Reviewer #1 (Public Review):

The authors build on their previous study that showed the midgut microbiome does not oscillate in Drosophila. Here, they focus on metabolites and find that these rhythms are in fact microbiome-dependent. Tests of time-restricted feeding, a clock gene mutant, and diet reveal additional regulatory roles for factors that dictate the timing and rhythmicity of metabolites. The study is well-written and straightforward, adding to a growing body of literature that shows the time of food consumption affects microbial metabolism which in turn could affect the host.

Some additional questions and considerations remain:

(1) The main finding that the microbiome promotes metabolite rhythms is very interesting. Which microbiota are likely to be responsible for these effects? The author's previous work in this area may shed light on this question. Are specific microbiota linked to some of the metabolic pathways investigated in Figure 5?

(2) TF increases the number of rhythmic metabolites in both microbiome-containing and abiotic flies in Figure 1. This is somewhat surprising given that flies typically eat during the daytime rather than at night, very similar to TF conditions. I would have assumed that in a clock-functioning animal, the effect of restricting food availability should not make a huge difference in the time of food consumption, and thus downstream impacts on metabolism and microbiome. Can the authors measure food intake directly to compare the ad-lib vs TF flies to see if there are changes in food intake? Would restricting feeding to other times of day shift the timing of metabolites accordingly?

(3) In Figure 2, Per loss of function reveals a change in the phase of rhythmic metabolites. In addition, the effect of the microbiome on these is very different = The per mutants show increased numbers of rhythmic metabolites when the microbiome is absent, unlike the controls. Is it possible that these changes are due to altered daily feeding rhythms in per mutants? Testing the time and amount of food consumed by the per mutant flies would address this question. Would TF in the per mutants rescue their metabolite rhythms and make them resemble clock-functioning controls?

(4) The calorie content of each diet - normal vs high protein vs high-sugar are different. The possibility of a calorie effect rather than a difference in nutrition (protein/carbohydrate) should be discussed. Another issue worth considering is the effect of high protein/sugar on the microbiome itself. While the microbiome doesn't seem to affect rhythms in the high-protein diet, the high-sugar diet seems highly microbiome-dependent in Supplementary Fig 8C vs D. Does the diet impact the microbiome and thus metabolite rhythmicity downstream?

(5) It would be good if a supplementary table was provided outlining the specific metabolites that are shown in the radial plots. It is not clear if the rhythms shown in the figures refer to the same metabolites peaking at the same time, or rather the overall abundance of completely different metabolites. This information would be useful for future research in this area.

Reviewer #1 (Public Review):

Summary:

In this work by Wang et al., the authors use single-molecule super-resolution microscopy together with biochemical assays to quantify the organization of Nipah virus fusion protein F (NiV-F) on cell and viral membranes. They find that these proteins form nanoscale clusters which favors membrane fusion activation, and that the physical parameters of these clusters are unaffected by protein expression level and endosomal cleavage. Furthermore, they find that the cluster organization is affected by mutations in the trimer interface on the NiV-F ectodomain and the putative oligomerization motif on the transmembrane domain, and that the clusters are stabilized by interactions among NiV-F, the AP2-complex, and the clathrin coat assembly. This work improves our understanding of the NiV fusion machinery, which may have implications also for our understanding of the function of other viruses.

Strengths:

The conclusions of this paper are well-supported by the presented data. This study sheds light on the activation mechanisms underlying the NiV fusion machinery.

Weaknesses:

The authors provide limited details of the convolutional neural network they developed in this work. Even though custom-codes are made available, a description of the network and specifications of how it was used in this work would aid the readers in assessing its performance and applicability. The same holds for the custom-written OPTICS algorithm. Furthermore, limited details are provided for the imaging setup, oxygen scavenging buffer, and analysis for the single-molecule data, which limits reproducibility in other laboratories. The claim of 10 nm resolution is not backed up by data and seems low given the imaging conditions and fluorophores used. Fourier Ring Correlation analysis would have validated this claim. If the authors refer to localization precision rather than resolution, then this should be specified and appropriate data provided to support this claim.

Reviewer #1 (Public Review):

Summary:

The study "Endogenous oligomer formation underlies DVL2 condensates and promotes Wnt/β-catenin signaling" by Senem Ntourmas et al. contributes to the understanding of phase separation in Dishevelled (DVL) proteins, specifically focusing on DVL2. It builds upon existing research by investigating the endogenous complexes of DVL2 using ultracentrifugation and contrasting them with DVL1 and DVL3 behavior. The study identifies a DVL2-specific region involved in condensate formation and introduces the "two-step" concept of DVL2 condensate formation, enriching the field's knowledge.

Strengths:

A notable strength of this study is the validation of endogenous DVL2 complexes, providing insights into its behavior compared to DVL1 and DVL3. The functional validation of the DVL C-terminus (here termed conserved domain 2 (CD2) and the identification of DVL2-specific regions (here termed LCR4) involved in condensate formation are significant contributions that complement the current knowledge on the importance of DVL DIX domain, DEP domain and intrinsically disordered regions between DIX and PDZ domains. Additionally, the introduction of the concept where oligomerization (step 1) precedes condensate formation (step 2) is an interesting hypothesis, which can be further experimentally challenged in the future.

Weaknesses:

However, the applicability of the findings to full-length DVL2 protein, hence the physiological relevance, is limited. This is mostly due to the fact that the authors almost completely depend on the set of DVL2 mutants, which lack the (i) DEP domain and (ii) nuclear export signal (NES). These variants fail to establish DEP domain-mediated interactions, including those with FZD receptors. Of note, the DEP domain itself represents a dimerization/tetramerization interface, which could affect the protein condensate formation of these mutants. Possibly even more importantly, the used mutants localize into the nucleus, which has different biochemical & biophysical properties than a cytoplasm, where DVL typically reside, which in turn affects the condensate formation. On top, in the nucleus, most of the DVL binding partners, including relevant kinases, which were reported to affect protein condensate formation, are missing.

Second, the use of an overexpression system, while suitable for comparing DVL2 protein condensate features, falls short in functional assays. The study could benefit from employing established "rescue systems" using DVL1/2/3 knockout cells and re-expression of DVL variants for more robust functional assessments.

Furthermore, the discussion and introduction overlook some essential aspects of DVL biology. One such example is the importance of the open/close conformation of DVL and its effects on DVL phase separation and activity. In the context of this study, it is important to say that this conformational plasticity is mediated by DVL C-terminus (CD2 in this study). The second example is the reported roles of DVL1 and DVL3, which can both mediate the Wnt3a signal. How this can be interpreted when DVL1 and DVL3 lack LCR4 and still form condensates?

In order to increase the physiological relevance of the study, I would recommend analyzing several key mutants in the context of the full-length DVL2 protein using the rescue/complementation system. Further, a more thorough discussion and connections with the existing literature on DVL protein condensates/puncta/LLPS can improve the impact of the study.

Reviewer #1 (Public Review):

This manuscript presents an extremely exciting and very timely analysis of the role that the nucleosome acidic patch plays in SWR1-catalyzed histone exchange. Intriguingly, SWR1 loses activity almost completely if any of the acidic patches are absent. To my knowledge, this makes SWR1 the first remodeler with such a unique and pronounced requirement for the acidic patch. The authors demonstrate that SWR1 affinity is dramatically reduced if at least one of the acidic patches is absent, pointing to a key role of the acidic patch in SWR1 binding to the nucleosome. The authors also pinpoint a specific subunit - Swc5 - that can bind nucleosomes and engage the acidic patch and obtain a cryo-EM structure of Swc5 bound to a nucleosome. They also identify a conserved arginine-rich motif in this subunit that is critical for nucleosome binding and histone exchange in vitro and for SWR1 function in vivo. The authors provide evidence that suggests a direct interaction between this motif and the acidic patch.

Strengths:

The manuscript is well-written and the experimental data are of outstanding quality and importance for the field. This manuscript significantly expands our understanding of the fundamentally important and complex process of H2A.Z deposition by SWR1 and would be of great interest for a broad readership.

Reviewer #1 (Public Review):

Summary:

Zhao and colleagues employ Drosophila nephrocytes as a model to investigate the effects of a high-fat diet on these podocyte-like cells. Through a highly focused analysis, they initially confirm previous research in their hands demonstrating impaired nephrocyte function and move on to observe the mislocalization of a slit diaphragm-associated protein (pyd). Employing a reporter construct, they identify the activation of the JAK/STAT signaling pathway in nephrocytes. Subsequently, the authors demonstrate the involvement of this pathway in nephrocyte function from multiple angles, using a gain-of-function construct, silencing of an inhibitor, and ectopic overexpression of a ligand. Silencing the effector Stat92E via RNAi or inhibiting JAK/STAT with Methotrexate effectively restored impaired nephrocyte function induced by a high-fat diet, while showing no impact under normal dietary conditions.

Strengths:

The findings establish a link between JAK/STAT activity and the impact of a high-fat diet on nephrocytes. This nicely underscores the importance of organ crosstalk for nephrocytes and supports a potential role for JAK/STAT in diabetic nephropathy, as previously suggested by other models.

Weaknesses:

The analysis is overly reliant on tracer endocytosis and single lines. Immunofluorescence of slit diaphragm proteins would provide a more specific assessment of the phenotypes.

Reviewer #1 (Public Review):

Summary:

TMEM16, OSCA/TMEM63, and TMC belong to a large superfamily of ion channels where TMEM16 members are calcium-activated lipid scramblases and chloride channels, whereas OSCA/TMEM63 and TMCs are mechanically activated ion channels. In the TMEM16 family, TMEM16F is a well-characterized calcium-activated lipid scramblase that plays an important role in processes like blood coagulation, cell death signaling, and phagocytosis. In a previous study, the group demonstrated that lysine mutation in TM4 of TMEM16A can enable the calcium-activated chloride channel to permeate phospholipids too. Based on this they hypothesize that the energy barrier for lipid scramblase in these ion channels is low, and that modification in the hydrophobic gate region by introducing a charged side chain between the TM4/6 interface in TMEM16 and OSCA/TMEM63 family can allow lipid scramblase. In this manuscript, using scramblase activity via Annexin V binding to phosphatidylserine, and electrophysiology, the authors demonstrate that lysine mutation in TM4 of TMEM16F and TMEM16A can cause constitutive lipid scramblase activity. The authors then go on to show that analogous mutations in OSCA1.2 and TMEM63A can lead to scramblase activity.

Strengths:

Overall, the authors introduce an interesting concept that this large superfamily can permeate ions and lipids.

Weaknesses:

The electrophysiology data does not entirely support their claims.

Reviewer #1 (Public Review):

Using a combination of cutting-edge high-resolution approaches (expansion microscopy, SIM, and CLEM) and biochemical approaches (in vitro translocation of actin filaments, cargo uptake assays, and drug treatment), the authors revisit previous results about TbMyo1 and TbACT in the bloodstream form (BSF) of Trypanosoma brucei. They show that a great part of the myosin motor is cytoplasmic but the fraction associated with organelles is in proximity to the endosomal system. In addition, they show that TbMyo1 can move actin filaments in vitro and visualize for the first time this actomyosin system using specific antibodies, a "classical" antibody for TbMyo1, and a chromobody for actin. Finally, using latrunculin A, which sequesters G-actin and prevents F-actin assembly, the authors show the delocalization and eventually the loss of the filamentous actin signal as well as the concomitant loss of the endosomal system integrity. However, they do not assess the localization of TbMyo1 in the same conditions.

Overall the work is well conducted and convincing. The conclusions are not over-interpreted and are supported by the experimental results.

Reviewer #1 (Public Review):

Summary:

The authors profile gene expression, chromatin accessibility, and chromosomal architecture (by Hi-C) in activated CD4 T cells and use this information to link non-coding variants associated with autoimmune diseases with putative target genes. They find over 1000 genes physically linked with autoimmune disease loci in these cells, many of which are upregulated upon T cell activation. Focusing on IL2, they dissect the regulatory architecture of this locus, including the allelic effects of GWAS variants. They also intersect their variant-to-gene lists with data from CRISPR screens for genes involved in CD4 T cell activation and expression of inflammatory genes, finding enrichments for regulators. Finally, they showed that pharmacological inhibition of some of these genes impacts T-cell activation.

This is a solid study that follows a well-established canvas for variant-to-gene prioritisation using 3D genomics, applying it to activated T cells. The authors go some way in validating the lists of candidate genes, as well as exploring the regulatory architecture of a candidate GWAS locus. Jointly with data from previous studies performing variant-to-gene assignment in activated CD4 T cells (and other immune cells), this work provides a useful additional resource for interpreting autoimmune disease-associated genetic variation.

Suggestions for improvement:

Autoimmune disease variants were already linked with genes in CD28-stimulated CD4 T cells using chromosome conformation capture, specifically Promoter CHi-C and the COGS pipeline (Javierre et al., Cell 2016; Burren et al., Genome Biol 2017; Yang et al., Nat Comms 2020). The authors cite these papers and present a comparative analysis of their variant-to-gene assignments (in addition to scRNA-seq eQTL-based assignments). Furthermore, they find that the Burren analysis yields a higher enrichment for gold standard genes.

The obvious question that the authors don't venture into is why the results are quite different. In principle, this could be due to the differences between:<br /> (a) the cell stimulation procedure<br /> (b) the GWAS datasets used<br /> (c) the types of assay (Hi-C vs Capture Hi-C)<br /> (d) approaches for defining gene-linked regions (loops vs neighbourhoods)<br /> (e) how the GWAS signals at gene-linked regions are aggregated (e.g., the flavours of COGS in Javierre and Burren vs the authors' approach).

Re (a), I'm not sure the authors make it explicitly clear in the main text that the Capture Hi-C-based studies also use *stimulated* CD4 T cells, particularly in the section "Comparative predictive power...". So the cells used are pretty much the same, and the differences likely arise from points (b) to (e).

It would be useful for the community to understand more clearly what is driving these differences, ideally with some added data. Could the authors, for example, take the PCHi-C data from Javierre/Burren and use their GWAS data and variant-to-gene assignment algorithms?

In addition, given that the authors use Hi-C, a popular method for V2G prioritisation for this type of data is currently ABC (Nasser et al, Nature 2021). Could the authors provide a comparative analysis with respect to the V2G assignments in the paper and, if they see it appropriate, also run ABC-based GWAS integration on their own Hi-C data?

Reviewer #1 (Public Review):

In this study, the authors examined the role of IBTK, a substrate-binding adaptor of the CRL3 ubiquitin ligase complex, in modulating the activity of the eiF4F translation initiation complex. They find that IBTK mediates the non-degradative ubiquitination of eiF4A1, promotes cap-dependent translational initiation, nascent protein synthesis, oncogene expression, and tumor cell growth. Correspondingly, phosphorylation of  IBTK by mTORC1/ S6K1 increases eIF4A1 ubiquitination and sustains oncogenic translation.

Strengths:

This study utilizes multiple biochemical, proteomic, functional and cell biology assays to substantiate their results.  Importantly, the work nominates IBTK as a unique substrate of mTORC1, and further validates eiF4A1 ( a crucial subunit of the ei44F complex) as a promising therapeutic target in cancer. Since IBTK interacts broadly with multiple members of the translational initial complex- it will be interesting to examine its role in eiF2alpha-mediated ER stress as well as eiF3-mediated translation. Additionally, since IBTK exerts pro-survival effects in multiple cell types, it will be of relevance to characterize the role of IBTK in mediating increased mTORC1 mediated translation in other tumor types, thus potentially impacting their treatment with eiF4F inhibitors.

Limitations/Weaknesses:

The findings are mostly well supported by data, but some areas need clarification and could potentially be enhanced with further experiments:

(1) Since eiF4A1 appears to function downstream of IBTK1, can the effects of IBTK1 KO/KD in reducing puromycin incorporation ( in Fig 3A),  cap-dependent luciferase reporter activity (Fig 3G), reduced oncogene expression ( Fig 4A) or 2D growth/ invasion assays (Fig 4) be overcome or bypassed by overexpressing eiF4A1? These could potentially be tested in future studies. <br /> (2) The decrease in nascent protein synthesis in puromycin incorporation assays in Figure 3A suggests that the effects of IBTK KO are comparable to and additive with silvesterol. It would be of interest to examine whether silvesterol decreases nascent protein synthesis or increases stress granules in the IBTK KO cells stably expressing IBTK as well. <br /> (3) The data presented in Figure 5 regarding the role of mTORC1 in IBTK-mediated eiF4A1 ubiquitination needs further clarification on several points:<br /> - It is not clear if the experiments in Figure 5F with Phos-tag gels are using the FLAG-IBTK deletion mutant or the peptide containing the mTOR sites as it is mentioned on line 517, page 19 "To do so, we generated an IBTK deletion mutant (900-1150 aa) spanning the potential mTORC1-regulated phosphorylation sites" This needs further clarification.<br /> -It may be of benefit to repeat the Phos tag experiments with full length FLAG-IBTK and/or endogenous IBTK with molecular weight markers indicating size of migrated bands.<br /> -Additionally, torin or Lambda phosphatase treatment may be used to confirm the specificity of the band in separate experiments.<br /> -Phos-tag gels with the IBTK CRISPR KO line would also help confirm that the non-phosphorylated band is indeed IBTK. <br /> -It is unclear why the lower, phosphorylated bands seem to be increasing ( rather than decreasing) with AA starvation/ Rapa in Fig 5H.

Reviewer #1 (Public Review):

Summary:

The authors used structural and biophysical methods to provide insight into Parkin regulation. The breadth of data supporting their findings was impressive and generally well-orchestrated. Still, the impact of their results builds on recent structural studies and the stated impact is based on these prior works.

Strengths:

(1) After reading through the paper, the major findings are:<br /> - RING2 and pUbl compete for binding to RING0.<br /> - Parkin can dimerize.<br /> - ACT plays an important role in enzyme kinetics.

(2) The use of molecular scissors in their construct represents a creative approach to examining inter-domain interactions.

