.

Two candidate IKK tyrosines were examined as the source of the phosphotyrosine immunoblotting signal. Activation loop residues Tyr169 and Tyr188 were each rendered non-phosphorylatable by mutation to Phe. The Tyr variants decreased both autophosphorylation and phosphotransfer to IκBα. Likewise, Y169F and Y188F IKK2 variants immunoprecipitated from TNFa-stimulated cells also exhibited reduced activity in vitro.

The authors further focus on Tyr169 phosphorylation, proposing a role as a phospho-sink capable of phosphotransfer to IκBα substrate. This model is reminiscent of the bacterial two-component signaling phosphotransfer from phosphohistidine to aspartate. Efforts are made to phosphorylate IKK2 and remove ATP to assess the capacity for phosphotransfer. Phosphorylation of IκBα is observed after ATP removal, although there are ambiguous requirements for ADP.

Strengths:

Ultimately, the authors draw together the lines of evidence for IKK2 phosphotyrosine and ATP-independent phosphotransfer to develop a novel model for IKK2-mediated phosphorylation of IκBα. The model suggests that IKK activation loop Ser phosphorylation primes the kinase for tyrosine autophosphorylation. With the assumption that IKK retains the bound ADP, the phosphotyrosine is conformationally available to relay the phosphate to IκBα substrate. The authors are clearly aware of the high burden of evidence required for this unusual proposed mechanism. Indeed, many possible artifacts (e.g., contaminating kinases or ATP) are anticipated and control experiments are included to address many of these concerns. The analysis hinges on the fidelity of pan-specific phosphotyrosine antibodies, and the authors have probed with two different anti-phosphotyrosine antibody clones. Taken together, the observations are thought-provoking, and I look forward to seeing this model tested in a cellular system.

Weaknesses:

Multiple phosphorylated tyrosines in IKK2 were apparently identified by mass spectrometric analyses. LC-MS/MS spectra are presented, but fragments supporting phospho-Y188 and Y325 are difficult to distinguish from noise. It is common to find non-physiological post-translational modifications in over-expressed proteins from recombinant sources. Are these IKK2 phosphotyrosines evident by MS in IKK2 immunoprecipitated from TNFa-stimulated cells? Identifying IKK2 phosphotyrosine sites from cells would be especially helpful in supporting the proposed model.

Reviewer #2 (Public review):

Brickwedde et al. investigate the role of alpha oscillations in allocating intermodal attention. A first EEG study is followed up with an MEG study that largely replicates the pattern of results (with small to be expected differences). They conclude that a brief increase in the amplitude of auditory and visual stimulus-driven continuous (steady-state) brain responses prior to the presentation of an auditory - but not visual - target speaks to the modulating role of alpha that leads them to revise a prevalent model of gating-by-inhibition.

Overall, this is an interesting study on a timely question, conducted with methods and analysis that are state-of-the-art. I am particularly impressed by the author's decision to replicate the earlier EEG experiment in MEG following the reviewer's comments on the original submission. Evidently, great care was taken to accommodate the reviewers suggestions.

In an earlier version, I was struggling with the report for two main reasons: It was difficult to follow the rationale of the study, due to structural issues with the narrative and missing information or justifications for design and analysis decisions, and I was not convinced that the evidence is strong, or even relevant enough for revising the mentioned alpha inhibition theory.

The authors have addressed my concerns through extensive revisions, and I find that it is now easier to follow, and makes a better case for rethinking how alpha may influence sensory processing through a clearer presentation of results and additional arguments.

Reviewer #3 (Public review):

In this study, the authors used RNAscope to explore the expression of RTN4RL2 RNA in hair cells and spiral ganglia. Through RTN4RL2 gene knockout mice, they demonstrated that the absence of RTN4RL2 leads to pre-synaptic changes of an increase in the size of presynaptic ribbons and a depolarized shift in the activation of calcium channels in inner hair cells. Additionally, they observed a post-synaptic reduction in GluA2-4 AMPA receptors and identified additional "orphan PSDs" not paired with presynaptic ribbons via immunostaining and an increased number of type I SGNs that are not connected with a ribbon synapse via serial block face imaging. These synaptic alterations ultimately resulted in an increased hearing threshold in mice, confirming that the RTN4RL2 gene is essential for normal hearing. These data are intriguing as they suggest that RTN4RL2 contributes to the proper formation and function of auditory afferent synapses and is critical for normal hearing. Most strikingly, the post-synaptic changes and hearing threshold changes are similar to recently published results by Carlton et al, 2024 on a mutation in Bai1, which is a potential binding partner for RTN4RL2. Overall this work provides some clues to the function of RTN4RL2 in the cochlea, but further studies are required to elucidate the function.