(3) From my assessment, the experiments are well-conceived and executed.

Weaknesses:

(1) The manuscript, as written, is NOT for a general audience. Admittedly, I am not an expert on Parkin structure and function, but I had to do a lot of homework to try to understand the underlying rationale and impact. This reflects, I think, that the work generally represents an incremental advance on recent structural findings.

(2) To this point, it is hard to understand the impact of this work without more information highlighting the novelty. There are several structures of Parkin in various auto-inhibited states, and it was hard to delineate how this is different.

(3) As noted, I appreciated the use of protease sites in the fusion protein construct. It is unclear how the loop region might affect the protein structure and function. The authors worked to demonstrate that this did not introduce artifacts, but the biological context is missing.

(4) While it is likely that the binding is competitive between the Ubl and RING2 domains, the data is not quantitative. Is it known whether the folding of the distinct domains is independent? Or are there interactions that alter folding? It seems plausible that conformational rearrangements may invoke an orientation of domains that would be incompatible. The biological context for the importance of this interaction was not clear to me.

(5) What is the rationale for mutating Lys211 to Asn? Were other mutations tried? Glu? Ala? Just missing the rationale. I think this may have been identified previously in the field, but not clear what this mutation represents biologically.

(6) I was confused about how the phospho-proteins were generated. After looking through the methods, there appear to be phosphorylation experiments, but it is unclear what the efficiency was for each protein (i.e. what % gets modified). In the text, the authors refer to phospho-Parkin (T270R, C431A), but not clear how these mutations might influence this process. I gather that these are catalytically inactive, but it is unclear to me how this is catalyzing the ubiquitination in the assay.

(7) The authors note that "ACT can be complemented in trans; however, it is more efficient in cis", but it is unclear whether both would be important or if the favored interaction is dominant in a biological context.

(8) The authors repeatedly note that this study could aid in the development of small-molecule regulators against Parkin to treat PD, but this is a long way off. And it is not clear from their manuscript how this would be achieved. As stated, this is conjecture.

Reviewer #1 (Public Review):

Summary:

In this paper, Bruter and colleagues report the effects of inducible deletion of the genes encoding the two paralogous kinases of the Mediator complex in adult mice. The physiological roles of these two kinases, CDK8 and CDK19, are currently rather poorly understood; although conserved in all eukaryotes, and among the most highly conserved kinases in vertebrates, individual knockouts of genes encoding CDK8 homologues in different species have revealed generally rather mild and specific effects, in contrast to Mediator itself. Here, the authors provide evidence that neither CDK8 nor CDK19 are required for adult homeostasis but they are functionally redundant for maintenance of reproductive tissue morphology and fertility in males.

Strengths:

The morphological data on the atrophy of the male reproductive system and the arrest of spermatocyte meiosis are solid and are reinforced by single-cell transcriptomics data, which is a challenging technique to implement in vivo. The main findings are important and will be of interest to scientists in the fields of transcription and developmental biology.

Weaknesses:

There are several major weaknesses.

The first is that data on the general health of mice with single and double knockouts is not shown, nor is there any data on effects in any other tissues. This gives the impression that the only phenotype is in the male reproductive system, which would be misleading if there were phenotypes in other tissues that are not reported. Furthermore, data for the genitourinary system in single knockouts are very sparse; data are described for fertility in Figure 1H, ploidy, and cell number in Figures 2B and C, plasma testosterone and luteinizing hormone levels in Figures 5C and 5D, and morphology of testis and prostate tissue for single Cdk8 knockout in Supplementary Figure 1C (although in this case the images do not appear very comparable between control and CDK8 KO, thus perhaps wider fields should be shown), but, for example, there is no analysis of different meiotic stages or of gene expression in single knockouts. It is worth mentioning that single knockouts seem to show a corresponding upregulation of the level of the paralogue kinase, indicating that any lack of phenotypes might be due to feedback compensation, which would be an interesting finding if confirmed; this has not been mentioned.

The second major weakness is that the correlation between double knockout and reduced expression of genes involved in steroid hormone biosynthesis is portrayed as a causal mechanism for the phenotypes observed. While this is a possibility, there are no experiments performed to provide evidence that this is the case. Furthermore, there is no evidence showing that CDK8 and/or CDK19 are directly responsible for the transcription of the genes concerned.

Finally, the authors propose that the phenotypes are independent of the kinase activity of CDK8 or CDK19 because treatment of mice for a month with an inhibitor does not recapitulate the effects of the knockout, and nor does expression of two steroidogenic genes change in cultured Leydig cells upon treatment with an inhibitor. However, there are no controls for effective target inhibition shown.

Reviewer #1 (Public Review):

Summary:

The authors presented here a novel 3D fibroblast culture and transdifferentiation approach for potential meat production with GelMA hydrogel.

Strengths:

(1) Reduced serum concentration for 3D chicken fibroblast culture and transdifferentiation is optimized.<br /> (2) Efficient myogenic transdifferentiation and lipogenesis as well as controlled fat deposition are achieved in the 3D GelMA.

Reviewer #1 (Public Review):

Summary:

The authors present a mean-field model that describes the interplay between (protein) aggregation and phase separation. Different classes of interaction complexity and aggregate dimensionality are considered, both in calculations concerning (equilibrium) phase behavior and kinetics of assembly formation.

Strengths:

The present work is, although purely theoretical, of high interest to understanding biological processes that occur as a result of a coupling between protein aggregation and phase separation. Of course, such processes are abundant, in the living cell as well as in in-vitro experiments. I appreciate the consideration of aggregates with various dimensionality, as well as the categorization into different "interaction classes", together with the mentioning of experimental observations from biology. The model is convincing and underlines the complexity associated with the distribution of proteins across phases and aggregates in the living cell.

Weaknesses:

There are a few minor weaknesses.

Reviewer #1 (Public Review):

Summary:

This paper provides a straightforward mechanism of how mycobacterial cAMP level is increased under stressful conditions and shows that the increase is important for the survival of the bacterium in animal hosts. The cAMP level is increased by decreasing the expression of an enzyme that degrades cAMP.

Strengths:

The paper shows that under different stresses the response regulator PhoP represses a phosphodiesterase (PDE) that degrades cAMP specifically. Identification of PhoP as a regulator of cAMP is significant progress in understanding Mtb pathogenesis, as an increase in cAMP apparently increases bacterial survival upon infection. On the practical side, reduction of cAMP by increasing PDE can be a means to attenuate the growth of the bacilli. The results have wider implications since PhoP is implicated in controlling diverse mycobacterial stress responses and many bacterial pathogens modulate host cell cAMP levels. The results here are straightforward, internally consistent, and of both theoretical and applied interests. The results also open considerable future work, especially how increases in cAMP level help to increase survival of the pathogen.

Weaknesses:

It is not clear whether PhoP-PDE Rv0805 is the only pathway to regulate cAMP level under stress.

Comments on revised submission:

The authors have addressed my comments adequately, actually except for all but one. I have only one comment to do with the last line of the abstract. First, "genetic manipulation" usually means changing DNA. In Mtb pathogenesis I hope there is no DNA modification or change in the bacterial DNA. Also, the authors did not really inactivate the whole PhoP- rv0805-cAMP pathway. It would be best if the last line is made more fact based: Thus, inactivation of PhoP decreases cAMP level, thereby stress tolerance and intracellular survival of the bacillus.

Reviewer #1 (Public Review):

This work presents CTFFIND5, a new version of the software for determination of the Contrast Transfer Function (CTF) that models the distortions introduced by the microscope in cryoEM images. CTFFIND5 can take acquisition geometry and sample thickness into consideration to improve CTF estimation.

To estimate tilt (tilt angle and tilt axis), the input image is split into tiles and correlation coefficients are computed between their power spectra and a local CTF model that includes the defocus variation according to a tilted plane. As a final step, by applying a rescaling factor to the power spectra of the tiles, an average tilt-corrected power spectrum is obtained and used for diagnostic purposes and to estimate the goodness of fit. This global procedure and the rescaling factor resemble those used in Bsoft, Warp, etc, with determination of the tilt parameters being a feature specific of CTFFIND5 (and formerly CTFTILT). The performance of the algorithm is evaluated with tilted 2D crystals and tilt-series, demonstrating accurate tilt estimation in some cases and some limitations in others. Further analysis of CTF determination with tilt-series, particularly showing whether there is accurate or stable estimation at high tilts, might be helpful to show the robustness of CTFFIND5 in cryoET.

CTFFIND5 represents the first CTF determination tool that considers the thickness-related modulation envelope of the CTF firstly described by McMullan et al. (2015) and experimentally confirmed by Tichelaar et al. (2020). To this end, CTFFIND5 uses a new CTF model that takes the sample thickness into account. CTFFIND5 thus provides more accurate CTF estimation and, furthermore, gives an estimation of the sample thickness, which may be a valuable resource to judge the potential for high resolution. To evaluate the accuracy of thickness estimation in CTFFIND5, the authors use the Lambert-Beer law on energy-filtered data and also tomographic data, thus demonstrating that the estimates are reasonable for images with exposure around 30 e/A2. While consideration of sample thickness in CTF determination sounds ideally suited for cryoET, practical application under the standard acquisition protocols in cryoET (exposure of 3-5 e/A2 per image) is still limited. In this regard, the authors are honest in the conclusions and clearly identify the areas where thickness-aware CTF determination will be valuable at present: e.g. in situ single particle analysis and in vitro single particle cryoEM of purified samples at low voltages.

In conclusion, the manuscript introduces novel methods inside CTFFIND5 that improve CTF estimation, namely acquisition geometry and sample thickness. The evaluation demonstrates the performance of the new tool, with fairly accurate estimates of tilt axis, tilt angle and sample thickness and improved CTF estimation. The manuscript critically defines the current range of application of the new methods in cryoEM.

Reviewer #1 (Public Review):

Summary:

The present work from Velloso and collaborators investigated the transcription profiles of resident and recruited hypothalamic microglia. They found sex-dependent differences between males and females and identified the protective role of chemokine receptor CXCR3 against diet-induced obesity.

Strengths:

(1) Novelty<br /> (2) Relevance, since this work provides evidence about a subset of recruited microglia that has a protective effect against DIO. This provides a new concept in hypothalamic inflammation and obesity.

Weaknesses:

(1) Lack of mechanistic insight into the sex-dependent effects.<br /> (2) Analysis of indirect calorimetry data requires more depth.<br /> (3) A deeper analysis of hypothalamic inflammation and ER stress pathways would strengthen the manuscript.

Reviewer #1 (Public Review):

Summary:

This is a follow-up study to the authors' previous eLife report about the roles of an alpha-arrestin called protein thioredoxin interacting protein (Txnip) in cone photoreceptors and in the retinal pigment epithelium. The findings are important because they provide new information about the mechanism of glucose and lactate transport to cone photoreceptors and because they may become the basis for therapies for retinal degenerative diseases.

Strengths:

Overall, the study is carefully done and, although the analysis is fairly comprehensive with many different versions of the protein analyzed, it is clearly enough described to follow. Figure 4 greatly facilitated my ability to follow, understand and interpret the study. The authors have appropriately addressed a few concerns about statistical significance and the relationship between their findings and previous studies of the possible roles of Txnip on GLUT1 expression and localization on the surfaces of RPE cells.

Joint Public Review:

In their revised manuscript additional experiments have been conducted competently, and the interpretation of experiments regarding exit from the ER are convincing. They collectively indicate that the phase partitioning behaviour of the TMDs do not have a significant effect on exit from the ER; they all exit the ER very slowly unless they carry a short cytoplasmic domain from LAT which is sufficient to accelerate ER exit. This data is consistent with available literature supporting a role for a ER-exit signal. Along with new experiments in their revision, they have also toned down the assertion that their data rule out a phase partitioning mechanism at the ER.

The authors, however, continue to interpret their experiments regarding Golgi exit of the transmembrane peptides (with luminal and cytoplasmic domains) as conclusive evidence of the role of lipid rafts in exit from the Golgi. This is once again based on correlation of the phase partitioning behaviour of these proteins in GPMVs, phase separated at low temperatures. They argue that this represents very strong evidence that trafficking out of the Golgi is driven by phase separation. The reviewers consider that there are a number of potential issues with the final model that need to be addressed.

We reiterate that:

(1) the phase segregation in the GPMV at low temperatures is dictated by thermodynamics given its composition and the measurement temperature. However at physiological temperatures at which membrane trafficking is taking place these GPMVs will not exhibit phase separation. Hence it is difficult to argue that a sorting mechanism based solely on the partitioning of the synthetic TM constructs into liquid ordered domain detected at low temperatures in GPMVs provide an explanation of the explanation of the differential kinetics of traffic of the LAT TMD and the allL-TMD constructs, although there is a strong correlation with its phase partitioning behaviour.

(2) The fluctuations of lipid composition resembling lo-domains if persisting at higher temperatures and its conversion into a sorting domain will require a cellular mechanism, that may or may not retain similar properties of these lipidic environments. Additionally, TMDs from TfR/VsVG and GPI prefer different domains in the GPMV assays (Table S1) yet they traffic to the cell surface equally rapidly.

(3) The authors fail to discuss the point raised about the relatively high colocalization of TfR with the GPI probe (seen in Fig 5E) in the Golgi. This is inconsistent with their explanation of traffic correlating with partitioning into distinct domains in the Golgi, since TfR and GPI probes show an opposite preference for lo versus ld domains in cooled GPMVs. TMD-allL and the LAT-allL are segregated from TfR in the Golgi, and end up in a different final destination (ie lysosomes). This could represent yet another membrane specialisation in the Golgi for lysosomal traffic. The segregation that the authors report in the Golgi is therefore not a convincing argument for phase preferences in GPMVs dictating the trafficking behaviour of these molecules towards the plasma membrane.

(4) Despite the authors' claim in their rebuttal that 'we feel that GPMVs are a useful tool for quantifying protein preference for ordered (raft) membrane domains and that this preference is a useful proxy for the raft-associated behavior ... biological membrane with a relevant and measurable cellular outcome, specifically inter-organelle trafficking rates." -several caveats for these observations need to be addressed before they constitute strong evidence for the raft model of membrane trafficking proposed. Phase partitioning in GPMVs is just another operational definition and while more refined (ie the data is derived from the membrane of interest, ie, the plasma membrane) it is not very different conceptually from quantitative measurements of detergent-insolubility.

(5) Further work is necessary to establish that ordered domains are formed at the Golgi at physiological temperatures, into which these proteins may partition; subsequently, there must be a mechanism that selectively traffics these proteins towards the cell surface.

(6) The authors continue to conflate thermodynamic phase separation mechanisms with the real possibility of the formation of functional sorting domains by cellular mechanisms that likely involve lipidic interactions, adding to the confusion in the literature.

Reviewer #1 (Public Review):

Summary:

This manuscript describes the identification and isolation of several phage from deep sea isolates of Lentisphaerae strains WC36 and zth2. The authors observe induction of several putative chronic phages with the introduction of additional polysaccharides to the media. The authors suggest that two of the recovered phage genomes encode AMGs associated with polysaccharide use. The authors also suggest that adding the purified phage to cultures of Pseudomonas stutzeri 273 increased the growth of this bacteria due to augmented polysaccharide use genes from the phage.

While the findings were of interest and relevance to the field, it is my opinion that several of the analysis fall short of supporting the key assertions presented.

Strengths:

Interesting isolate pf deep sea Lentisphaerae strains which will undoubtedly further our understanding of deep sea microbial life.

Weaknesses:

Many of the findings are consistent with a phage contamination in the polysaccharide stock solution.

The genes presented as AMGs are largely well known and studied phage genes which play a role in infection cycles.

The evidence that the isolated phage can infect Pseudomonas stutzeri 273 is lacking, putting into question the dependent results.

Reviewer #1 (Public Review):

This manuscript examines the individual and dual effects of CHIP and LOY in MI employing a cohort of ~460 individuals. CHIP is assessed by NGS and LOY is assessed by PCR. The threshold for CHIP is set at 2% (an arbitrary cutoff that is often used) and LOY at 9% (according to the Discussion text - this reviewer may have missed the section that describes why this threshold was employed). The investigation assessed whether LOY could modulate inflammation, atherosclerotic burden, or MI risk associated with CHIP. Neither CHIP nor LOY independently affected hsCRP, atherosclerotic burden, or MI incidence, nor did LOY presence diminish these outcomes in CHIP+ male subjects.

This study represents the first dual analysis of CHIP and LOY on CVD outcomes. The results are largely negative, contradictory to other studies (many with much larger sample sizes). I would attribute the limitation of sample size as a major contributor to the negative data. While the negative data are suspect, the "positive" finding that LOY abolishes the prognostic significance of CHIP on MI is of interest (and consistent with what is understood from mechanistic studies).

Overall, I enjoyed reading the paper, and it is of interest to the research community. However, I disagree with some of the authors' interpretations of the data. Generally, many conclusions on CHIP interpretation are based on the comparison of findings from very large datasets that have been evaluated by shallow NGS DNA sequencing. These studies lack sensitivity and accuracy, but this is counterbalanced by their very large sample sizes. Thus, they draw conclusions from the sickest individuals (ICD codes) with the largest clones (explaining the 10% VAF threshold). Here, the study has a well-phenotyped cohort, but as far as this reviewer can tell, the DNA sequencing is "shallow" NGS. Typically, to assess smaller datasets, investigators employ an error-correction method (DNA barcodes, duplex sequencing, etc.) for the sensitivity and accuracy of calling variants. Thus, the current study appears to suffer from this limitation (small sample sizes combined with NGS).

While the "negative" data from this study are inconclusive, the positive data (i.e. CHIP being prognostic for MI in the absence but not presence of MI) is of interest. Thus, the investigators may want to consider a shorter report that largely focuses on this finding.

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors investigate the role of BEND2, a novel regulator of meiosis, in both male and female fertility. Huang et al have created a mouse model where the full-length BEND2 transcript is depleted but the truncated BEND2 version remains. This mouse model is fertile, and the authors used it to study the role of BEND2 on both male and female meiosis. Overall, the full-length BEND2 appears dispensable for male meiosis. The more interesting phenotype was observed in females. Females exhibit a lower ovarian reserve suggesting that full-length BEND2 is involved in the establishment of the primordial follicle pool.

Strengths:

The authors generated a mouse model that enabled them to study the role of BEND2 in meiosis. The role of BEND2 in female fertility is novel and enhances our knowledge of genes involved in the establishment of the primordial follicle pool.

Weaknesses:

The manuscript extensively explores the role of BEND2 in male meiosis; however, a more interesting result was obtained from the study of female mice. Only a few experiments were performed using female mice, therefore, more experiments should be performed to complete the story of the role of BEND2 on female fertility. In addition, the title and abstract of the manuscript do not align with the story, as female fertility is only a small portion of the data compared to the male fertility section.

Reviewer #1 (Public Review):

This study by Porter et al reports on outcomes from a small, open-label, pilot randomized clinical trial comparing dornase-alfa to best available care in patients hospitalized with COVID-19 pneumonia. As the number of randomized participants is small, investigators describe also a contemporary cohort of controls and the study concludes with decrease of inflammation (reflected by CRP levels) after 7 days of treatment but no other statistically significant clinical benefit.

I read with interest this manuscript and I find the idea about treatment of COVID-19 patients with dornase-alfa novel and inspiring. I have some major concerns about the methodology the authors followed in this RCT.

My major concerns are:

(1) The authors have chosen a primary outcome that cannot be at least considered as clinically relevant or interesting. After 3 years of the pandemic with so much research, why investigate if a drug reduces CRP levels as we already have marketed drugs that provide beneficial clinical outcomes such as dexamethasone, anakinra, tocilizumab and baricitinib.

(2) ΙΤΤ analysis is not followed

Reviewer #1 (Public Review):

I have reviewed, with interest, the manuscript "Psychological stress disturbs bone metabolism via miR-335-3p/Fos signaling in osteoclast". The described findings are relevant and useful for daily practice in periodontology. The paper is concise, professionally written, and easy to read. In this study, Jiayao et al. revealed the role of miR-335-3p in psychological stress-induced osteoporosis. CUMS mice were constructed to observe the femur phenotype, osteoclasts were identified as the primary research object, and miRNA-seq was used to find the key miRNAs linking the brain and peripheral tissues. This study showed that the expression of miR-335-3p was simultaneously reduced in mice's NAC, serum, and bone under psychological stress. The miR-335-3p/Fos/NFATC1 signaling pathway was validated in osteoclasts to reveal the potential mechanism of enhanced osteoclast activity under psychological stress. From a new perspective of miRNAs, this study indicates a possible cause of disturbed bone metabolism due to psychological stress and may suggest a new approach to treating osteoporosis.

Reviewer #1 (Public Review):

Existing literature suggests that brain structures implicated in memory such as the hippocampus, and reward/punishment processing such as the striatal regions are also engaged in learning and value-based decision-making. However, how the contributions of these regions to learning and value-based decision-making change over time, particularly in children where these neural systems show protracted maturation was not studied systematically. This is the question the authors are aiming to address in this work in which children 6-to-7-years-old were recruited for a neuroimaging study that involves taking structural scans from this cohort to investigate how they correlate with changes in the way children approach a reinforcement learning task in which they learn to identify the better shape between 2 options through trial-and-error.

Particular strengths of the paper are longitudinally following up a cohort of small children and engaging them in a value-based decision-making task so that the relationship between neural maturation and improvements in reinforcement learning can be studied reliably. Towards this end, the authors make use of well-established computational modelling approaches to extract key parameters such as learning rates (which designate the speed of learning from expected versus actual outcomes) or choice stochasticity (which designate the inherent variation in people's decisions and the tendency to explore between the options) from children's choices so that their structural neural correlates can be established. As a part of this endeavour, the authors rely on methodological choices which do not warrant much criticism. Their data visualization choices are particularly spot-on and highly informative about the details of the raw data.

Also considering the importance of the hippocampal system in human memory, the key contribution of the paper is that the volumetric increases in hippocampus size between 2 assessment points correlated selectively with the delayed, but not immediate, learning score which refers to the learning condition in which the outcome feedback is given to the children after a 5-seconds delay. Although the authors also demonstrate evidence to suggest that changes in the striatal volume are also implicated in learning performance, this was more general as associations were found for both immediate and delayed feedback conditions. Thus, the paper makes an important contribution to the fields of developmental and decision neuroscience. An important question arising from the authors' findings could be that, whether the hippocampus maintains this selective role in value-based learning during the course of neuronal development, for example, whether a similar association would be found in children 8-to-9 years old. A better understanding of how these developmental trajectories map onto changes in learning and decision-making can inform fields outside neuroscience, for example tailoring educational approaches onto neural development pathways to boost learning efficiency in young children.

Reviewer #1 (Public Review):

Summary:

The authors goal here was to explore how a non hebbian form of plasticity, heterosynaptic LTP, could shape neuronal responses and learning. They used several conceptually and technically innovative approaches to answer this. First, they identified a behavioral paradigm that was a subthreshold training paradigm (stimulation of thalamic inputs with a footshock), which could be 'converted' to a memory via homosynaptic LTP (HFS of thalamic inputs). They then find that stimulation of 'cortical' inputs could also convert the subthreshold stimulation to a lasting memory, and that this was associated with a change in neuronal response, akin to LTP. Finally, they provide some slice work which demonstrated that stimulation of cortical inputs could stabilize LTP at thalamic inputs.

Strengths:

(1) The approach was innovative and asked an important question in the field.

(2) The studies are, for the most part, quite rigorous, using a novel dual opsin approach to probe multiple inputs in vivo.

(3) The authors explore neural responses both in vivo and ex vivo, as well as leveraging a 'simple' behavior output of freezing.

Reviewer #1 (Public Review):

Summary:

Li and colleagues describe an experiment whereby sequences of dots in different locations were presented to participants while electroencephalography (EEG) was recorded. By presenting fixed sequences of dots in different locations repeatedly to participants, the authors assumed that participants had learned the sequences during the experiment. The authors also trained classifiers using event-related potential (ERP) data recorded from separate experimental blocks of dots presented in a random (i.e., unpredictable) order. Using these trained classifiers, the authors then assessed whether patterns of brain activity could be detected that resembled the neural response to a dot location that was expected, but not presented. They did this by presenting an additional set of sequences whereby only one of the dots in the learned sequence appeared, but not the other dots. They report that, in these sequences with omitted stimuli, patterns of EEG data resembled the visual response evoked by a dot location for stimuli that could be expected, but were not presented. Importantly, this only occurred for an omitted dot stimulus that would be expected to appear immediately after the dot that was presented in these partial sequences.

This exciting finding complements previous demonstrations of the ability to decode expected (but not presented) stimuli in Blom et al. (2020) and Robinson et al. (2020) that are cited in this manuscript. It suggests that the visual system is able to generate patterns of activity that resemble expected sensory events, approximately at times at which an observer would expect them.

Strengths:

The experiment was carefully designed and care was taken to rule out some confounding factors. For example, gaze location was tracked over time, and deviations from fixation were marked, in order to minimise the contributions of saccades to above-chance decoding of dot position. The use of a separate block of dots (with unpredictable locations) to train the classifiers was also useful in isolating visual responses evoked by each dot location independently of any expectations that might be formed during the experiment. A large amount of data was also collected from each participant, which is important when using classifiers to decode stimulus features from EEG data. This careful approach is commendable and draws on best practices from existing work.

Weaknesses:

While there was clear evidence of careful experiment design, there are some aspects of the data analysis and results that significantly limit the inferences that can be drawn from the data. Both issues raised here relate to the use of pre-stimulus baselines and associated problems. As these issues are somewhat technical and may not be familiar to many readers, I will try to unpack each line of reasoning below. Here, it should be noted that these problems are complex, and similar issues often go undetected even by highly experienced EEG researchers.

Relevant to both issues, the authors derived segments of EEG data relative to the time at which each dot was presented in the sequences (or would have appeared when the stimuli were omitted in the partial sequences). Segments were derived that spanned -100ms to 300ms relative to the actual or expected onset of the dot stimulus. The 300ms post-stimulus time period corresponds to the duration of each dot in the sequence (100ms) plus the inter-stimulus interval (ISI) that was 200ms in duration before the next dot appeared (or would be expected to appear in the partial sequences). Importantly, a pre-stimulus baseline was applied to each of these segments of data, meaning that the average amplitude at each electrode between -100ms and 0ms relative to (actual or expected) stimulus onset was subtracted from each segment of data (i.e., each epoch in common EEG terminology). While this type of baseline subtraction procedure is commonplace in EEG studies, in this study design it is likely to cause problematic effects that could plausibly lead to the patterns of results reported in this manuscript.

First of all, the authors compare event-related potentials (ERPs) evoked by dots in the full as compared to partial sequences, to test a hypothesis relating to attentional tuning. They reported ERP amplitude differences across these conditions, for epochs corresponding to when a dot was presented to a participant (i.e., excluding epochs time-locked to omitted dots). However, these ERP comparisons are complicated by the fact that, in the full sequences, dot presentations are preceded by the presentation of other dots in the sequence. This means that ERPs evoked by the preceding dots in the full sequences will overlap in time with the ERPs corresponding to the dots presented at the zero point in the derived epochs. Importantly, this overlap would not occur in the partial sequence conditions, where only one dot was presented in the sequence. This essentially makes any ERP comparisons between full and partial sequences very difficult to interpret, because it is unclear if ERP differences are simply a product of overlapping ERPs from previously presented dots in the full sequence conditions. For example, there are statistically significant differences observed even in the pre-stimulus baseline period for this ERP analysis, which likely reflects the contributions ERPs evoked by the preceding dots in the full sequences, which are absent in the partial sequences.

The problems with interpreting this data are also compounded by the use of pre-stimulus baselines as described above. Importantly, the use of pre-stimulus baselines relies on the assumption that the ERPs in the baseline period (here, the pre-stimulus period) do not systematically differ across the conditions that are compared (here, the full vs. partial sequences). This assumption is violated due to the overlapping ERPs issue described just above. Accordingly, the use of the pre-stimulus baseline subtraction can produce spurious effects in the time period after stimulus onset (for examples see Feuerriegel & Bode, 2022, Neuroimage). This also makes it very difficult to meaningfully compare the ERPs following dot stimulus onset in these analyses.

The second issue relates to the use of pre-stimulus baselines and concerns the key finding reported in the paper: that EEG patterns corresponding to expected but omitted events can be decoded in the partial sequences. In the partial sequences, there are two critical epochs that were derived: One time-locked to the presentation of the dot, and another that was time-locked to 300ms after the dot was presented (i.e. when the next dot would be expected to appear). The latter epoch was used to test for representations of expected, but omitted, stimulus locations.

For the epochs in which the dots were presented, above-chance decoding can be observed spanning a training time range from around 100-300ms and a testing time range of a similar duration (see the plot in Figure 4b). This plot indicates that, during the time window of around 200-300ms following dot stimulus onset, the position of the dot can be decoded not only from trained classifiers using the same time windows spanning 200-300ms, but also using classifiers trained using earlier time windows of around 100-200ms.

This is important because the 200-300ms time period after dot onset in the partial sequences is the window used for pre-stimulus baseline subtraction when deriving epochs corresponding to the first successor representation (i.e., the first stimulus that might be expected to follow from the presented dot, but did not actually appear). In other words, the 200-300ms time window from dot onset corresponds to the -100 to 0 ms time window in the first successor epochs. Accordingly, the pattern that is indicative of the preceding, actually presented dot position would be subtracted from the EEG data used to test for the successor representation. Notably, the first successor condition would always be in another visual field quadrant (90-degree rotated or the opposite quadrant) as stated in the methods. In other words, the omitted stimulus would be expected to appear in the opposite vertical and/or horizontal visual hemifield as compared to the previously presented dot in these partial sequences.

This is relevant because ERPs tend to show reversed polarity across hemifields. For example, a stimulus presented in the right hemifield will have reversed polarity patterns at the same electrode as compared to an equivalent stimulus presented in the left hemifield (e.g., Supplementary Figure 3 in the comparable study of Blom et al., 2020). By subtracting the ERP patterns evoked by the presented dot in the partial sequences during the time period of 200-300ms (corresponding to the -100 to 0ms baseline window), this would be expected to bias patterns of EEG data in the first successor epochs to resemble stimulus positions in opposite hemifields. This could plausibly produce above-chance decoding accuracy in the time windows identified in Figure 5a, where the training time windows broadly correspond to the periods of above-chance decoding during 200-300ms from dot stimulus onset in Figure 4b.

In other words, the above-chance decoding of the first successor representation may plausibly be an artefact of the pre-stimulus baseline subtraction procedure used when deriving the epochs. This casts some doubt as to whether genuine successor representations were actually detected in the study. Additional tests for successor representations using ERP baselines prior to the presented dot in the partial sequences may be able to get around this, but such analyses were not presented, and the code and data were not accessible at the time of this review.

Although the study is designed well and a great amount of care was taken during the analysis stage, these issues with ERP overlap and baseline subtraction raise some doubts regarding the interpretability of the findings in relation to the analyses currently presented.

Reviewer #1 (Public Review):

Summary:

This is a strong paper that sets the foundation for future work that will explore the innervation of the giant fiber, allowing experiments that will link molecular/developmental mechanisms to circuit function at a level of resolution that has not previously been possible. In the course of this work the investigators discover an axon-axon competition that reflects the order of innervation of the target. In addition, a host of reagents are developed that will be of wide use in dissecting this system.

Strengths:

(1) The developmental, functional and connectomic characterization of the wiring pattern to be dissected is impressively thorough and quantitative.<br /> (2) The reagents that the authors establish will be foundational to subsequent effort.<br /> (3) The discovery that axon-axon competition is involved in patterning this system, and might combined with innervation order to give a deterministic outcome is an interesting one (and might be useful to address variation in cell number (see below)!

Weaknesses:

(1) In my opinion, the authors miss an opportunity to leverage their connectomics characterization somewhat more. That is, from characterization of the connectomes of two flies, the authors describe substantial variation in the number of pre-synaptic cells providing inputs (for example, in FAFB, there are 55 LC4 cells, while in the hemibrain, there are 71 - almost 30 percent more), yet the number of total synapses provided by each class of cell types is remarkably stereotyped 2442 synapses versus 2290 synapses). And the ratio of LC4 to LPLC2 synapses is even more stereotyped... As this kind of stereotypy would be consistent with the authors competition model, but inconsistent with a model in which each cell makes a similar number of synapses (which would be the model from the periphery of the visual system), the authors should comment a bit more on what they see. Perhaps the wiring model the authors advocate for compensates for what appears to be quite significant variation in the numbers of LC neurons?

(2) I appreciate how the authors pivoted to interpreting their results using Kir2.1 to reflect the effects of cell ablation. However, I worry that since the mechanism behind Kir2.1 mediated ablation is unknown, there could be other effects associated with this perturbation, creating indirect effects that alter LPLC2 cells somehow. I would therefore ask that the authors repeat these experiments with a more standard cell ablation strategy (such as a light gated caspase, or ricin). More crucially, the author's model that arrival order is functionally important would be greatly strengthened if they did the reciprocal ablation of LPLC2 and asked what happens to LC4. One could easily imagine a model in which these two cell types mutually compete for real estate, after an initial bias is set by arrival order.

Reviewer #1 (Public Review):

The manuscript by Poltavski and colleagues describes the discovery of previously unreported enteric neural crest-derived cells (ENCDC) which are marked by Pax2 and originating from the Placodes. By creating multiple conditional mouse mutants, the authors demonstrate these cells are a distinct population from the previously reported ENCDCs which originate from the Vagal neural crest cells and express Wnt1.

These Pax2-positive ENCDCs are affected due to the loss of both Ret and Ednrb highlighting that these cells are also ultimately part of the canonical processes governing ENCDC and enteric nervous system (ENS) development. The authors also make explant cultures from the mouse GI tract to detect how Ednrb signaling is important for Ret signaling pathways in these cells and rediscovers the interactions between these 2 pathways. One important observation the authors make is that CGRP-positive neurons in the adult distal colon seem to be primarily derived from these Pax2-positive ENCDCs, which are significantly reduced in the Ednrb mutants, thus highlighting the role of Ednrb in maintaining this neuronal type.

I appreciate the amount of work the authors have put into generating the mouse models to detect these cells, but there isn't any new insight on either the nature of ENCDC development or the role of Ret and Ednrb. Also, there are sophisticated single-cell genomics methods to detect rare cell type/states these days and the authors should either employ some of those themselves in these mouse models or look at extensively publicly available single-cell datasets of the developing wildtype and mutant mouse and human ENS to map out the global transcriptional profile of these cells. A more detailed analysis of these Pax2-positive cells would be really helpful to both the ENS community as well as researchers studying gut motility disorders.

Reviewer #1 (Public Review):

The manuscript entitled "A septo-hypothalamic-medullary circuit directs stress-induced analgesia" by Shah et al., showed that the dLS-to-LHA circuit is sufficient and necessary for stress-induced analgesia (SIA), which is mediated by the rostral ventromedial medulla (RVM) in a opioid-dependent manner. This study is interesting and important and the conclusions are largely supported by the data. I have a few concerns as follows:

(1) The present data show that activation of dLS neurons produces SIA, however, this manipulation is non-specific. It may be better to see the effect of specific manipulation of stress-activated c-Fos positive neurons in the dLS using a combination of the Tet-Off system and chemogenetic/optogenetic tools.

(2) Depending on its duration, and intensity, stress can exert potent and bidirectional modulatory effects on pain, either reducing pain (SIA) or exacerbating it (stress-induced hyperalgesia, SIH). Is the circuit in the manuscript involved in SIH?