A few points would improve the manuscript and the strength of the data presented.

(1) A quantitative assessment is necessary in Figure 1 when discussing RNA scope data. It would be beneficial to show that expression levels are quantitatively reduced in KO mice compared to wild-type mice. This suggestion also applies to Figure 3D, which examines expression levels of Gria2. Data is provided for KO reduction in SGN, but not showing that hair cell labeling is specific. If slides are not available for the young ages, showing hair cell expression at P40 would be sufficient along with a loss of labeling at in the KO at P40.

(2) In Figure 2, the authors present a morphological analysis of synapses and discuss the presence of "orphan PSDs." I agree that Homer1 not juxtaposed with Ctbp2 is increased in KO mice compared to the control group. However, in quantifying this, they opted to measure the number of Ctbp2 puncta with Homer 1 juxtaposed, which indicates the percentages of orphan ribbons rather than directly quantifying the number of Homer1 not juxtaposed with Ctbp2. Quantifying the number of Homer1 not juxtaposed with Ctbp2 would more clearly represent "orphan PSDs" and provide stronger support for the discussion surrounding their presence. A measurement of these was provided in the rebuttal letter, and while this number much more clearly demonstrates the increase in the number of orphan puncta, this analysis is not provided in the manuscript. This number also suggests the number of orphan receptors may be quite high, outnumbering ribbons 2:1.

(3) In Figure 3, the authors discuss GluA2/3 puncta reduction and note that Gria2 RNA expression remains unchanged. However, the GluA2/3 labeling is done at 1-1.5 months, whereas the Gria2 RNAscope is done at P4. Additionally, there is a lack of quantification for Gria2 RNA expression due to their tissue being processed separately. RNA scope at a comparable age to the GluA2/3 would be stronger support for their statement that Gria2 expression is comparable despite a reduction in GluA2/3 puncta.

(4) In Figure 4, the authors indicate that RTN4RL2 deficiency reduces the number of type 1 SGNs connected to ribbons. Given that the number of ribbons remains unchanged (Figure 2), it is important to clearly explain the implications of this finding. It is already known that each type I SGN forms a single synaptic contact with a single IHC. The fact that the number of ribbons remains constant while additional "orphan PSDs" are present suggests that the overall number of SGNs might need to increase to account for these findings, however, the authors noted no change in the number of SGN soma. This discrepancy is important to point out.

Reviewer #2 (Public review):

Summary:

The authors had two aims: First, to decompose the attentional blink (AB) deficit into the two components of signal detection theory: sensitivity and bias. Second, the authors aimed to assess the two subcomponents of sensitivity: detection and discrimination. They observed that the AB is only expressed in sensitivity. Furthermore, detection and discrimination were doubly dissociated. Detection modulated N2p and P3 ERP amplitude, but not frontoparietal beta-band coherence, whereas this pattern was reversed for discrimination.

Strengths:

The experiment is elegantly designed, and the data -both behavioral and electrophysiological- are aptly analyzed. The outcomes, in particular the dissociation between detection and discrimination blinks, are very consistently and clearly supported by the results. The discussion of the results is also appropriately balanced.

Weaknesses:

The lack of an effect of stimulus contrast does not seem very surprising from what we know of the nature of AB already. Low-level perceptual factors are not thought to cause the AB. This is fine, as there are also other, novel findings reported. In their revision, the authors have bolstered the importance of these (null) findings by referring to AB-specific papers that would have predicted different outcomes in this regard.

The ERP analyses are extended in the revised manuscript, including those of the N1 component, which is now more appropriately analyzed at more lateral electrode sites.

Impact & Context:<br /> The results of this study will likely influence how we think about selective attention in the context of the AB phenomenon. In their revision, the authors have further extended their theoretical framing by referring to recent work on the nature of the AB deficit, showing that it can be discrete (all-or-none) and gradual.

Reviewer #2 (Public review):

Summary:

The authors aimed to explore and better understand the complex topographical organization of the human pulvinar, a brain region crucial for various high-order functions such as perception and attention. They sought to move beyond traditional histological subdivisions by investigating continuous 'gradients' of cortical connections along the dorsoventral and mediolateral axes. Using advanced imaging techniques and a comprehensive PET atlas of neurotransmitter receptors, the study aimed to identify and characterize these gradients in terms of structural connections, functional coactivation, and molecular binding patterns. Ultimately, the authors targeted to provide a more nuanced understanding of pulvinar anatomy and its implications for brain function in both healthy and diseased states.

Strengths:

A key strength of this study lies in the authors' effort to comprehensively combine multimodal data, encompassing both functional and structural connectomics, alongside the analysis of major neurotransmitter distributions. This approach enabled a more nuanced understanding of the overarching organizational principles of the pulvinar nucleus within the broader context of whole-brain connectivity. By employing cortex-wide correlation analyses of multimodal embedding patterns derived from 'gradients,' which provide spatial maps reflecting the underlying connectomic and molecular similarities across voxels, the study offers a thorough characterization of the functional neuroanatomy of the pulvinar.

Weaknesses:

Despite its strengths, the current manuscript falls short in presenting the authors' unique perspectives on integrating the diverse biological principles derived from the various neuroimaging modalities. The findings are predominantly reported as correlations between different gradient maps, without providing the in-depth interpretations that would allow for a more comprehensive understanding of the pulvinar's role as a central hub in the brain's network.

Reviewer #2 (Public review):

This work describes the single-cell expression profiling of thousands of cells of recombinant genotypes from a model natural-variation system, a cross between two divergent yeast strains.

I appreciate the addition of lines 282-291, which now makes the authors' point about one advantage of the single-cell technique for eQTL mapping clearly: the authors don't need to normalize for culture-to-culture variation the way standard bulk methods do (e.g. in Albert et al., 2018 for the current yeast cross), and without this normalization, they can integrate analyses of expression with those of estimates of growth behaviors from the abundance of a genotype in the pool. The main question the manuscript addresses with the latter, in Figure 3, is how much variation in growth appears to have nothing to do with expression, for which the answer the authors given is 30%. I agree that this represents a novel finding. The caveats are (1) the particular point will perhaps only be interesting to a small slice of the eQTL research community; (2) the authors provide no statistical controls/error estimate or independent validation of the variance partitioning analysis in Figure 3, and (3) the authors don't seem to use the single-cell growth/fitness estimates for anything else, as Figure 4 uses loci mapped to growth from a previously published, standard culture-by-culture approach. It would be appropriate for the manuscript to mention these caveats.

I also think it is not appropriate for the manuscript to avoid a comparison between the current work and Boocock et al., which reports single-cell eQTL mapping in the same yeast system. I recommend a citation and statement of the similarities and differences between the papers.

I appreciate the new statement about the single-cell technique affording better power in eQTL mapping (lines 445-453).

Reviewer #2 (Public review):

Summary:

Whole-brain network modeling is a common type of dynamical systems-based method to create individualized models of brain activity incorporating subject-specific structural connectome inferred from diffusion imaging data. This type of model has often been used to infer biophysical parameters of the individual brain that cannot be directly measured using neuroimaging but may be relevant to specific cognitive functions or diseases. Here, Ziaeemehr et al introduce a new toolkit, named "Virtual Brain Inference" (VBI), offering a new computational approach for estimating these parameters using Bayesian inference powered by artificial neural networks. The basic idea is to use simulated data, given known parameters, to train artificial neural networks to solve the inverse problem, namely, to infer the posterior distribution over the parameter space given data-derived features. The authors have demonstrated the utility of the toolkit using simulated data from several commonly used whole-brain network models in case studies.

Strengths:

(1) Model inversion is an important problem in whole-brain network modeling. The toolkit presents a significant methodological step up from common practices, with the potential to broadly impact how the community infers model parameters.

(2) Notably, the method allows the estimation of the posterior distribution of parameters instead of a point estimation, which provides information about the uncertainty of the estimation, which is generally lacking in existing methods.

(3) The case studies were able to demonstrate the detection of degeneracy in the parameters, which is important. Degeneracy is quite common in this type of model. If not handled mindfully, they may lead to spurious or stable parameter estimation. Thus, the toolkit can potentially be used to improve feature selection or to simply indicate the uncertainty.

(4) In principle, the posterior distribution can be directly computed given new data without doing any additional simulation, which could improve the efficiency of parameter inference on the artificial neural network if well-trained.

Weaknesses:

(1) While the posterior estimator was trained with a large quantity of simulated data, the testing/validation is only demonstrated with a single case study (one point in parameter space) per model. This is not sufficient to demonstrate the method's accuracy and reliability, but only its feasibility. Demonstrating the accuracy and reliability of the posterior estimation in large test sets would inspire more confidence.

(2) The authors have only demonstrated validation of the method using simulated data, but not features derived from actual EEG/MEG or fMRI data. So, it is unclear if the posterior estimator, when applied to real data, would produce results as sensible as using simulated data. Human data can often look quite different from the simulated data, which may be considered out of distribution. Thus, the authors should consider using simulated test data with out-of-distribution parameters to validate the method and using real human data to demonstrate, e.g., the reliability of the method across sessions.