(3) It is well-accepted that opioid and cannabinoid receptors participate in the SIA, and the evidence is especially strong for the RVM endocannabinoid system. Given this, why did the authors focus their study on the opioid system?

(4) Does silencing of the dLS neurons affect stress-induced anxiety-like behaviors? Alternatively, what is the relationship between SIA and the level of stress-induced anxiety?

(5) Direct electrophysiological evidence should be provided to confirm the efficacy of the MP-CNO.

(6) Is the LHA a specific downstream target for SIA, and is the LHA involved in stress-induced anxiety-like behaviors?

(7) Do LHA neurons have direct projections to the RVM? If yes, what is its role in the SIA?

Joint Public Review:

Summary:

This manuscript investigates how energetic demands affect the sleep-wake cycle in Drosophila larvae. L2 stage larvae do not show sleep rhythm and long-term memory (LTM), however, L3 larvae do. The authors manipulate food content to provide insufficient nutrition, which leads to more feeding, no LTM, and no sleep even in older larvae. Similarly, activation of NPF neurons suppresses sleep rhythm. Furthermore, they try to induce a sleep-like state using pharmacology or genetic manipulations in L2 larvae, which can mimic some of the L3 behaviours. A key experimental finding is that activation of DN1a neurons activate the downstream DH44 neurons, as assayed by GCaMP calcium imaging. This occurs only in third instar and not in second instar, in keeping with the development of sleep-wake and feeding separation. The authors also show that glucose metabolic genes are required in Dh44 neurons to develop sleep rhythm and that DH44 neurons respond differently in malnutrition or younger larvae.

Strengths:

Previous studies from the same lab have shown the sleep is required for LTM formation in the larvae, and that this requires DN1a and DH44 neurons. The current work builds upon this observation and addresses in more detail when and how this might develop. The authors can show that low quality food exposure and enhanced feeding during larval stage of Drosophila affects the formation of sleep rhythm and long-term memory. This suggests that the development of sleep and LTM are only possible under well fed and balanced nutrition in fly larvae. Non-sleep larvae were fed in low sugar conditions and indeed, the authors also find glucose metabolic genes to be required for a proper sleep rhythm. The paper presents precise genetic manipulations of individual classes of neurons in fly larvae followed by careful behavioural analysis. The authors also combine thermogenetic or peptide bath application experiments with direct calcium imaging of specific neurons.

Weaknesses:

The authors tried to induce sleep in younger L2 larvae, however the behavioral results suggest that they were not able to induce proper sleep behaviour as in normal L3 larvae. Thus, they cannot show that sleep during L2 stage would be sufficient to form LTM.<br /> The authors suggest that larval Dh44 neurons may integrate "information about the nutritional environment through the direct sensing of glucose levels to modulate sleep-wake rhythm development". They identify glucose metabolism genes (e.g., Glut1) in the downstream DH44 neurons as being required for the organization of the sleep-wake-feeding rhythm, and that CCHa signaling in DN1a signaling to the DH44 cells via the receptor. However, how this is connected is not well explained. Do the authors think that the nutrient sensing is only occurring in the DH44 neurons and not in DN1a or other neurons? Would not knocking down glucose metabolism in any neuron lead to a functional defect? What is the evidence that Dh44 neurons are specific sensors of nutritional state? For example, do the authors think that e.g. the overexpression of Glut1 in Dh44 neurons, a manipulation that can increase transport of glucose into cells, would rescue the effects of low-sugar food?<br /> Some of the genetic controls seem to be inconsistent suggesting some genetic background effects. In Figure 2B, npf-gal4 flies without the UAS show no significant circadian change in sleep duration, whereas UAS-TrpA flies do. The genetic control data in Figure 2D are also inconsistent. Npf-Gal4 seems to have some effect by itself without the UAS. The same is not seen with R76G11-Gal4. Suppl Fig 2: Naïve OCT and AM preference in L3 expressing various combinations of the transgenes show significant differences. npf-Gal4 alone seems to influence preference.<br /> The sleep duration and bout number/length data are highly variable.

Reviewer #1 (Public Review):

Summary:

The manuscript gives a broad overview of how to write NeuroML, and a brief description of how to use it with different simulators and for different purposes - cells to networks, simulation, optimization, and analysis. From this perspective, it can be an extremely useful document to introduce new users to NeuroML.

However, the manuscript itself seems to lose sight of this goal in many places, and instead, the description at times seems to target software developers. For example, there is a long paragraph on the board and user community. The discussion on simulator tools seems more for developers, not users. All the information presented at the level of a developer is likely to be distracting to readers..

Strengths:

The modularity of NeuroML is indeed a great advantage. For example, the ability to specify the channel file allows different channels to be used with different morphologies without redundancy. The hierarchical nature of NeuroML also is commendable, and well illustrated in Figures 2a through c.

The number of tools available to work with NeuroML is impressive.

The abstract, beginning, and end of the manuscript present and discuss incorporating NeuroML into research workflows to support FAIR principles.

Having a Python API and providing examples using this API is fantastic. Exporting to NeuroML from Python is also a great feature.

Weaknesses:

Though modularity is a strength, it is unclear to me why the cell morphology isn't also treated similarly, i.e., specify the morphology of a multi-compartmental model in a separate file, and then allow the cell file to specify not only the files containing channels, but also the file containing the multi-compartmental morphology, and then specify the conductance for different segment groups. Also, after pynml_write_neuroml2_file, you would not have a super long neuroML file for each variation of conductances, since there would be no need to rewrite the multi-compartmental morphology for each conductance variation.

This would be especially important for optimizations, if each trial optimization wrote out the neuroML file, then including the full morphology of a realistic cell would take up excessive disk space, as opposed to just writing out the conductance densities. As long as cell morphology must be included in every cell file, then NeuroML is not sufficiently modular, and the authors should moderate their claim of modularity (line 419) and building blocks (551). In addition, this is very important for downloading NeuroML-compliant reconstructions from NeuroMorpho.org. If the cell morphology cannot be imported, then the user has to edit the file downloaded from NeuroMorpho.org, and provenance can be lost. Also, Figure 2d loses the hierarchical nature by showing ion channels, synapses, and networks as separate main branches of NeuroML.

In Figure 5, the difference between the core and native simulator is unclear. What is involved in helper scripts? I thought neurons could read NeuroML? If so, why do you need the export simulator-specific scripts? In addition, it seems strange to call something the "core" simulation engine, when it cannot support multi-compartmental models. It is unclear why "other simulators" that natively support NeuroML cannot be called the core. It might be more helpful to replace this sort of classification with a user-targeted description. The authors already state which simulators support NeuroML and which ones need code to be exported. In contrast, lines 369-370 mention that not all NeuroML models are supported by each simulator. I recommend expanding this to explain which features are supported in each simulator. Then, the unhelpful separation between core and native could be eliminated.

The body of the manuscript has so much other detail that I lose sight of how NeuroML supports FAIR. It is also unclear who is the intended audience. When I get to lines 336-344, it seems that this description is too much detail for the audience. The paragraph beginning on line 691 is a great example of being unclear about who is the audience. Does someone wanting to develop NeuroML models need to understand XSD schema? If so, the explanation is not clear. XSD schema is not defined and instead explains NeuroML-specific aspects of XSD. Lines 734-735 are another example of explaining to code developers (not model developers).

Reviewer #2 (Public Review):

Summary:

While a significant portion of immunotherapy research has focused on the pivotal role of T cells in tumor immunity, their effectiveness may be limited by the suppressive nature of the tumor environment. On the other hand, myeloid cells are commonly found within tumors and can withstand these adverse conditions. However, these cells often adopt an immunosuppressive phenotype when infiltrating tumors. Therefore, manipulating myeloid cells could potentially enhance the anti-tumor potential of immunotherapy.<br /> In this manuscript, Farhat-Younes and colleagues have demonstrated that activating the IgM receptor signaling in myeloid cells induces an oxygen burst, the secretion of Granzyme B, and the lysis of adjacent tumor cells. Furthermore, they have outlined a strategy to utilize these features to generate CAR macrophages. However, they have identified a limitation: the expression of scFv in myeloid cells induces ER stress and the degradation of misfolded proteins. To address this issue, chimeric receptors were designed based on the high-affinity FcγRI for IgG. When macrophages transfected with these receptors were exposed to tumor-binding IgG, extensive tumor cell killing, and the release of reactive oxygen species and Granzyme B were observed.

Strengths:

In general, I consider this work to be significant, and the results are compelling. It emphasizes the specific considerations and requirements for successful manipulation in myeloid cells, which could further advance the field of cellular engineering for the benefit of immunotherapy

Following the revision of the original manuscript, I can clearly state that my concerns have been addressed and the article has been improved.

Reviewer #1 (Public Review):

Summary:

The work from Petazzi et al. aimed at identifying novel factors supporting the differentiation of human hematopoietic progenitors from induced pluripotent stem cells (iPSCs). The authors developed an inducible CRISPR-mediated activation strategy (iCRISPRa) to test the impact of newly identified candidate factors on the generation of hematopoietic progenitors in vitro. They first compared previously published transcriptomic data of iPSC-derived hemato-endothelial populations with cells isolated ex vivo from the aorta-gonad-mesonephros (AGM) region of the human embryo and they identified 9 transcription factors expressed in the aortic hemogenic endothelium that were poorly expressed in the in vitro differentiated cells. They then tested the activation of these candidate factors in an iPSC-based culture system supporting the differentiation of hematopoietic progenitors in vitro. They found that the IGF binding protein 2 (IGFBP2) was the most upregulated gene in arterial endothelium after activation and they demonstrated that IGFBP2 promotes the generation of functional hematopoietic progenitors in vitro.

Strengths:

The authors developed an extremely useful doxycycline-inducible system to activate the expression of specific candidate genes in human iPSC. This approach allows us to simultaneously test the impact of 9 different transcription factors on in vitro differentiation of hematopoietic cells, and the system appears to be very versatile and applicable to a broad variety of studies.

The system was extensively validated for the expression of 1 transcription factor (RUNX1) in both HeLa cells and human iPSC, and a detailed characterization of this test experiment was provided.

The authors exhaustively demonstrated the role of IGFBP2 in promoting the generation of functional hematopoietic progenitors in vitro from iPSCs. Even though the use of IGFBP2-interacting proteins IGF1 and IGF2 have been previously reported in human iPSC-derived hematopoietic differentiation in vitro (Ditadi and Sturgeon, Methods 2016; Ng et al., Nature Biotechnology 2016), and IGFBP-2 itself has been shown to promote adult HSC expansion ex vivo (Zhang et al., Blood 2008), its role on supporting in vitro hematopoiesis was demonstrated here for the first time.

Weaknesses:

Although the authors performed a very thorough characterization of the system in proof-of-principle experiments activating a single transcription factor, the data provided when 9 independent factors were used is not sufficient to fully validate the experimental strategy. Indeed, in the current version of the manuscript, it is not clear whether the results presented in both the scRNAseq analysis and the functional assays are the consequence of the simultaneous activation of all 9 TF or just a subset of them. This is essential to establish whether all the proposed factors play a role during embryonic hematopoiesis, and a more complete analysis of the scRNAseq dataset could help clarify this aspect.

Similarly, the data presented in the manuscript are not sufficient to clarify at what stage of the endothelial-to-hematopoietic transition (EHT) the TF activation has an impact. Indeed, even though the overall increase of functional hematopoietic progenitors is fully demonstrated, the assays proposed in the manuscript do not clarify whether this is due to a specific effect at the endothelial level or to an increased proliferation rate of the generated hematopoietic progenitors. Similar conclusions can be applied to the functional validation of IGFBP2 in vitro.

The overall conclusions are sometimes vague and not always supported by the data. For instance, the authors state that the CRISPR activation strategy resulted in transcriptional remodeling and a steer in cell identity, but they do not specify which cell types are involved and at what level of the EHT process this is happening. In the discussion, the authors also claim that they provided evidence to support that RUNX1T1 could regulate IGFBP2 expression. However, this is exclusively based on the enrichment of RUNX1T1 gRNA in cells expressing higher levels of IGFBP2 and it does not demonstrate any direct or indirect association of the two factors.

Reviewer #2 (Public Review):

This preprint by Pokrovsky and coworkers is a descriptive study reporting on non-breeding itinerant behaviour of an intrapalearctic migratory raptor, the rough-legged buzzard, and relating such non-breeding movements to snow cover across the European non-breeding range. The article is based on long-term GPS tracking data from a relatively large (n=43) sample of individuals that were equipped with state-of-the-art tracking devices in the Russian Arctic during 2013-2019. The results show that, upon breeding, buzzards migrated rapidly to southern non-breeding areas, located in open areas north of the Black and Caspian seas, where they perform continuous directional movements at a slower pace, initially moving SW (Oct to Jan) and then progressively moving NE (Feb to Apr) before embarking on rapid spring migration. It is suggested that such itinerant behaviour follows variation (expansion and retreat) of snow cover across the non-breeding range.

The results are potentially useful for researchers investigating the ecological drivers of bird movement patterns. The analytical framework appears solid, although some details on the analyses (requested during the previous review round) are still unclear and have not been modified despite explicit requests. Significant weaknesses in the theoretical framework persist in the revised version, including unwarranted claiming of novelty, overselling of importance of the study, and overinterpretation of the data. Below are key points that the authors did not consider when revising their manuscript.

(1) The authors underemphasize the fact that what they term 'fox-trot' migration is actually a well-known patterns for many other migratory species, both in the Nearctic and in the Afro-Palearctic migration systems. Such behaviour has previously been identified as 'itinerant' or 'non-breeding itinerancy', involving an alternation of stopovers and movements between different short-term non-breeding residency areas, or even slow continuous movements, and it seems that the pattern the authors report for this particular species is perfectly in line with such previous evidence. For instance, this is well-documented among migratory raptors, such as the Montagu's harrier, lesser kestrels or black kites, that exploit Sahelian savannahs, where large spatio-temporal variation in greenness and hence resource availability occurs. And, besides the mentioned cuckoos and nightingales, there are studies of red-backed shrikes suggesting the same, as well as of tree swallows in the Nearctic. Therefore, the authors should avoid claiming novelty for this study and introducing unnecessary and confusing new terms in the literature (i.e. the 'fox-trot' migration patterns) when these are definitely not strictly needed as they have been previously observed and defined otherwise. Sentences such as 'We used the rough-legged buzzard as a model..." are also similarly unwarranted. This is simply a descriptive studies reporting on such behaviour in yet another migratory species. The whole introduction is pervaded by a faulty logic. The authors first introduce a new (unwarranted) term based on previous evidence from other studies (none of which felt any need to introduce and describe it); then they assume, for unclear reasons, that the species they are studying should behave in that way; even more worryingly, based on these assumptions, they make specific predictions on how this species should behave, without any sound biological reason for these predictions. I admit I hardly see any scientific logic in this approach.

(2) The species has a very standard migration for a short-distance migrant, by all means. It moves to non-breeding areas, and once there it slowly moves towards the south in autumn and back again in spring, until it departs for pre-breeding migration. This is no different from other trans-Saharan migratory raptor species that spend the non-breeding period in the Sahel. Whether the species perform any short/medium term stopover (frequenting the same are for some days) during the non-breeding stage (between end of autumn migration and onset of spring migration), as is the case of most species showing non-breeding itinerancy, is not reported. The authors only show a slower pace of movement during the non-breeding period compared to autumn and spring migration, without providing any further details. This hinders comparisons with other previous studies.

(3) The current title is unnecessarily general (it may recall rather a review or meta-analysis) and not adequately describing the content of the manuscript. In order to be informative, the title should more tightly reflect the content of the article. A valid alternative would be 'Itinerant non-breeding behaviour of an intra-Palaearctic migratory raptor', as it would be far more adequate and informative.

(4) The text, particularly the Introduction and the Discussion, would greatly benefit from profound reframing in light of the above comments. Upon reading the first sentence of the introduction, it looks surprising that the authors based their suggestion for 'fox-trot' migration based on a very outdated article on the migration of Montagu's harrier based on sparse ring recovery data which merely suggests the existence of 'movements' within the non-breeding areas (i.e. non-breeding itinerancy), while subsequent large scale satellite tracking studies of this species provided compelling evidence for non-breeding area itinerancy (and again, no mention of 'fox-trot' whatsoever). The discussion is entirely framed around potential issues related to accurate monitoring of population size and trends, which the author surprisingly refer to 'conservation implications'. As I already mentioned in my previous review, the 'conservation implications' of this study are nearly negligible. At best, it suggests that caution should be applied when interpreting population trends of migratory species based on non-breeding area counts only, a pattern that is already well known for decades (consider the long-running IWC coordinated by Wetlands International!). In addition, Christmas Bird Count, a long-term monitoring program of AOS, is mentioned without any accurate reference to what it actually is, assuming that any reader would be familiar with a very peculiar monitoring scheme of the Nearctic region.

The final paragraph epitomizes how authors are overstating the importance of this study, claiming for non-existent novelty and even 'discovery': "Our study has identified and characterized a new pattern of migratory behavior, the 'foxtrot migration', along with the associated concept of 'dynamic range'. This discovery has significant implications for conservation strategies and adequate representation of non-breeding habitats".

Reviewer #1 (Public Review):

Summary:

For many years, there has been extensive electrophysiological research investigating the relationship between local field potential patterns and individual cell spike patterns in the hippocampus. In this study, using state-of-the-art imaging techniques, they examined spike synchrony of hippocampal cells during locomotion and immobility states. In contrast to conventional understanding of the hippocampus, the authors demonstrated that hippocampal place cells exhibit prominent synchronous spikes locked to theta oscillations.

Strengths:

The voltage imaging used in this study is a highly novel method that allows recording not only suprathreshold-level spikes but also subthreshold-level activity. With its high frame rate, it offers time resolution comparable to electrophysiological recordings. Moreover, it enables the visualization of actual cell locations, allowing for the examination of spatial properties (e.g., Figure 4G).