(3) The z-scores used to measure prediction error are generally between 1-3, which seems quite large to me. It would give readers a better sense of the utility of the method if comparisons to simpler methods, such as k-nearest neighbor methods, are provided in terms of accuracy.

(4) A lot of simulations are required to train the posterior estimator, which seems much more than existing approaches. Inferring from Figure S1, at the required order of magnitudes of the number of simulations, the simulation time could range from days to years, depending on the hardware. Although once the estimator is well-trained, the parameter inverse given new data will be very fast, it is not clear to me how often such use cases would be encountered. Because the estimator is trained based on an individual connectome, it can only be used to do parameter inversion for the same subject. Typically, we only have one session of resting state data from each participant, while longitudinal resting state data where we can assume the structural connectome remains constant, is rare. Thus, the cost-efficiency and practical utility of training such a posterior estimator remains unclear.

Reviewer #2 (Public review):

Summary:

The cerebellum is known to be vulnerable to aging, yet specific cell type vulnerability remains understudied. This important study convincingly demonstrates that the normal aged mouse cerebellum exhibits Purkinje cell loss, and that the vulnerable PCs to age are arranged on the basis of the known zebrin stripe pattern that represents a particular subtype of the PCs. Although the patterns of PC loss were analyzed qualitatively, the phenotype is robust enough to clearly appreciate that PC loss occurs predominantly in zebrin-negative regions when combined with zebrin immunohistochemistry. Interestingly, the authors demonstrate that this phenotype appears stochastically even within the inbred C57BL/6J mouse strain examined, though the mechanisms behind this individual variability remain unexplored. In contrast to the expectation that the PC loss could account for age-related motor decline, the authors did not find any correlation between them. While the authors attempt to draw parallels with normal human aging, the human phenotypes have not been conclusively shown to match those in mice beyond the occurrence of potentially age-related PC loss. Future studies should investigate why this PC loss phenotype occurs stochastically across the population and whether these findings parallel human cerebellar aging.

Strength:

(1) Banding pattern of PC loss is very clearly demonstrated by combining immunostaining for zebrin.

(2) A critical methodological concern that a standard PC marker, calbindin, could be compromised in aging has been addressed by performing control experiments with appropriate counterstaining.

(3) Parallels with neurodegenerative phenotype would be helpful to understand the mechanisms of PC loss in the future.

Weakness:

(1) Limited strain diversity: The study exclusively uses C57BL/6J mice despite known genetic and motor differences even the closely related strains like C57BL/6N.

(2) No correlation quantified between the degree of PC loss, aging, and motor performance.

(3) It has not been demonstrated whether the neurodegenerative changes are indeed observed in zebrin-negative PCs.

(4) The mechanisms of why only a subset of mice show PC loss remain unexplored and not discussed.

(5) Linkages with normal human aging and cerebellar function are not well supported. While motor behavioral assays captured phenotypes that mimic aged people, correlation with PC loss is demonstrated to be absent in mice. It remains unclear whether this PC loss phenomenon is universal or specific to a particular individual; and whether specific to a human PC subtype.

(6) Analyses in the paraflocculus are currently not easy to understand. This lobule has heterogeneous PC subtypes, developmentally or molecularly. Zebrin-weak and Zebrin-intense PCs are known to be arranged in stripes, which resembles the pattern of developmentally defined PC subsets (Fujita et al., 2014, Plos one; Fujita et al., 2012, J Neurosci). In the data presented, it is hard to appreciate whether the viewing angle is consistent relative to the angle of the paraflocculus. This may be a limitation of the analysis of the paraflocculus in general, that the orientation of this lobule is so susceptible to fixation and dissection. Discrepancy between PC loss stripe and zebrin pattern may be an overstatement, because appropriate analyses on the paraflocculus would require a rigorously standardized analytic method.

Reviewer #2 (Public review):

Summary:

The authors were building on prior research linking cortical norepinephrine in a test of attentional set shifting. They extended prior research by assessing the effects of excitatory or inhibitory DREADDs prior to the test of attentional set shifting.

Strengths:

The use of DREADDs in the previously validated test of attentional set shifting improves temporal control of corticopetal, noradrenergic pathways during behavior. While mice typically require multiple intradimensional shifts to form an attentional set, the subjects in the current study perform a variant of the task similar to that used in humans, improving the translational validity of the work.

Weaknesses:

A critical piece of evidence needed to support the behavioral claim that mice form an attentional set is a statistically significant difference between the number of trials to reach criterion at the intradimensional vs. the extradimensional stage of the task. Based on prior literature, this could be done as a planned comparison, which would improve the power to detect differences beyond that found using an HSD test. An additional methodological ambiguity is the amount of time between the administration of CNO and the performance of the ED. As reported, it seems likely that the DREADDS were impacting performance at multiple stages of the test.

Overall, the authors seem to have achieved their aims, but have failed to provide critical statistical support for claims made.

The work is likely to be of interest to the burgeoning number of scientists investigating the role of cortical norepinephrine in cognitive flexibility.

Reviewer #2 (Public review):

Summary:

This Phase II clinical trial investigates the combination of Gamma Knife Stereotactic Body Radiation Therapy (SBRT) with Tislelizumab for the treatment of metastatic colorectal cancer (mCRC) in patients with proficient mismatch repair (pMMR). The study addresses a critical clinical challenge in the management of pMMR CRC, focusing on the selection of appropriate candidates. The results suggest that the combination of Gamma Knife SBRT and Tislelizumab provides a safe and potent treatment option for patients with pMMR/MSS/MSI-L mCRC who have become refractory to first- and second-line chemotherapy. The study design is rigorous, and the outcomes are promising.

Advantage:

The trial design was meticulously structured, and appropriate statistical methods were employed to rigorously analyze the results. Bioinformatics approaches were utilized to further elucidate alterations in the patient's tumor microenvironment and to explore the underlying factors contributing to the observed differences in treatment efficacy. The conclusions drawn from this trial offer valuable insights for managing advanced colorectal cancer in patients who have not responded to first- and second-line therapies.

Weakness:

(1) Clarity and Structure of the Abstract<br /> - Results Section: The results section should contain important data, I suggest some important sequencing data should be shown to enhance understanding.<br /> (2) As the author using the NanoString assay for transcriptome analysis, more detail should be shown such as the version of R, and the bioinformatics analysis methods.<br /> (3) It is interesting for included patients that PD-L1 increase expression after Gamma Knife Stereotactic Body Radiation Therapy (SBRT) treatment, How to explain it?<br /> (4) It would be helpful to include a brief discussion of the limitations of the study, such as sample size constraints and their impact on the generalizability of the results. This will give readers a more comprehensive understanding of the findings.<br /> (5) Language Accuracy: There are a few instances where wording could be more professional or precise.

Revision comment:

The author had responded to all questions and improved the manuscript. The author's answers and revisions are very satisfactory to me. I believe it is an important study for the immunotherapy of colorectal cancer.

Reviewer #2 (Public review):

Summary:

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

Layer 4 - Yakoubi et al 2019 eLife<br /> Layer 5 - Yakoubi et al 2019 Cerebral Cortex,<br /> Layer 6 - Schmuhl-Giesen et al 2022 Cerebral Cortex

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

Strengths:

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

Weaknesses:

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

Reviewer #2 (Public review):

The authors hypothesized that chemostat propagated viromes could modulate the GM and reduce NEC lesions while avoiding potential side effects, such as the earlier onset of diarrhea. This is interesting.

Major Comments:

(1) As the authors state that the aim of the research is 'We hypothesized that chemostat propagated viromes could modulate the GM and reduce NEC lesions while avoiding potential side effects, such as earlier onset of diarrhea'.<br /> a) For the efficacy, in Figure 5, there is no significance in stomach pathology and enterocolitis between groups, even between the control group and experimental groups, is it because of the low incidence of NEC? This may affect the statistical power of the conclusions. Therefore, it is unclear how one can draw the conclusion that chemostat can reduce NEC lesions?<br /> b) Convincing pathology images would be helpful.<br /> c) For the safety, such as body weight development, FVT had no statistical significance difference from control, CVT, and CVT-MO. So how can the authors draw the conclusion that chemostat can avoid potential side effects?<br /> d) There is a lack of evidence to convince the reader that there is a decrease in eukaryotic viruses. More quantitative data here would be useful.

(2) Questions regarding Figure 3F:<br /> a) How can the medium have 'the baseline viral content'?<br /> b) What is the statistical significance of the relative abundance of specific eukaryotic viruses?<br /> c) The hosts for some of the listed eukaryotic viruses are neither pigs or humans, as such, the significance of a decrease in these viruses to humans is unclear.

(3) In this study, pH 6.5 was selected as the pH value for chemostat cultivation, but considering the different adaptability of different bacteria to pH, it is recommended to further explore the effect of pH on bacteria and virus groups. In particular, it was optimized to maintain the growth of beneficial bacteria such as Lactobacillaceae and Bacteroides in order to improve the effect of chemostat cultivation.

(4) Please improve the quality of the images, charts, error bars, and statistical significance markers throughout and mark the n's. used in each experiment.