Weaknesses:

There is a notable deviation from several observations obtained through conventional electrophysiological recordings. Particularly, as mentioned below in detail, the considerable differences in baseline firing rates and no observations of ripple-triggered firing patterns raise some concerns about potential artifacts from imaging and analsyis, such as cell toxicity, abnormal excitability, and false detection of spikes. While these findings are intriguing if the validity of these methods is properly proven, accepting the current results as new insights is challenging.

Reviewer #1 (Public Review):

Granados-Aparici et al., investigate somatic-germline interactions in female mice. Mammalian oocytes are nurtured in multi-cellular ovarian follicles and communication with surrounding somatic cells is critical for oocyte development. This study focused on transzonal projections (TZP) extending from granulosa cells to the surface of oocytes and document the importance of SMAD4, a TGF- β mediator, in regulating the TZPs. They propose a model in which individual TZPs contact the surface of the oocyte and stably attaches if there is sufficient N-cadherin. In SMAD4-depleted cells, there is insufficient N-cadherin to stabilize the attachment. The TZP continues to elongate but eventually retracts. Their model is well supported by their experimental evidence and the manuscript is both well-formulated and written.

Comments on revised version:

The authors have addressed the issues raised in the original review.

Reviewer #1 (Public Review):

Recognition of bacterial lipopolysaccharide by Toll-like Receptor 4 is an essential molecular event triggering inflammation and overcoming Recognition of bacterial lipopolysaccharide by Toll-like Receptor 4 is an essential molecular event in triggering inflammation and overcoming infection by gram-negative bacteria. However, TLR4 has recently been found to respond to other endogenously derived ligands. This has implicated TLR4 signaling in the development of disease pathology, for example, Alzheimer's disease, through the recognition of amyloid-beta. Intriguingly, the signaling response to these non-bacterial-derived ligands differs from that of bacterial-derived LPS, suggesting mechanistic differences between endogenous and bacterial-derived agonists. In this work, the authors set out to characterize these mechanistic differences. TLR4 signals through two large macromolecular complexes that assemble at activated receptors: the Myddosome and Triffosome. One hypothesis the authors aimed to test was that different ligands alter these signaling complexes' kinetics and nano-scale features. The authors focused on testing this hypothesis by examining the formation of the Myddosome in live cells. A significant strength of the paper is that the authors developed technological innovations to address this problem. Using a nanopipette delivery mechanism combined with light sheet microscopy, the authors could observe Myddosome signaling in the whole cell volume of live macrophages. This allowed them to accurately quantify the Myddosome number, size, and kinetics of complex formation and compare cells stimulated with amyloid-beta and LPS. The authors discovered differences in Myddosomes formed under LPS versus amyloid-beta stimulation. In general, amyloid-beta TLR4 stimulation resulted in slower Myddosome formation with altered morphology. One limitation of the work, which the authors point out in the discussion, is that they could not distinguish signaling-competent Myddosomes. Future work will be needed to understand whether these amyloid beta induced Myddosomes assembly have a similar or altered complement of downstream signaling proteins (such as the IRAK4/1 and TRAF6). Secondly, the structural basis for how TLR4 would distinguish between different radically agonists remains speculative, and will need further investigation. Nonetheless, this paper is important for the technological innovation to look at the molecular dynamics of signal transduction, a technology that could be adapted to study other receptor signaling pathways.

It is already known that the subcellular location of intracellular TLRs is important for limiting the recognition of self-derived ligands and maintaining tolerance. This work hints at another possible layer of regulation: that a cell surface TLR (TLR4) generates diverse signaling outcomes to extrinsic or intrinsically derived agonists by changing the dynamic behavior of signaling proteins. If correct (and much further work is required to understand endogenous TLR ligands better), it might suggest that the innate immune system employs the same molecular hardware but with altered kinetics to distinguish between exogenous and endogenous inflammatory signals. Thus, pathological aggregates or markers of sterile inflammation might be recognized and responded to by a specific signaling program that is defined kinetically. It will be an interesting direction for future studies to investigate whether and how diverse pathogen and endogenous inflammatory signals modulate the dynamics of signaling complexes.

Reviewer #1 (Public Review):

Summary:

The paper carries out an impressive and exhaustive non-sense mutagenesis using deep mutational scanning (DMS) of the gonadotropin-releasing hormone receptor for the WT protein and two single point mutations that I) influences TM insertion (V267T) and ii) influences protein stability (W107A) and then measures the effect of these mutants on correct plasma membrane expression (PME).

Overall, most mutations decreased mGnRHR PME levels in all three backgrounds, indicating poor mutational tolerance under these conditions. The W107A variant wasn't really recoverable with low levels of plasma membrane localisation. For the V267T variant, most additional mutations were more deleterious than WT based on correct trafficking, indicating a synergistic effect. As one might expect, there was a higher degree of positive correlation between V267T/W107A mutants and other mutants located in TM regions, confirming that improper trafficking was a likely consequence of membrane protein co-translational folding. Nevertheless, context is important, as positive synergistic mutants in the V27T could be negative in the W107A background and vice versa. Taken together, this important study highlights the complexity of membrane protein folding in dissecting the mechanism-dependent impact of disease-causing mutations related to improper trafficking.

Strengths:

This is a novel and exhaustive approach to dissect how receptor mutations under different mutational backgrounds related to co-translational folding, could influence membrane protein trafficking.

Weaknesses:

The premise for the study requires an in-depth understanding of how the single point mutations analysed effect membrane protein folding in context of DMS, but the single point mutants used could do with further validation. The V267T mutant only reduced MP insertion by 10% and the effect of W107A on protein stability was not assessed. Furthermore, plasma membrane expression has been used as a proxy for incorrect membrane protein folding, but this not necessarily be the case, as even correctly folded membrane proteins may not be trafficked correctly, at least, under heterologous expression conditions. In addition, mutations can effect trafficking and potential post-translational modifications, like glycosylation.

Reviewer #1 (Public Review):

This manuscript describes soluble Uric Acid (sUA) as an endogenous inhibitor of CD38, affecting CD38 activity and NAD+ levels both in vitro and in vivo. Importantly, the inhibition constants calculated support the claim that sUA inhibits CD38 under physiological conditions. These findings are of extreme importance to understanding the regulation of an enzyme that has been shown to be the main NAD+/NMN-degrading enzyme in mammals, which impacts several metabolic processes and has major implications for understanding aging diseases. The manuscript is well written, the figures are self-explanatory, and in the experiments presented, the data is very solid. The authors discuss the main limitations of the study, especially in regard to the in vivo results. As a whole, I believe that this is a very interesting manuscript that will be appreciated by the scientific community and that opens a lot of new questions in the field of metabolism and aging. I found some issues that I believe constitute a weakness in the manuscript, and although they do not require new experiments, they may be considered by the authors for discussion in the final version of the manuscript.

The authors acknowledge the existence of several previous papers involving pharmacological inhibition of CD38 and their impact on several models of metabolism and aging. However, they only cite reviews. Given the focus of the manuscript, I believe that the seminal original papers should be cited.

Related to the previous comment, the authors show that they have identified the functional group on sUA that inhibits CD38, 1,3-dihydroimidazol-2-one. How does this group relate with previous structures that were shown to inhibit CD38 and do not have this chemical structure? Is sUA inhibiting CD38 in a different site? A crystallographic structure of CD38-78c is available in PDB that could be used to study or model these interactions.

Although the mouse model used to manipulate sUA levels is not ideal, the authors discuss its limitations, and importantly, they have CD38 KO mice as control. However, all the experiments were performed in very young mice, where CD38 expression is low in most tissues (10.1016/j.cmet.2016.05.006). This point should be mentioned in the discussion and maybe put in the context of variations of sUA levels during aging.

Reviewer #1 (Public Review):

Summary:

In "1 Exploring the Spatial Distribution of Persistent SARS-CoV-2 Mutations -Leveraging mobility data for targeted sampling" Spott et al. combine SARS-CoV-2 genomic data alongside granular mobility data to retrospectively evaluate the spread of SARS-CoV-2 alpha lineages throughout Germany and specifically Thuringia. They further prospectively identified districts with strong mobility links to the first district in which BQ.1.1 was observed to direct additional surveillance efforts to these districts. The additional surveillance effort resulted in the earlier identification of BQ.1.1 in districts with strong links to the district in which BQ.1.1 was first observed.

Strengths:

There are two important strengths of this work. The first is the scale and detail in the data that has been generated and analyzed as part of this study. Specifically, the authors use 6,500 SARS-CoV-2 sequences and district-level mobility data within Thuringia. I applaud the authors for making a subset of their analyses public e.g. on the associated micro react page.

Further, the main focus of the article is on the potential utility of mobility-directed surveillance sequences. While I may certainly be mistaken, I have not seen this proposed elsewhere, at least in the context of SARS-CoV-2. The authors were further able to test this concept in a real-world setting during the emergence of BQ.1.1. This is a unique real-world evaluation of a novel surveillance sequencing strategy and there is considerable value in publishing this analysis.

Weaknesses:

The article is quite strong and I find the analyses to generally be rigorous. However, there are places where I believe the text should be modified to slightly weaken the conclusions drawn from the presented analyses. Specific examples include:

- It seems the mobility-guided increased surveillance included only districts with significant mobility links to the origin district and did not include any "control" districts (those without strong mobility links). As such, you can only conclude that increasing sampling depth increased the rate of detection for BQ.1.1., not necessarily that doing so in a mobility-guided fashion provided an additional benefit. I absolutely understand the challenges of doing this in a real-world setting and think that the work remains valuable even with this limitation, but I would like the lack of control districts to be more explicitly discussed.

- Line 313: While this work has reliably shown that the spread of Alpha was slower in Thuringia, I don't think there have been sufficient analyses to conclude that this is due to the lack of transportation hubs. My understanding is that only mobility within Thuringia has been evaluated here and not between Thuringia and other parts of Germany.

- Line 333 (and elsewhere): I'm not convinced, based on the results presented in Figure 2, that the authors have reliably identified a sampling bias here. This is only true if you assume (as in line 235) that the variant was in these districts, but that hasn't actually been demonstrated here. While I recognize that for high-prevalence variants there is a strong correlation between inflow and variant prevalence, low-prevalence variants by definition spread less and may genuinely be missing from some districts. To support this conclusion that they identified a bias, I'd like to see some type of statistical model that is based e.g. on the number of sequences, prevalence of a given variant in other districts, etc. Alternatively, the language can be softened ("putative sampling bias").

Reviewer #1 (Public Review):

Summary:

Zanzibar archipelago is close to achieve malaria elimination, but despite the implementation of effective control measures there is still low level seasonal malaria transmission. This could be due to the frequent importation of malaria from the mainland Tanzania and Kenya, reservoir of asymptomatic infections and competent vectors. To investigate population structure and gene flow of P. falciparum in Zanzibar and mainland Tanzania, they used 178 samples from mainland Tanzania and 213 from Zanzibar that were previously sequenced using molecular inversion probes (MIPs) panels targeting single nucleotide polymorphisms (SNPs). They performed Principal Component Analysis (PCA) and identity by descent (IBD) analysis to assess genetic reladness between isolates. Parasites from coastal mainland Tanzania contribute for the genetic diversity in parasite population in Zanzibar. Despite this, there is a pattern of isolation by distance and microstructure within the achipelago, and evidence of local sharing of highly related strains sustaining malaria transmission in Zanzibar that are important targets for interventions such as mass drug administration and vector control, in addition to measures against imported malaria.

Strengths:

This study presents important samples to understand population structure and gene flow between mainland Tanzania and Zanzibar, especially from rural Bagamoyo District, where malaria transmission persists and there is a major port of entry to Zanzibar. In addition, this study includes a larger set of SNPs, providing more robustness for analyzes such as PCA and IBD. Therefore, the conclusions of this paper are well supported by data.

Comments on revised version:

The authors answered all my questions.

Reviewer #1 (Public Review):

Summary:

The presented study focuses on the role of formin-like 2 (FMNL2) in oocyte meiosis. The authors assessed FMNL2 expression and localization in different meiotic stages and subsequently, by using siRNA, investigated the role of FMNL2 in spindle migration, polar body extrusion, and distribution of mitochondria and endoplasmic reticulum (ER) in mouse oocytes.

Strengths:

Novelty in assessing the role of formin-like 2 in oocyte meiosis

Weaknesses:

Overstating some of the presented data

Unconvincing analysis of the endoplasmic reticulum and mitochondria distribution

The authors addressed all my comments. The section materials and methods was improved. However, some statements still need to be clarified, as they seem to be overstated. I'm still not convinced about the main findings. For example, the analysis of ER and mitochondria distribution was based on a subjective assessment of clustering in meiosis I oocytes, and it's missing objective parameters and timing of the analysis.

Comments on revised version:

The authors addressed all my comments. The section materials and methods was improved. However, some statements still need to be clarified, as they seem to be overstated.

Reviewer #1 (Public Review):

Summary:

HIV associated nephropathy (HIVAN) is a rapidly progressing form of kidney disease that manifests secondary to untreated HIV infection, and is predominantly seen in individuals of African descent. Tg26 mice carrying an HIV transgene lacking gag and pol exhibit high levels of albuminuria and rapid decline in renal function that recapitulates many features of HIVAN in humans. HIVAN is seen predominantly in individuals carrying two copies of missense variants in the APOL1 gene, and the authors have previously shown that APOL1 risk variant mRNA induces activity of the double strand RNA sensor kinase PKR. Because of the tight association between the APOL1 risk genotype and HIVAN, the authors hypothesized that PKR activation may mediate the renal injury in Tg26 mice, and tested this hypothesis by treating mice with a commonly used PKR inhibitory compound called C16. Treatment with C16 substantially attenuated renal damage in the Tg26 model as measured by urinary albumin/creatinine ratio, urinary NGAL/creatinine ratio and improvement in histology. The authors then performed bulk and single-nucleus RNAseq on kidneys from mice from different treatment groups to identify pathways and patterns of cell injury associated with HIV transgene expression as well as to determine the mechanistic basis for the effect of C16 treatment. They show that proximal tubule nuclei from Tg26 mice appear to have more mitochondrial transcripts which was reversed by C16 treatment and suggest that this may provide evidence of mitochondrial dysfunction in this model. They explore this hypothesis by showing there is a decrease in the expression of nuclear encoded genes and proteins involved in oxidative phosphorylation as well as a decrease in respiratory capacity via functional assessment of respiration in tubule and glomerular preparations from these mouse kidneys. All of these changes were reversed by C16 treatment. The authors propose the existence of a novel injured proximal tubule cell-type characterized by the leak of mitochondrial transcripts into the nucleus (PT-Mito). Analysis of HIV transgene expression showed high level expression in podocytes, consistent with the pronounced albuminuria that characterizes this model and HIVAN, but transcripts were also detected in tubular and endothelial cells. Because of the absence of mitochondrial transcripts in the podocytes, the authors speculate that glomerular mitochondrial dysfunction in this model is driven by damage to glomerular endothelial cells.

Strengths:

The strengths of this study include the comprehensive transcriptional analysis of the Tg26 model, including an evaluation of HIV transgene expression, which has not been previously reported. This data highlights that HIV transcripts are expressed in a subset of podocytes, consistent with the highly proteinuric disease seen in mouse and humans. However, transcripts were also seen in other tubular cells, notably intercalated cells, principal cells and injured proximal tubule cells. Though the podocyte expression makes sense, the relevance of the tubular expression to human disease is still an open question.

The data in support of mitochondrial dysfunction are also robust and rely on combined evidence from downregulation of transcripts involved in oxidative phosphorylation, decreases in complex I and II as determined by immunoblot, and assessments of respiratory capacity in tubular and glomerular preparations. These data are largely consistent with other preclinical renal injury model reported in the literature as well as previous, less thorough assessments in the Tg26 model.

Weaknesses:

The key weakness of the study lies in the use of a PKR inhibitor with questionable specificity. C16 has been reported to inhibit numerous other kinases including cyclin CDKs and GSK3α and -β, and this means that the conclusions of this study with respect to the role of PKR are highly questionable. The rationale for the dose used was not provided (and is lower than used in other publications with C16), and in the absence of drug exposure data and assessment of target engagement, it is difficult to ascertain whether substantial inhibition of PKR was achieved.

A second key weakness lies in the identification of the PT-Mito cell cluster. Though the authors provide some rationale for the identification of this specific cell type, it seems equally plausible the cells merely reflect a high background capture of mitochondria in a subset of droplets. The IHC analysis that was provided is not convincing enough to support the claim and more careful high resolution imaging and in situ hybridization (with appropriate quantitation) will be needed to provide substantive support for the presence of a proximal tubule cell type with mitochondrial transcript that are trafficked to the nucleus.

Revision summary:

The authors have revised the manuscript to acknowledge the potential limitations of the C16 tool compound used and have performed some additional analyses that suggest the PT-Mito population can be identified in samples from KPMP. The authors added some control images for the in situ hybridizations, which are helpful, though they don't get to the core issue of limited resolution to determine whether mitochondrial RNA is present in the nuclei of injured PT cells. Some additional work has been done to show that C16 treatment results in a decrease in phospho-PKR, a readout of PKR inhibition. These changes strengthen the manuscript by providing some evidence for the translatability of the PT-mito cluster to humans and some evidence for on-target activity for C16. It would be helpful if the authors could quantify the numbers of cells in IHC with nuclear transcripts as well as pointing out some specific examples in the images provided, as comparator data for the snRNAseq studies in which 3-6% of cortex cells had evidence of nuclear mitochondrial transcripts.

Reviewer #1 (Public Review):

Summary:

The authors constructed a live-attenuated vaccine candidate, BK2102, combining naturally occurring virulence-attenuating mutations in the key coding regions. They showed that intranasal inoculation with the candidate vaccine-induced humoral and cellular immune responses in Syrian hamsters without apparent tissue damage in the lungs and protected against a wild-type SARS-CoV-2 strain with D614G mutation and the latest Omicron subvariant (BA.5) strain. The neutralizing antibodies induced by BK2102 persisted for the long term (up to 364 days). Furthermore, they confirmed the safety of the proposed vaccine using transgenic (Tg) mice expressing human ACE2 (hACE2).