Reviewer #2 (Public review):

Trac, Huang, et al used the AZ Drug Combination Prediction DREAM challenge data to make a new random forest-based model for drug synergy. They make comparisons to the winning method and also show that their model has some predictive capacity for a completely different dataset. They highlight the ability of the model to be interpretable in terms of pathway and target interactions for synergistic effects.

In their revised manuscript and response, the authors have tried to address all points. I do not fully agree with them about the definition of overfitting still. If the objective it to identify synergies given any 2 drugs, not just those in a dataset at different doses, then the results certainly appear overfit to the training set given the performance degradation. However, at this time, I cannot add any useful suggestions to improve performance.

Reviewer #2 (Public review):

The manuscript by Liu and colleagues applied Mendelian Randomization (MR) techniques to study the causal relationship of atherosclerosis (categorized into four subtypes) and intracranial aneurysms (classified as unruptured or ruptured), as well as the potential mediation by 12 plasma matrix metalloproteinase (MMP) levels. The authors have followed rigorous MR analysis guidelines by using multiple analytical approaches, implementing strict selection criteria, and employing comprehensive sensitivity analyses. One of the strengths is the lack of overlapping samples in their two-sample MR analysis. This approach helps mitigate potential biases and increases the reliability of their causal inference. The analysis is fundamentally sound, but there are still several nuanced areas where the methodology could be strengthened. Given that most of the identified causal associations do not hold after correcting for multiple tests, the conclusions should be carefully reviewed to be fully supported by the results.

The recommendations below are meant to enhance the already robust approach.

(1) The selection of 12 MMPs lacks a clear, explicit rationale in the provided excerpt. A more detailed explanation of why these specific MMPs were chosen would strengthen the methodological rigor.

(2) Adjusting p-value for multiple testing using Bonferroni correction needs to be elucidated better.

(3) The authors should provide a more robust explanation of why they shifted from 5×10-9 to 5×10-6 to select genomic instruments.

(4) Egger's intercept may be a more robust approach for this study to test horizontal pleiotropy rather than MR-PRESSO.

Reviewer #2 (Public review):

Summary

The report by Dalas and colleagues introduces a significant novelty in the field of pentameric ligand-gated ion channels (pLGICs). Within this family of receptors, numerous structures are available, but a widely recognised problem remains in assigning structures to functional states observed in biological membranes. Here, the authors obtain both structural and functional information of a pLGIC in a liposome environment. The model receptor ELIC is captured in the resting, desensitized, and open states. Structures in large nanodiscs, possibly biased by receptor-scaffold protein interactions, are also reported. Altogether, these results set the stage for the adoption of liposomes as a proxy for the biological membranes, for cryoEM studies of pLGICs and membrane proteins in general.

Strengths

The structural data is comprehensive, with structures in liposomes in the 3 main states (and for each, both inward-facing and outward-facing), and an agonist-bound structure in the large spNW25 nanodisc (and a retreatment of previous data obtained in a smaller disc). It adds up to a series of work from the same team that constitutes a much-needed exploration of various types of environment for the transmembrane domain of pLGICs. The structural analysis is thorough.

The tone of the report is particularly pleasant, in the sense that the authors' claims are not inflated. For instance, a sentence such as "By performing structural and functional characterization under the same reconstitution conditions, we increase our confidence in the functional annotation of these structures." is exemplary.

Weaknesses

Core parts of the method are not described and/or discussed in enough detail. While I do believe that liposomes will be, in most cases, better than, say, nanodiscs, the process that leads from the protein in its membrane down to the liposome will play a big role in preserving the native structure, and should be an integral part of the report. Therefore, I strongly felt that biochemistry should be better described and discussed. The results section starts with "Optimal reconstitution of ELIC in liposomes [...] was achieved by dialysis". There is no information on why dialysis is optimal, what it was compared to, the distribution of liposome sizes using different preparation techniques, etc... Reading the title, I would have expected a couple of paragraphs and figure panels on liposome reconstitution. Similarly, potential biochemical challenges are not discussed. The methods section mentions that the sample was "dialyzed [...] over 5-7 days". In such a time window, most of the members of this protein family would aggregate, and it is therefore a protocol that can not be directly generalised. This has to be mentioned explicitly, and a discussion on why this can't be done in two days, what else the authors tested (biobeads? ... ?) would strengthen the manuscript.

To a lesser extent, the relative lack of both technical details and of a broad discussion also pertains to the cryoEM and thallium flux results. Regarding the cryoEM part, the authors focus their analysis on reconstructions from outward-facing particles on the basis of their better resolutions, yet there was little discussion about it. Is it common for liposome-based structures? Are inward-facing reconstructions worse because of the increased background due to electrons going through two membranes? Are there often impurities inside the liposomes (we see some in the figures)? The influence of the membrane mimetics on conformation could be discussed by referring to other families of proteins where it has been explored (for instance, ABC transporters, but I'm sure there are many other examples). If there are studies in other families of channels in liposomes that were inspirational, those could be mentioned. Regarding thallium flux assays, one argument is that they give access to kinetics and set the stage for time-resolved cryoEM, but if I did not miss it, no comparison of kinetics with other techniques, such as electrophysiology, nor references to eventual pioneer time-resolved studies are provided.

Altogether, in my view, an updated version would benefit from insisting on every aspect of the methodological development. I may well be wrong, but I see this paper more like a milestone on sample prep for cryoEM imaging than being about the details of the ELIC conformations.

Reviewer #2 (Public review):

Summary:

This Phase II clinical trial investigates the combination of Gamma Knife Stereotactic Body Radiation Therapy (SBRT) with Tislelizumab for the treatment of metastatic colorectal cancer (mCRC) in patients with proficient mismatch repair (pMMR). The study addresses a critical clinical challenge in the management of pMMR CRC, focusing on the selection of appropriate candidates. The results suggest that the combination of Gamma Knife SBRT and Tislelizumab provides a safe and potent treatment option for patients with pMMR/MSS/MSI-L mCRC who have become refractory to first- and second-line chemotherapy. The study design is rigorous, and the outcomes are promising.

Advantage:

The trial design was meticulously structured, and appropriate statistical methods were employed to rigorously analyze the results. Bioinformatics approaches were utilized to further elucidate alterations in the patient's tumor microenvironment and to explore the underlying factors contributing to the observed differences in treatment efficacy. The conclusions drawn from this trial offer valuable insights for managing advanced colorectal cancer in patients who have not responded to first- and second-line therapies.

Weakness:

(1) Clarity and Structure of the Abstract<br /> - Results Section: The results section should contain important data, I suggest some important sequencing data should be shown to enhance understanding.<br /> (2) As the author using the NanoString assay for transcriptome analysis, more detail should be shown such as the version of R, and the bioinformatics analysis methods.<br /> (3) It is interesting for included patients that PD-L1 increase expression after Gamma Knife Stereotactic Body Radiation Therapy (SBRT) treatment, How to explain it?<br /> (4) It would be helpful to include a brief discussion of the limitations of the study, such as sample size constraints and their impact on the generalizability of the results. This will give readers a more comprehensive understanding of the findings.<br /> (5) Language Accuracy: There are a few instances where wording could be more professional or precise.

Reviewer #2 (Public review):

Summary:

Ning and colleagues present studies supporting a role for breast carcinoma amplified sequence 2 (Bcas2) in positively regulating primitive wave hematopoiesis through amplification of beta-catenin-dependent (canonical) Wnt signaling. The authors present compelling evidence that zebrafish bcas2 is expressed at the right time and place to be involved in primitive hematopoiesis, that there are primitive hematopoietic defects in hetero- and homozygous mutant and knockdown embryos, that Bcas2 mechanistically positively regulates canonical Wnt signaling, and that Bcas2 is required for nuclear retention of B-cat through physical interaction involving armadillo repeats 9-12 of B-cat and the coiled-coil domains of Bcas2. Overall, the data and writing are clean, clear, and compelling. This study is a first rate analysis of a strong phenotype with highly supportive mechanistic data. The findings shed light on the controversial question of whether, when, and how canonical Wnt signaling may be involved in hematopoietic development.

In the revised version of their previous work, they have included responses to some of our suggestions for minor experiments and edits. We previously suggested they examine the structural compatibility of a Bcas2/beta-catenin dimer with binding to the DNA-binding protein Tcf7l1 (previously Tcf3), which would be expected for a beta-catenin nuclear-retention factor that potentiates canonical Wnt signaling responses. Although the authors did not test compatibility of Bcas2 with Tcf3 binding to beta-catenin, they show that a three-way complex with the family member Tcf4 is possible (Fig. S12), which suggests that Lef/Tcf family binding in general is plausible.