Strengths:

The authors followed a robust methodology to establish the proposed vaccine's protective effect and safety profile in the hamsters and transgenic mice expressing human ACE2.

Weaknesses:

(1) A comparative safety assessment of the available m-RNA and live attenuated vaccines will be necessary. The comparison should include details of the doses, neutralizing antibody titers with duration of protection, tissue damage in the various organs, and other risks, including virulence reversal.

(2) The vaccine's effect on primates is doubtful. The study fails to explain why only two of four monkeys developed neutralizing antibodies. Information about the vaccine's testing in monkeys is also missing: What was the level of protection and duration of the persistence of neutralizing antibodies in monkeys? Were the tissue damages and other risks assessed?

(3) The vaccine's safety in immunosuppressed individuals or individuals with chronic diseases should be assessed. Authors should make specific comments on this aspect.

(4) The candidate vaccine has been tested with a limited number of SARS-CoV-2 strains. Of note, the latest Omicron variants have lesser virulence than many early variants, such as the alfa, beta, and delta strains.

(5) Limitations of the study have not been discussed.

Reviewer #1 (Public Review):

Overview:

The authors construct a pair of E. coli populations that differ by a single gene duplication in a selectable fluorescent protein. They then evolve the two populations under differing selective regimes to assess whether the end result of the selective process is a "better" phenotype when starting with duplicated copies. Importantly, their starting duplicated population is structured to avoid the duplication-amplification process often seen in bacterial artificial evolution experiments. They find that while duplication increases robustness and speed of adaptation, it does not result in more highly adapted final states, in contrast to Ohno's hypothesis.

Major comments:

This is an excellent study with a very elegant experimental setup that allows a precise examination of the role of duplication in functional evolution, exclusive of other potential mechanisms. My main concern is to clarify some of the arguments relating to Ohno's hypothesis.

I think my main confusion on first reading the manuscript was in the precise definition of Ohno's hypothesis. I think this confusion was mine and not the authors, but it is likely common and could be addressed.

Most evolutionary biologists think of gene duplication as making neofunctionalization "easier" by providing functional redundancy and a larger mutational target, such that the evolutionary process of neofunctionalization is faster (as the authors observed). In this framework, the final evolved state might not differ when selection is applied to duplicated copies or a single-copy gene. Ohno's hypothesis, by contrast, argues that there generally exist adaptive conflicts between the ancestral function and the "desired" novel function, such that strong selection on a single-copy gene cannot produce the evolutionary optima that selection on two copies would. This idea is hinted at in the quotation from Ohno in paragraph 2 of the introduction. However, the sentences that follow I don't think reinforce this concept well enough and lead to some confusion.

With that definition in mind, I agree with the authors' conclusion that these data do not support Ohno's hypothesis. My quibble would be that what is actually shown here is that adaptive conflict in function is not universal: there are cases where a single gene can be optimized for multiple functions just as well as duplicated copies. I do not think the authors have, however, refuted the possibility that such adaptive conflicts are nonetheless a significant barrier to evolutionary innovation in the absence of gene duplication generally. Perhaps just a sentence or two to this effect might be appropriate.

I also think the authors need to clarify their approach to normalizing fluorescence between the two populations to control for the higher relative protein expression of the population with a duplicated gene. Since each population was independently selected with the highest fluorescing 60% (or less) of the cells selected, I think this normalization is appropriate. Of course, if the two populations were to compete against each other, this dosage advantage of the duplicates would itself be a selective benefit. Even as it is, the dosage advantage should be a source of purifying selection on the duplication, and perhaps this should be noted.

Finally, I am slightly curious about the nature of the adaptations that are evolving. The authors primarily discuss a few amino-acid changing mutations that seem to fix early in the experiment. Looking at Figure 3, it however, appears that the populations are still evolving late in the experiment, and so presumably other changes are occurring later on. Do the authors believe that perhaps expression changes to increase protein levels are driving these later changes?

Reviewer #1 (Public Review):

In their manuscript "Spindle assembly checkpoint-dependent mitotic delay is required for cell division in absence of centrosomes," Farrell and colleagues employ carefully chosen approaches to assay mitotic timeliness in the absence of centrosomes in mammalian culture cells, namely the mechanism behind it and its function. The authors acknowledge prior work well and present their data succinctly, clearly, and with a clear logical flow of experiments. The experiments are thoughtfully designed and presented, with appropriate controls in place.

The authors' model whereby centrosome separation and its early definition of poles mediates timely mitosis without relying on a SAC-dependent delay is compelling, and the authors' data are consistent with it. They show using two different MPS1 inhibitors that acentrosomal cell division fails, supporting their claims that a SAC-dependent delay is required in these instances. Furthermore, they show that reintroducing a time delay is sufficient to restore cell division, but inhibiting a different aspect of SAC function does not restore cell division. Next, the authors rule out polyploidy as a potential confounding factor for requiring a SAC-dependent delay, and instead demonstrate that inhibiting centrosome separation by Eg5 inhibition is sufficient to require this delay for mitotic progression. The authors' findings overall support their proposed model.

Probing what aspects of centrosomes protect against a requirement for SAC-dependent delays would strengthen the work and specifically the conclusion that centrosomes provide "two-ness". For example, the authors could examine division in a population of cells with only one centrosome. Seeing some restoration of mitotic progression in the absence of SAC-dependent delays would suggest that even one centrosome with uninhibited Eg5 is sufficient to negate SAC-dependent delays, and would limit models for what exactly centrosomes contribute. This would help disentangle the roles of actual centrosomes and their biochemical cues, Eg5-driven centrosome separation, and early definition of poles on mitotic progression in the absence of SAC-dependent delays. Making a high fraction of cells with one centrosome could be achieved by using centrinone for a shorter time.

Comments on revised version:

The main point from the initial review does not appear to be addressed in the revised version. As such, the comments on the revised version remain the same.

Reviewer #1 (Public Review):

Summary:

Zanetti et al. use biophysical and cellular assays to investigate the interaction of the birnavirus VP3 protein with the early endosome lipid PI3P. The major novel finding is that the association of the VP3 protein with an anionic lipid (PI3P) appears to be important for viral replication, as evidenced through a cellular assay on FFUs.

Strengths:

Supports previously published claims that VP3 may associate with early endosomes and bind to PI3P-containing membranes. The claim that mutating a single residue (R200) critically affects early endosome binding and that the same mutation also inhibits viral replication suggests a very important role for this binding in the viral life cycle.

Weaknesses:

The manuscript is relatively narrowly focused: one bimolecular interaction between a host cell lipid and one protein of an unusual avian virus (VP3-PI3P). Aspects of this interaction have been described previously. Additional data would strengthen claims about the specificity and some technical issues should be addressed. Many of the core claims would benefit from additional experimental support to improve consistency.

Reviewer #1 (Public Review):

Summary:

This is an important work showing that loss of LRRK function causes late-onset dopaminergic neurodegeneration in a cell-autonomous manner. One of the LRRK members, LRRK2, is of significant translational importance as mutations in LRRK2 cause late-onset autosomal dominant Parkinson's disease (PD). While many in the field assume that LRRK2 mutant causes PD via increased LRRK2 activity (i.e., kinase activity), it is not a settled issue as not all disease-causing mutant LRRK2 exhibits increased activity. Further, while LRRK2 inhibitors are under clinical trials for PD, the consequence of chronic, long-term LRRK2 inhibition is unknown. Thus, studies evaluating the long-term impact of LRRK deficit have important translational implications. Moreover, because LRRK proteins, particularly LRRK2, are known to modulate immune response and intracellular membrane trafficking, the study's results and the reagents will be valuable for others interested in LRRK function.

Strengths:

This report describes a mouse model where LRRK1 and LRRK2 genes are conditionally deleted in dopaminergic neurons. Previously, this group showed that while loss of LRRK2 expression does not cause brain phenotype, loss of both LRRK1 and LRRK2 causes a later onset, progressive degeneration of catecholaminergic neurons, dopaminergic (DAergic) neurons in the substantia nigra (SN) and noradrenergic neurons in the Locus Coeruleus (LC). However, because LRRK genes are widely expressed with some peripheral phenotypes, it was unknown if the neurodegeneration in LRRK double Knock Out (DKO) was cell autonomous. To rigorously test this question, the authors generated a double conditional KO allele where both LRRK1 and LRRK2 genes were targeted to contain loxP sites. This was beyond what is usually required as most investigators might just have combined one KO allele with another floxed allele. The authors provide a rigorous validation showing that the Driver (DAT-Cre) is expressed in most DAergic neurons in SN and that LRRK levels are decreased selectively in the ventral midbrain. Using these mice, the authors show that the number of DA neurons is average at 15 but significantly decreased at 20 months of age. Moreover, the authors show that the number of apoptotic neurons is increased by ~2X in aged SN, demonstrating increased ongoing cell death and activated microglia. The degeneration is limited to DA neurons as LC neurons are not lost, and this population does not express DAT. Overall, the mouse genetics and experimental analysis were performed rigorously, and the results were statistically sound and compelling.

Weakness:

I only have a few minor comments. First, in PD and other degenerative conditions, axon and terminal loss occur prior to cell bodies. It might be beneficial to show the status of DAergic markers in the striatum. Second, previous studies indicate that very little, if any, LRRK1 is expressed in SN DAergic neurons. This also the case with the Allen Brain Atlas profile. Thus, the authors should discuss the discrepancy, as they imply significant LRRK1 expression in DA neurons.

Revision:

I appreciate the authors revising the manuscript with additional data that have clarified my prior comments. They now show that TH levels in the striatum decrease with SNpc neurons. Further, while there is some disagreement regarding the expression LRRK1 in SNpc, the authors provide a convincing case that LRRK1 function is important in SNpc DA neurons.

Reviewer #1 (Public Review):

Summary:

The authors present a detailed study of a nearly complete Entomophthora muscae genome assembly and annotation, along with comparative analyses among related and non-related entomopathogenic fungi. The genome is one of the largest fungal genomes sequenced, and the authors document the proliferation and evolution of transposons and presence/absence of related genetic machinery to explore how this may have occurred. There has also been an expansion in gene number, which appears to contain many "novel" genes unique to E. muscae. Functionally, the authors were interested in CAZymes, proteases, circadian clock related genes (due to entomopathogenicity/ host manipulation), other insect pathogen specific genes, and secondary metabolites. There are many interesting findings including expansions in trahalases, unique insulinase and another peptidase, and some evidence for RIP in Entomophthoralean fungi. The authors performed a separate study examining E. muscae species complex and related strains. Specifically, morphological traits were measured for strains and then compared to the 28S+ITS-based phylogeny, showing little informativeness of these morpho characters with high levels of overlap.

This work represents a big leap forward in genomics of non-Dikarya fungi and large fungal genomes. Most of the gene homologs have been studied in species that diverged hundreds of millions of years ago, and therefore using standard comparative genomic approaches are not trivial and still relatively little is known. This paper provides many new hypotheses and potential avenues of research about fungal genome size expansion, entomopathogenesis in zygomycetes, and cellular functions like RIP and circadian mechanisms.

Strengths:

There are many strengths to this study. It represents a massive amount of work and a very thorough functional analysis of the gene content in these fungi (which are largely unsequenced and definitely understudied). Too often comparative genomic work will focus on one aspect and leave the reader wondering about all the other ways genome(s) are unique or different from others. This study really dove in and explored the relevant aspects of the E. muscae genome.

The authors used both a priori and emergent properties to shape their analyses (by searching for specific genes of interest and by analyzing genes underrepresented, expanded, or unique to their chosen taxa), enabling a detailed review of the genomic architecture and content. Specifically, I'm impressed by the analysis of missing genes (pFAMs) in E. muscae, none of which are enriched in relatives, suggesting this fungus is really different not by gene loss, but by its gene expansions.

Analyzing species-level boundaries and the data underlying those (genetic or morphological) is not something frequently presented in comparative genomic studies, however, here it is a welcome addition as the target species of the study is part of a species complex where morphology can be misleading and genetic data is infrequently collected in conjunction with the morphological data.

Weaknesses:

The conclusions of this paper are well supported, and I think the clarifications and improvements made to the manuscript in the revision process have greatly improved the paper.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Day et al. present a high-throughput version of expansion microscopy to increase the throughput of this well-established super-resolution imaging technique. Through technical innovations in liquid handling with custom-fabricated tools and modifications to how the expandable hydrogels are polymerized, the authors show robust ~4-fold expansion of cultured cells in 96-well plates. They go on to show that HiExM can be used for applications such as drug screens by testing the effect of doxorubicin on human cardiomyocytes. Interestingly, the effects of this drug on changing DNA organization were only detectable by ExM, demonstrating the utility of HiExM for such studies.

Overall, this is a very well-written manuscript presenting an important technical advance that overcomes a major limitation of ExM - throughput. As a method, HiExM appears extremely useful, and the data generally support the conclusions.

Strengths:

Hi-ExM overcomes a major limitation of ExM by increasing the throughput and reducing the need for manual handling of gels. The authors do an excellent job of explaining each variation introduced to HiExM to make this work and thoroughly characterize the impressive expansion isotropy. The dox experiments are generally well-controlled and the comparison to an alternative stressor (H2O2) significantly strengthens the conclusions.

Weaknesses:

(1) Based on the exceedingly small volume of solution used to form the hydrogel in the well, there may be many unexpanded cells in the well and possibly underneath the expanded hydrogel at the end of this. How would this affect the image acquisition, analysis, and interpretation of HiExM data?

(2) It is unclear why the expansion factor is so variable between plates (e.g., Figure 2H). This should be discussed in more detail.

(3) The authors claim that CF dyes are more resistant to bleaching than other dyes. However, in Figure. S3, it appears that half of the CF dyes tested still show bleaching, and no data is shown supporting the claim that Alexa dyes bleach. It would be helpful to include data supporting the claim that Alexa dyes bleach more than CF dyes and the claim that CF dyes in general are resistant to bleaching should be modified to more accurately reflect the data shown.

(4) Related to the above point, it appears that Figure S11 may be missing the figure legend. This makes it hard to understand how HiExM can use other photo-inducible polymerization methods and dyes other than CF dyes.

(5) The use of automated high-content imaging is impressive. However, it is unclear to me how the increased search space across the extended planar area and focal depths in expanded samples is overcome. It would be helpful to explain this automated imaging strategy in more detail.

(6) The general method of imaging pre- and post-expansion is not entirely clear to me. For example, on page 5 the authors state that pre-expansion imaging was done at the center of each gel. Is pre-expansion imaging done after the initial gel polymerization? If so, this would assume that the gelation itself has no effect on cell size and shape if these gelled but not yet expanded cells are used as the reference for calculating expansion factor and isotropy.

(7) In the dox experiments, are only 4 expanded nuclei analyzed? It is unclear in the Figure 3 legend what the replicates are because for the unexpanded cells, it says the number of nuclei but for expanded it only says n=4. If only 4 nuclei are analyzed, this does not play to the strengths of HiExM by having high throughput.

(8) I am not sure if the analysis of dox-treated cells is accurate for the overall phenotype because only a single slice at the midplane is analyzed. It would be helpful to show, at least in one or two example cases, that this trend of changing edge intensity occurs across the whole 3D nucleus.

(9) It would be helpful to provide an actual benchmark of imaging speed or throughput to support the claims on page 8 that HiExM can be combined with autonomous imaging to capture thousands of cells a day. What is the highest throughput you have achieved so far?

Reviewer #1 (Public Review):

Summary:

Zheng and colleagues assessed the real-world efficacy of SARS-CoV-2 vaccination against re-infection following the large omicron wave in Shanghai in April 2022. The study was performed among previously vaccinated individuals. The study successfully documents a small but real added protective benefit of re-vaccination, though this diminishes in previously boosted individuals. Unsurprisingly, vaccine preventative efficacy was higher if the vaccine was given in the month before the 2nd large wave in Shanghai. The re-infection rate of 24% suggests that long-term anti-COVID immunity is very difficult to achieve. The conclusions are largely supported by the analyses. These results may be useful for planning the timing of subsequent vaccine rollouts.

Strengths:

The strengths of the study are a very large and unique cohort based on synchronously timed single infection among individuals with well-documented vaccine histories. Statistical analyses seem appropriate. As with any cohort study, there are potential confounders and the possibility of misclassification and the authors outline limitations nicely in the discussion.

Weaknesses:

(1) Partially and fully vaccinated are never defined and it is difficult to understand how this differs from single, and double, booster vaccines. The figures including all of these groups are a bit confusing for this reason.

(2) Figure 3 is a bit challenging to interpret because it is a bit atypical to compare each group to a different baseline (ie 2V-I-V vs 2V-I). I would label the y-axis 2V-I-V vs 2V-I (change all of the labels) to make this easier to understand.

(3) A 15% reduction in infection is quite low. It would be helpful to discuss if any quantitative or qualitative signals suggest at least a reduction in severe outcomes such as death, hospitalization, ER visits, or long COVID. I am not sure that a 15% reduction in cases supports extra vaccination without some other evidence of added benefit.

(4) Why exclude the 74962 unvaccinated from the analysis. it would be interesting to see if getting vaccinated post-infection provides benefits to this group

(5) Pudong should be defined for those who do not live in China.

(6) The discussion about healthcare utilization bias is welcomed and well done. It would be great to speculate on whether this bias might favor the null or alternative hypothesis.