The authors' acceptance of our suggestion to evaluate cdx and hox gene expression is welcome, as these genes have previously been defined as canonical Wnt targets (Lengerke et al., 2009) that regionalize the lateral plate mesoderm (LPM) and confer pre-hematopoietic identity there (Davidson et al., 2003; Davidson and Zon, 2004). The authors' finding that cdx4 and hoxa9a are diminished in the bcas2 mutants (Fig. S7) validates this suggestion and seem to imply that the primary defect here is specification of the early hematopoietic field in the LPM, however the results are a little confusing or surprising given that scl - which is unaffected in the bcas2 mutant (Fig. 2A) - is a downstream target of Cdx4 (Davidson et al., 2003, Fig. 1b, 3d). The results in the current submission imply that early maintenance of pre-hematopoietic competence in the LPM is a canonical-Wnt-directed phenomenon separable from the earliest specification of the hematopoietic field. We believe it would be of value to further evaluate regulation of cdx1, which has been shown to cooperate with cdx4 in regulation of the LPM hematopoietic field, as well as analyze some of the putative downstream hox family targets.

We previously reviewed the article as suitable for publication and we continue to support our prior assessment. The authors have presented strong data supporting a role for Bcas2 in hematopoietic development across phyla and a mechanistic involvement in promoting canonical Wnt signaling.

Strengths:

(1) The study features clear and compelling phenotypes and results.<br /> (2) The manuscript narrative exposition and writing are clear and compelling.<br /> (3) The authors have attended to important technical nuances sometimes overlooked, for example, focusing on different pools of cytosolic or nuclear b-catenin.<br /> (4) The study sheds light on a controversial subject: regulation of hematopoietic development by canonical Wnt signaling and presents clear evidence of a role.<br /> (5) The authors present evidence of phylogenetic conservation of the pathway.

Reviewer #2 (Public review):

This paper describes an interesting observation that ER-targeted misfolded proteins are trapped within vesicles inside nucleus to facilitate quality control during cell division. This work supports the concept that transient sequestration of misfolded proteins is a fundamental mechanism of protein quality control. The authors satisfactorily addressed several points asked in the review of first submission. The manuscript is improved but still unable to fully address the mechanisms.

Strengths:

The observations in this manuscript are very interesting and open up many questions on proteostasis biology.

Weaknesses:

Despite inclusions of several protein-level experiments, the manuscript remained a microscopy-driven work and missed the opportunity to work out the mechanisms behind the observations.

Reviewer #2 (Public review):

Summary

The study is an innovative and fundamental study that clarified important aspects of brain processes for integration of information from speech and iconic gesture (i.e., gesture that depicts action, movement, and shape), based on tDCS, TMS and EEG experiments. They evaluated their speech and gesture stimuli in information-theoretic ways and calculated how informative speech is (i.e., entropy), how informative gesture is, and how much shared information speech and gesture encode. The tDCS and TMS studies found that the left IFG and pMTG, the two areas that were activated in fMRI studies on speech-gesture integration in the previous literature, are causally implicated in speech-gesture integration. The size of tDC and TMS effects are correlated with entropy of the stimuli or mutual information, which indicates that the effects stems from the modulation of information decoding/integration processes. The EEG study showed that various ERP (event-related potential, e.g., N1-P2, N400, LPC) effects that have been observed in speech-gesture integration experiments in the previous literature are modulated by the entropy of speech/gesture and mutual information. This makes it clear that these effects are related to information decoding processes. The authors propose a model of how speech-gesture integration process unfolds in time, and how IFG and pMTG interact with each other in that process.

Strengths

The key strength of this study is that the authors used information-theoretic measures of their stimuli (i.e., entropy and mutual information between speech and gesture) in all of their analyses. This made it clear that the neuro-modulation (tDCS, TMS) affected information decoding/integration and ERP effects reflect information decoding/integration. This study used tDCS and TMS methods to demonstrate that left IFG and pMTG are causally involved in speech-gesture integration. The size of tDCS and TMS effects are correlated with information-theoretic measures of the stimuli, which indicate that the effects indeed stem from disruption/facilitation of information decoding/integration process (rather than generic excitation/inhibition). The authors' results also showed correlation between information-theoretic measures of stimuli with various ERP effects. This indicates that these ERP effects reflect the information decoding/integration process.

Weakness

The "mutual information" cannot capture all types of interplay of the meaning of speech and gesture. The mutual information is calculated based on what information can be decoded from speech alone and what information can be decoded from gesture alone. However, when speech and gesture are combined, a novel meaning can emerge, which cannot be decoded from a single modality alone. When example, a person produce a gesture of writing something with a pen, while saying "He paid". The speech-gesture combination can be interpreted as "paying by signing a cheque". It is highly unlikely that this meaning is decoded when people hear speech only or see gestures only. The current study cannot address how such speech-gesture integration occur in the brain, and what ERP effects may reflect such a process. The future studies can classify different types of speech-gesture integration and investigate neural processes that underlie each type. Another important topic for future studies is to investigate how the neural processes of speech-gesture integration change when the relative timing between the speech stimulus and the gesture stimulus changes.

Comments on revisions: The authors addressed my concerns well.