Reviewer #1 (Public Review):

Summary:

In this study, Franke et al. explore and characterize color response properties across primary visual cortex, revealing specific color opponent encoding strategies across the visual field. The authors use awake 2P imaging to define the spectral response properties of visual interneurons in layer 2/3. They find that opponent responses are more pronounced at photopic light levels, and that diversity in color opponent responses exists across the visual field, with green ON/ UV OFF responses more strongly represented in the upper visual field. This is argued to be relevant for the detection of certain features that are more salient when using chromatic space, possibly due to noise reduction. In the revised version, Franke et al. have addressed the potential pitfalls in the discussion, which is an important point for the non-expert reader. Thus, this study provides a solid characterization of the color properties of V1 and is a valuable addition to visual neuroscience research.

My remaining concerns are based more on the interpretation. I'm still not convinced by the statement "This type of color-opponency in the receptive field center of V1 neurons was not present in the receptive field center of retinal ganglion cells and, therefore, is likely computed by integrating center and surround information downstream of the retina." and I would suggest rewording it in the abstract.

As discussed previously and now nicely added to the discussion, it is difficult to make a direct comparison given the different stimulus types used to characterize the retina and V1 recordings and the different levels of adaptation in both tissues. I will leave this point to the discussion, which allows for a more nuanced description of the phenomenon. Why do I think this is important? In the introduction, the authors argue that "the discrepancy [of previous studies] may be due to differences in stimulus design or light levels." However, while different light levels can be tested in V1, this cannot be done properly in the retina with 2P experiments. To address this, one would have to examine color-opponency in RGC terminals in vivo, which is beyond the scope of this study. Addressing these latter points directly in the discussion would, in my opinion, only strengthen the study.

Reviewer #1 (Public Review):

Summary:

Willems and colleagues test whether unexpected shock omissions are associated with reward-related prediction errors by using an axiomatic approach to investigate brain activation in response to unexpected shock omission. Using an elegant design that parametrically varies shock expectancy through verbal instructions, they see a variety of responses in reward-related networks, only some of which adhere to the axioms necessary for prediction error. In addition, there were associations between omission-related responses and subjective relief. They also use machine learning to predict relief-related pleasantness, and find that none of the a priori "reward" regions were predictive of relief, which is an interesting finding that can be validated and pursued in future work.

Strengths:

The authors pre-registered their approach and the analyses are sound. In particular, the axiomatic approach tests whether a given region can truly be called a reward prediction error. Although several a priori regions of interest satisfied a subset of axioms, no ROI satisfied all three axioms, and the authors were candid about this. A second strength was their use of machine learning to identify a relief-related classifier. Interestingly, none of the ROIs that have been traditionally implicated in reward prediction error reliably predicted relief, which opens important questions for future research.

Weaknesses:

To ensure that the number of omissions is similar across conditions, the task employs inaccurate verbal instructions; i.e. 25% of shocks are omitted, regardless of whether subjects are told that the probability is 100%, 75%, 50%, 25%, or 0%. Given previous findings on interactions between verbal instruction and experiential learning (Doll et al., 2009; Li et al., 2011; Atlas et al., 2016), it seems problematic a) to treat the instructions as veridical and b) average responses over time. Based on these prior work, it seems reasonable to assume that participants would learn to downweight the instructions over time through learning (particularly in the 100% and 0% cases); this would be the purpose of prediction errors as a teaching signal. The authors do recognize this and perform a subset analysis in the 21 participants who showed parametric increases in anticipatory SCR as a function of instructed shock probability, which strengthened findings in the VTA/SN; however given that one third of participants (n=10) did not show parametric SCR in response to instructions, it seems like some learning did occur. As prediction error is so important to such learning, a weakness of the paper is that conclusions about prediction error might differ if dynamic learning were taken into account.

Reviewer #1 (Public Review):

Summary:

The process of taste perception is significantly more intricate and complex in Lepidopteran insects. This investigation provides valuable insights into the role of Gustatory receptors and their dynamics in the sensation of sucrose, which serves as a crucial feeding cue for insects. The article highlights the differential sensitivity of Grs to sucrose and their involvement in feeding and insect behavior.

Strengths:

To support the notion of the differential specificity of Gr to sucrose, this study employed electrophysiology, ectopic expression of Grs in Xenopus, genome editing, and behavioral studies on insects. This investigation offers a fundamental understanding of the gustation process in lepidopteran insects and its regulation of feeding and other gustation-related physiological responses. This study holds significant importance in advancing our comprehension of lepidopteran insect biology, gustation, and feeding behavior.

Weaknesses:

While this manuscript demonstrates technical proficiency, there exists an opportunity for additional refinement to optimize comprehensibility for the intended audience. Several crucial sugars have been overlooked in the context of electrophysiology studies and should be incorporated. Furthermore, it is imperative to consider the potential off-target effects of Gr knock-out on other Gr expressions. This investigation focuses exclusively on Gr6 and Gr10, while neglecting a comprehensive narrative regarding other Grs involved in sucrose sensation.

Reviewer #1 (Public Review):

Summary

This study identifies a family of solute transports in the enteric protist, Blastocystis, that may mediate the transport of glycolytic intermediates across the mitochondrial membrane. The study builds on previous observations suggesting that Blastocystis (and other Stramenopiles) are unusual in having a compartmentalized glycolytic pathway with enzymes involved in upper and lower glycolysis being located in the cytosol and mitochondria, respectively. In this study, the authors identified two putative Stamenopile metabolite transporters that are related to plant di/tricarboxylic acid transporters that might mediate the transport of glycolytic intermediates across the mitochondrial membrane. These GIC-transporters were localized to the Blastocystis mitochondrion using specific rabbit antibodies and shown to bind several glycolytic intermediates (including GAP, DHAP and PEP) based on thermostability shift assays. Direct evidence for transport activity was obtained by reconstituting native proteins in proteoliposomes and measuring uptake of 14C-malate or 35S-sulphate against unlabelled substrates. This assay showed that GIC-2 transported DHAP, GAP and PEP. However, significant transport activity was not observed for bGIC-2. Overall, the study provides strong, but not conclusive evidence that bGIC-2 is involved in transporting glycolytic intermediates across the inner membrane of the mitochondria, while the function of GIC-1 remains unclear, despite exhibiting the same metabolite binding properties as bGIC-2 n thermostability assays.

Strengths:

Overall, the findings are of interest in the context of understanding the diversity of core metabolic pathways in evolutionarily diverse eukaryotes, as well as the process by which cytosolic glycolysis evolved in most eukaryotes. The experiments are carefully performed and clearly described.

Weaknesses:

The main weakness of the study is the lack of direct evidence that either bGIC-1 and/or bGIC2 are active in vivo. While it is appreciated that the genetic tools for disrupting GIC genes in Blastocystis are limited/lacking, are there opportunities to ectopically express or delete these genes in other genetically tractable Stamenopiles, such as Phaeodactylum triconuteum?

The authors demonstrate that both bGIC-1 and bGIC-2 are targeted to the mitochondrion, based on immunofluorescence studies. However, the precise localization and topology of these carriers in the inner or outer membrane is not defined. The conclusions of the study would be strengthened if the authors could show that one/both transporters are present in the inner membrane using protease protection experiments following differential solubilization of the outer and inner mitochondrial membranes.

It is not clear why hetero-exchange reactions were not performed for bGIC-1 (only for bGIC-2).

In both their previous study (Bartulos et al (2018) and the current study, the authors have shown that Blastocystis express a TPI-GAPDH fusion protein which is located to the mitochondrion. The presence of the TPI domain in the mitochondrial matrix would obviate the need for bGIC-1/2 triose transporters and decrease their value as drug targets. It is noted that Blastocystis still retains some TPI activity in the cytosol, presumably due to expression of a second cytoplasmic isoform, which could account for the presence of the bGIC transporters. However, some discussion on the role of this mitochondrial TPI-GAPDG fusion protein in Blastocystis and other Stramenopiles would be useful.

The summary slide (Fig 7) in the revised manuscript no longer shows PEP being used as a countersolute for the import of G3P and DHAP. Although it complicates the story, the role of PEP as a counter solute should be shown for completeness and also to make sense of some of the statements in the discussion. In particular, as noted by the authors, mitochondrial PEP could be exported back to the cytsol and converted to pyruvate and/or lactate to generate ATP and NAD, although at the expense of ATP synthesis in the mitochondria.

Reviewer #1 (Public Review):

Summary:

Previous work demonstrated a strong bias in the percept of an ambiguous Shepard tone as either ascending or descending in pitch, depending on the preceding contextual stimulus. The authors recorded human MEG and ferret A1 single-unit activity during presentation of stimuli identical to those used in the behavioral studies. They used multiple neural decoding methods to test if context-dependent neural responses to ambiguous stimulus replicated the behavioral results. Strikingly, a decoder trained to report stimulus pitch produced biases opposite to the perceptual reports. These biases could be explained robustly by a feed-forward adaptation model. Instead, a decoder that took into account direction selectivity of neurons in the population was able to replicate the change in perceptual bias.

Strengths:

This study explores an interesting and important link between neural activity and sensory percepts, and it demonstrates convincingly that traditional neural decoding models cannot explain percepts. Experimental design and data collection appear to have been executed carefully. Subsequent analysis and modeling appear rigorous. The conclusion that traditional decoding models cannot explain the contextual effects on percepts is quite strong.

Weaknesses:

Beyond the very convincing negative results, it is less clear exactly what the conclusion is or what readers should take away from this study. The presentation of the alternative, "direction aware" models is unclear, making it difficult to determine if they are presented as realistic possibilities or simply novel concepts. Does this study make predictions about how information from auditory cortex must be read out by downstream areas? There are several places where the thinking of the authors should be clarified, in particular, around how this idea of specialized readout of direction-selective neurons should be integrated with a broader understanding of auditory cortex.

Reviewer #1 (Public Review):

Summary:

Zhao et al. perform a series of experiments aimed at identifying the role of the preoptic area (POA) in controlling the impact of social isolation on same-sex female social behavior. They focus their manuscript on the effects of short-term (3d) isolation and females, both of which have been relatively understudied, making the overall topic of the manuscript exciting and important.

Strengths:

The work highlighted is well designed, the experiments original, and the manuscript is elegant and clearly written. The strengths of the manuscript lie in the attention to multiple facets of social behavior (investigation, mounting, USVs), sex differences, and the use of multiple loss- and gain-of-function approaches.

Weaknesses:

The main weaknesses of the paper are a lack of significance in key findings, and relatedly, concluding effects from insignificant findings. Additional elements could be improved to help strengthen this overall well-rounded and intriguing set of results.

Reviewer #1 (Public Review):

This experiment sought to determine what effect congenital/early-onset hearing loss (and associated delay in language onset) has on the degree of inter-individual variability in functional connectivity to the auditory cortex. Looking at differences in variability rather than group differences in mean connectivity itself represents an interesting addition to the existing literature. The sample of deaf individuals was large, and quite homogeneous in terms of age of hearing loss onset, which are considerable strengths of the work. The experiment appears well conducted and the results are certainly of interest. I do have some concerns with the way that the project has been conceptualized, which I share below.

The authors should provide careful working definitions of what exactly they think is occurring in the brain following sensory deprivation. Characterizing these changes as 'large-scale neural reorganization' and 'compensatory adaptation' gives the impression that the authors believe that there is good evidence in support of significant structural changes in the pathways between brain areas - a viewpoint that is not broadly supported (see Makin and Krakauer, 2023). The authors report changes in connectivity that amount to differences in coordinated patterns of BOLD signal across voxels in the brain; accordingly, their data could just as easily (and more parsimoniously) be explained by the unmasking of connections to the auditory cortex that are present in typically hearing individuals, but which are more obvious via MR in the absence of auditory inputs.

I found the argument that the deaf use a single modality to compensate for hearing loss, and that this might predict a more confined pattern of differential connectivity than had been previously observed in the blind to be poorly grounded. The authors themselves suggest throughout that hearing loss, per se, is likely to be driving the differences observed between deaf and typically-hearing individuals; accordingly, the suggestion that the modality in which intentional behavioral compensation takes place would have such a large-scale effect on observed patterns of connectivity seems out of line.

The analyses highlighting the areas observed to be differentially connected to the auditory cortex and areas observed to be more variable in their connectivity to the auditory cortex seem somewhat circular. If the authors propose hearing loss as a mechanism that drives this variability in connectivity, then it is reasonable to propose hypotheses about the directionality of these changes. One would anticipate this directionality to be common across participants and thus, these areas would emerge as the ones that are differently connected when compared to typically hearing folks.

While the authors describe collecting data on the etiology of hearing loss, hearing thresholds, device use, and rehabilitative strategies, these data do not appear in the manuscript, nor do they appear to have been included in models during data analysis. Since many of these factors might reasonably explain differences in connectivity to the auditory cortex, this seems like an omission.

Reviewer #1 (Public Review):

In this manuscript, by using simulation, in vitro and in vivo electrophysiology, and behavioral tests, Peng et al. nicely showed a new approach for the treatment of neuropathic pain in mice. They found that terahertz (THz) waves increased Kv conductance and decreased the frequency of action potentials in pyramidal neurons in the ACC region. Behaviorally, terahertz (THz) waves alleviated neuropathic pain in the mouse model. Overall, this is an interesting study. The experimental design is clear, the data is presented well, and the paper is well-written. I have a few suggestions.

(1) The authors provide strong theoretical and experimental evidence for the impact of voltage-gated potassium channels by terahertz wave frequency. However, the modulation of action potential also relies on non-voltage-dependent ion channels. For example, I noticed that the RMP was affected by THz application (Figure 3F) as well. As the RMP is largely regulated by the leak potassium channels (Tandem-pore potassium channels), I would suggest testing whether terahertz wave photons have also any impact on the Kleak channels as well.

(2) The activation curves of the Kv currents in Figure 2h seem to be not well-fitted. I would suggest testing a higher voltage (>100 mV) to collect more data to achieve a better fitting.

(3) In the part of behavior tests, the pain threshold increased after THz application and lasted within 60 mins. I suggest conducting prolonged tests to determine the end of the analgesic effect of terahertz waves.

(4) Regarding in vivo electrophysiological recordings, the post-HFTS recordings were acquired from a time window of up to 20 min. It seems that the HFTS effect lasted for minutes, but this was not tested in vitro where they looked at potassium currents. This long-lasting effect of HFTS is interesting. Can the authors discuss it and its possible mechanisms, or test it in slice electrophysiological experiments?

(5) How did the authors arrange the fiber for HFTS delivery and the electrode for in vivo multi-channel recordings? Providing a schematic illustration in Figure 4 would be useful.

(6) Some grammatical errors should be corrected.

Reviewer #1 (Public Review):

Summary:

In their paper, Hou and co-workers explored the use of a FRET sensor for endogenous g-sec activity in vivo in the mouse brain. They used AAV to deliver the sensor to the brain for neuron specific expression and applied NIR in cranial windows to assess FRET activity; optimizing as well an imaging and segmentation protocol. In brief they observe clustered g-sec activity in neighboring cells arguing for a cell non-autonomous regulation of endogenous g-sec activity in vivo.

Weaknesses:

Overall the authors provide a very limited data set and in fact only a proof of concept that their sensor can be applied in vivo. This is not really a research paper, but a technical note. With respect to their observation of clustered activity, the images do not convince me as they show only limited areas of interest: from these examples (for instance fig 5) one sees that merely all neurons in the field show variable activity and a clustering is not really evident from these examples. Even within a cluster, there is variability. With r values between 0.23 to .36, the correlation is not that striking. The authors herein do not control for expression levels of the sensor: for instance, can they show that in all neurons in the field, the sensor is equally expressed, but FRET activity is correlated in sets of neurons? Or are the FRET activities that are measured only in positively transduced neurons, while neighboring neurons are not expressing the sensor? Without such validation, it is difficult to make this conclusion.

Secondly, I am lacking some more physiological relevance for this observation. The experiments are performed in wild-type mice, but it would be more relevant to compare this with a fadPSEN1 KI or a PSEN1cKO model to investigate the contribution of a gain of toxic function or LOF to the claimed cell non-autonomous activations. Or what would be the outcome if the sensor was targeted to glial cells?

For this reviewer it is not clear what resolution they are measuring activity, at cellular or subcellular level? In other words are the intensity spots neuronal cell bodies? Given g-sec activity are in all endosomal compartments and at the cell surface, including in the synapse, does NIR imaging have the resolution to distinguish subcellular or surface localized activities? If cells 'communicate' g-sec activities, I would expect to see hot spots of activity at synapses between neurons: is this possible to assess with the current setup?

Without some more validation and physiological relevant studies, it remains a single observation and rather a technical note paper, instead of a true research paper.

Reviewer #1 (Public Review):

Summary:

Zhang et al. demonstrate that CD4+ single positive (SP) thymocytes, CD4+ recent thymic emigrants (RTE), and CD4+ T naive (Tn) cells from Cd11c-p28-flox mice, which lack IL-27p28 selectively in Cd11c+ cells, exhibit a hyper-Th1 phenotype instead of the expected hyper Th2 phenotype. Using IL-27R-deficient mice, the authors confirm that this hyper-Th1 phenotype is due to IL-27 signaling via IL-27R, rather than the effects of monomeric IL-27p28. They also crossed Cd11c-p28-flox mice with autoimmune-prone Aire-deficient mice and showed that both T cell responses and tissue pathology are enhanced, suggesting that SP, RTE, and Tn cells from Cd11c-p28-flox mice are poised to become Th1 cells in response to self-antigens. Regarding mechanism, the authors demonstrate that SP, RTE, and Tn cells from Cd11c-p28-flox mice have reduced DNA methylation at the IFN-g and Tbx21 loci, indicating 'de-repression', along with enhanced histone tri-methylation at H3K4, indicating a 'permissive' transcriptional state. They also find evidence for enhanced STAT1 activity, which is relevant given the well-established role of STAT1 in promoting Th1 responses, and surprising given IL-27 is a potent STAT1 activator. This latter finding suggests that the Th1-inhibiting property of thymic IL-27 may not be due to direct effects on the T cells themselves.

Strengths:

Overall the data presented are high quality and the manuscript is well-reasoned and composed. The basic finding - that thymic IL-27 production limits the Th1 potential of SP, RTE, and Tn cells - is both unexpected and well described.

Weaknesses:

A credible mechanistic explanation, cellular or molecular, is lacking. The authors convincingly affirm the hyper-Th1 phenotype at epigenetic level but it remains unclear whether the observed changes reflect the capacity of IL-27 to directly elicit epigenetic remodeling in developing thymocytes or knock-on effects from other cell types which, in turn, elicit the epigenetic changes (presumably via cytokines). The authors propose that increased STAT1 activity is a driving force for the epigenetic changes and resultant hyper-Th1 phenotype. That conclusion is logical given the data at hand but the alternative hypothesis - that the hyper-STAT1 response is just a downstream consequence of the hyper-Th1 phenotype - remains equally likely. Thus, while the discovery of a new anti-inflammatory function for IL-27 within the thymus is compelling, further mechanistic studies are needed to advance the finding beyond phenomenology.

Reviewer #1 (Public Review):

Summary:

Inflammatory T cells have been recognized to play an important role in human COPD lung tissue and animal models of emphysema. The authors have previously identified that Th17 cells regulate chronic inflammatory diseases, including in mice exposed to smoke or nanoparticulate carbon black (nCB). Here, the authors interrogate the role of Tc17 cells using similar mouse models. Investigating let-7 miRNA, which induces antigen-presenting cell activation and T cell-mediated Th17a inflammation, they show that the master regulator of Tc17/Th17 differentiation, RAR-related orphan receptor gamma t (RORγt), is a direct target of let-7 miRNA in T cells. Because RORγt expression is elevated in COPD patients and in mouse models of COPD, the authors generate a Let-7 overexpressing mouse in T cells and reduce RORγt expression and Th17 and Tc17 cell recruitment in nCB-exposed mice.

Strengths:

The authors use a previously published RNA-seq dataset (GSE57148) from lungs of control and COPD subjects to explore the involvement of Let-7 in emphysema. They further evaluate Let-7a expression by qPCR in lung tissue samples of smokers with emphysema and non-emphysema controls. Moreover, expression of Let-7a, Let-7b, Let-7d, and Let-7f in purified CD4+ T cells were inversely correlated with emphysema severity lungs. Similar findings were found in their mouse models (CS or nCB) in both lung tissue and isolated lung CD4+ and CD8+ T cells, with reduced let-7afd and let-7bc2 expression.

Using mice harboring a conditional deletion of the let-7bc2 cluster in all T cells (let-7bc2LOF) derived from the CD4+CD8+ double-positive stage, the authors show enhanced emphysema in nCB- or CS-exposed mice with enhanced recruitment of macrophages and neutrophils to the lung. While CD8+IL17a+ Tc17 cells and CD4+ IL17a+ Th17 cells were increased in nCB-exposed control animals, only let-7bc2LOF mice showed an increase in CD8+IL17a+ Tc17 cells. Further, unexposed let-7bc2LOF and let-7afdLOF mice expressed greater RORγt expression in both CD8+ and CD4+ T cells.

Generating a let-7 gain of function mouse with overexpression of let-7g in thymic double-positive-derived T cells, protein levels of RORγt were suppressed in CD8+ and CD4+ T cells of let-7GOF mice relative to controls. Let-7GOF mice treated with nCB showed similar lung alveolar distension as controls suggesting that increased let-7 expression does not protect the lung from emphysema. However, let-7GOF mice showed reduced lung Tc17 and Th17 cell populations and were resistant to the induction of RORγt after nCB exposure.

Weaknesses:

Limited data is shown on the let-7afdLOF mice. Does this mouse respond similarly to nCB as the let-7bc2LOF.<br /> Because the authors validate their findings from a previously published RNA-seq dataset in subjects with and without emphysema, the authors should include patient demographics from the data presented in Figure 1C-D.<br /> To validate their mouse models, the absence of Let-7 or enhanced Let-7 expression needs to be shown in isolated T cells from exposed mice.<br /> In Figure 3, the authors are missing the unexposed let-7bc2LOF group from all panels. This is again an issue in Figure 6 with the let-7GOF.<br /> Because the GOF mouse enhances Let-7g within T cells, the importance of Let-7g should be determined in human subjects. Why did the authors choose to overexpress Let-7g, the rationale is not clear.<br /> The purity of the CD4+ and CD8+ T cells is not shown and the full gating strategy should be included.<br /> The authors indicate that Tc17 and Th17 T cells were reduced in the GOF mouse, it remains unclear if macrophage or neutrophil recruitment is altered in GOF mice.

Reviewer #1 (Public Review):

Klupt, Fam, Zhang, Hang and colleagues present a novel study examining the function of sagA in E. faecium, including impacts on growth, peptidoglycan cleavage, cell separation, antibiotic sensitivity, NOD2 activation and modulation of cancer immunotherapy. This manuscript represents a substantial advance over their prior work, where they found that sagA-expressing strains (including naturally-expressing strains and versions of non-expressing strains forced to overexpress sagA) were superior in activating NOD2 and improving cancer immunotherapy. Prior to the current study, an examination of sagA mutant E. faecium was not possible and sagA was thought to be an essential gene.

The study is overall very carefully performed with appropriate controls and experimental checks, including confirmation of similar densities of ΔsagA throughout. Results are overall interpreted cautiously and appropriately.

Reviewer #1 (Public Review):

This study presents a genetic and molecular analysis of the role of the cytoplasmic ub ligase Deltex (Dx) in regulating the Drosophila Wingless (Wg) pathway in the larval wing disc. The study exploits the strength of the fly system to uncover a series of genetic interactions between dx and wg and fz allele that support a role for Dx upstream of the Wg pathway. These are paired with molecular evidence that dx lof alleles lower Wg protein in 'source' cells at the DV margin, and that Dx associates with Arm and lowers its levels in a manner that can be rescued by pharmacological inhibition of the proteasome. The genetic data are solid but subject to alternative explanations based on the authors' model that Dx both inhibits and activates the pathway. The molecular data are suggestive, but need follow up tests of how Dx affects Wg spread, and how Dx mediates poly-ub of Arm, and the degree to which Dx shares this role with the validated Arm E3 ligase Slmb. Overall, the story is very interesting but has mechanistic gaps that lead to speculative models that require more rigorous study to clarify mechanism. Dx sharing a role in Arm degradation with the Slmb/APC destruction would have important implications for the many Wg/Wnt regulated processes in development and disease.

Reviewer #1 (Public Review):

Summary:

This is an interesting study that utilizes a novel epigenome profiling technology (single molecule imaging) in order to demonstrate its utility as a readout of therapeutic response in multiple DIPG cell lines. Two different drugs were evaluated, singly and in combination. Sulfopin, an inhibitor of a component upstream of the MYC pathway, and Vorinostat, an HDAC inhibitor. Both drugs sensitised DIPG cells, but high (>10 micromolar) concentrations were needed to achieve half-maximal effects. The combination seemed to have some efficacy in vivo, but also produced debilitating side-effects that precluded the measurement of any survival benefit.

Strengths:

Interesting use of a novel epigenome profiling technology (single molecule imaging).

Weaknesses:

The use of this novel imaging technology ultimately makes up only a minor part of the study. The rest of the results, i.e. DIPG sensitivity to HDAC and MYC pathway inhibition, have already been demonstrated by others (Grasso Monje 2015; Pajovic Hawkins 2020, among others). The drugs have some interesting opposing effects at the level of the epigenome, demonstrated through CUT&RUN, but this is not unexpected in any way. The drugs evaluated here also didn't have higher efficacy, or efficacy at especially low concentrations, than inhibitors used in previous reports. The combination therapy attempted here also caused severe side effects in mice (dehydration/deterioration), such that an effect on survival could not be determined. I'm not sure this study advances knowledge of targeted therapy approaches in DIPGs, or if it iterates on previous findings to deliver new, or more efficient, mechanistic or therapeutic/pharmaclogic insights. It is a translational report evaluating two drugs singly and in combination, finding that although they sensitise cells in vitro, efficacy in vivo is limited at best, as this particular combination cannot progress to human translation.

Reviewer #1 (Public Review):

The study provides strong evidence that some genes are conditionally essential in urine because of iron limitation.

The authors raise the intriguing possibility that some mutants can "cheat" by benefitting from the surrounding cells that are phenotypically wild-type. The authors make it clear that the proposed cheating mechanism is speculation, but there is a missed opportunity to test this hypothesis. I did not understand the authors' rationale for not doing this experiment.

In cases where there are disparities between studies, e.g., for genes inferred to be essential for serum resistance, it would be informative to test individual deletions for genes described as essential in only one study. The authors argue this is beyond the scope of the study. Their conclusions of the study are not impacted by the absence of these experiments, but readers will be left wondering which lists of conditionally essential genes are correct, or whether there are strain-dependent or condition-dependent contexts that influence conditional essentiality.

Reviewer #2 (Public Review):

This manuscript illustrates the power of "combined" research, incorporating a range of tools, both old and new to answer a question. This thorough approach identifies a novel target in a well-established signalling pathway and characterises a new player in Drosophila CNS development.

Largely, the experiments are carried out with precision, meeting the aims of the project, and setting new targets for future research in the field. It was particularly refreshing to see the use of multi-omics data integration and Targeted DamID (TaDa) findings to triage scRNA-seq data. Some of the TaDa methodology was unorthodox, however, this does not affect the main finding of the study. The authors (in the revised manuscript) have appropriately justified their TaDa approaches and mentioned the caveats in the main text.

Their discovery of Spar as a neuropeptide precursor downstream of Alk is novel, as well as its ability to regulate activity and circadian clock function in the fly. Spar was just one of the downstream factors identified from this study, therefore, the potential impact goes beyond this one Alk downstream effector.

Reviewer #1 (Public Review):

Summary:

This paper explores the contribution of transgenerational effects to phenotypic variation in twenty-five phenotypes and transcript variation in the heart, liver, pituitary, whole embryo, and placenta. The authors use a powerful design, exploiting the use of consomics, and argue that there are no observable changes attributable to the differences in the parental origin of the four chromosomes they examine.

Strengths:<br /> It's good to see a use for consomics. This is a powerful and useful design to address the problem they are tackling.

Weaknesses:<br /> The difficulty faced by the authors is that they have interrogated only a small portion of the genome, using bulk RNA sequencing and a set of correlated phenotypes, thus restricting the conclusions they can draw from the absence of significant findings.

Joint Public Review:

This manuscript by Yue et al. aims to understand the molecular mechanisms underlying the better reproductive outcomes of Tibetans at high altitude by characterizing the transcriptome and histology of full-term placenta of Tibetans and compare them to those Han Chinese at high elevations.

The approach is innovative, and the data collected are valuable for testing hypotheses regarding the contribution of the placenta to better reproductive success of populations that adapted to hypoxia. The authors identified hundreds of differentially expressed genes (DEGs) between Tibetans and Han, including the EPAS1 gene that harbors the strongest signals of genetic adaptation. The authors also found that such differential expression is more prevalent and pronounced in the placentas of male fetuses than those of female fetuses, which is particularly interesting, as it echoes with the more severe reduction in birth weight of male neonates at high elevation observed by the same group of researchers (He et al., 2022).

This revised manuscript addressed several concerns raised by reviewers in last round. However, we still find the evidence for natural selection on the identified DEGs--as a group--to be very weak, despite more convincing evidence on a few individual genes, such as EPAS1 and EGLN1.

The authors first examined the overlap between DEGs and genes showing signals of positive selection in Tibetans and evaluated the significance of a larger overlap than expected with a permutation analysis. A minor issue related to this analysis is that the p-value is inflated, as the authors are counting permutation replicates with MORE genes in overlap than observed, yet the more appropriate way is counting replicates with EQUAL or MORE overlapping genes. Using the latter method of p-value calculation, the "sex-combined" and "female-only" DEGs will become non-significantly enriched in genes with evidence of selection, and the signal appears to solely come from male-specific DEGs. A thornier issue with this type of enrichment analysis is whether the condition on placental expression is sufficient, as other genomic or transcriptomic features (e.g., expression level, local sequence divergence level) may also confound the analysis.

The authors next aimed to detect polygenic signals of adaptation of gene expression by applying the PolyGraph method to eQTLs of genes expressed in the placenta (Racimo et al 2018). This approach is ambitious but problematic, as the method is designed for testing evidence of selection on single polygenic traits. The expression levels of different genes should be considered as "different traits" with differential impacts on downstream phenotypic traits (such as birth weight). As a result, the eQTLs of different genes cannot be naively aggregated in the calculation of the polygenic score, unless the authors have a specific, oversimplified hypothesis that the expression increase of all genes with identified eQTL will improve pregnancy outcome and that they are equally important to downstream phenotypes. In general, PolyGraph method is inapplicable to eQTL data, especially those of different genes (but see Colbran et al 2023 Genetics for an example where the polygenic score is used for testing selection on the expression of individual genes).

We would recommend removal of these analyses and focus on the discussion of individual genes with more compelling evidence of selection (e.g., EPAS1, EGLN1)

Reviewer #1 (Public Review):

In the study described in the manuscript, the authors identified Mecp2, a methyl-CpG binding protein, as a key regulator involved in the transcriptional shift during the exit of quiescent cells into the cell cycle. Their data show that Mecp2 levels were remarkably reduced during the priming/initiation stage of partial hepatectomy-induced liver regeneration and that altered Mecp2 expression affected the quiescence exit. Additionally, the authors identified Nedd4 E3 ligase that is required for downregulation of Mecp2 during quiescence exit. This is an interesting study with well-presented data that supports the authors' conclusions regarding the role of Mecp2 in transcription regulation during the G0/G1 transition. However, the significance of the study is limited by a lack of mechanistic insights into the function of Mecp2 in the process. This weakness can be addressed by identifying the signaling pathway(s) that trigger Mecp2 degradation during the quiescence exit.

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors reported that miR-199b-5p is elevated in osteoarthritis (OA) patients. They also found that overexpression of miR-199b-5p induced OA-like pathological changes in normal mice and inhibiting miR-199b-5p alleviated symptoms in knee OA mice. They concluded that miR-199b-5p is not only a potential micro target for knee OA, but also provides a potential strategy for future identification of new molecular drugs.

Strengths:

The data are generated from both human patients and animal models. The data presented in this revised manuscript is solid and support their conclusions. The questions from reviewers are also properly addressed and the quality of this manuscript has been significantly improved.

There are no significant weaknesses identified in this revised manuscript.

Reviewer #1 (Public review):

The key discovery of the manuscript is that the authors found that genetically wild type females descended from Khdc3 mutants shows abnormal gene expression relating to hepatic metabolism, which persist over multiple generations and pass through both female and male lineages. They also find dysregulation of hepatically-metabolized molecules in the blood of these wild type mice with Khdc3 mutant ancestry. These data provide solid evidence further support that phenotype can be transmitted to multiple generations without altering DNA sequence, supporting the involvement of epigenetic mechanisms. The authors further performed exploratory studies on the small RNA profiles in the oocytes of Khdc3-null females, and their wild type descendants, suggesting that altered small RNA expression could be a contributor of the observed phenotype transmission, although this has not been functionally validated.

Reviewer #1 (Public Review):

Summary:

In their article entitled "Formin-like 1 beta phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse", Javier Ruiz-Navarro and co-workers address the question of the mechanisms regulating the polarization of the microtubule organizing center (MTOC) and of the multivesicular bodies (MVB) at the immunological synapse (IS) in T lymphocytes.

This work is a follow-up of previous studies published by the same team showing that TCR-stimulated protein kinase C delta(PKCdelta) phosphorylates FMNL1beta, which plays a crucial role in cortical actin reorganization at the IS, and controls MTOC/MVB polarization and thus exosome secretion by T lymphocytes at the IS.

The authors first compare the amino acid sequences of FMNL2 and of FMNL1beta, to seek similarities in the DID-DAD auto-inhibition sequences and find that the sequence surrounding S1086 in the arginine-rich DAD of FMNL1beta displays high similarity to that around S1072 in FMNL2 which is phosphorylated by PKCdelta. They then interrogate the role of the phosphorylation of S1086 in the arginine-rich DAD of FMNL1betaby introducing S1086A and S1086D mutations that, respectively, cannot be phosphorylated or mimic the phosphorylated form of FMNL1beta, in cells expressing an FMNL1 shRNA.

Using these tools, they show that:

- FMNL1beta is phosphorylated by PMA an activator of PKCs.

- The S1086A mutant of FMNL1beta does not restore the defect in MTOC and MVB polarization at the IS present in FMNL1 deficient T cells, whereas the phosphomimetic mutant does.

- Although FMNL1betaphosphorylation at S1086 is necessary, it is not sufficient for MTOC polarization, since it does not restore the defect of polarization observed in PKCdelta deficient T cells.

- FMNL1b translocates to the IS. This neither requires PKC expression nor phosphorylation of S1086.

- Phosphorylation of FMNL1betaon S1086 regulates actin remodeling at the immune synapse.

- Phosphorylation of FMNL1betaon S1086 regulates secretion of extracellular vesicles containing CD63 by T lymphocytes.

Strengths:

This work shows for the first time the role of the phosphorylation of FMNL1beta on S1086 on the regulation of the IS formation and secretion of extracellular vesicles by T lymphocytes.

Weaknesses:

Although of interest, this work has several weaknesses. First, all the experiments are performed in Jurkat T cells that may not recapitulate the regulation of polarization in primary T cells. Moreover, all the experiments analyzing the role of PKCdelta are performed in one clone of wt or PKCdelta KO Jurkat cells. This is problematic since clonal variation has been reported in Jurkat T cells. Moreover, the remodeling of F-actin at the IS lacks careful quantification as well as detailed analysis of the actin structure in mutant cells. Finally, although convincing, the defect in the secretion of vesicles by T cells lacking phosphorylation of FMNL1beta on S1086 is preliminary. It would be interesting to analyze more precisely this defect. The expression of the CD63-GFP in mutants by WB is not completely convincing. Are other markers of extracellular vesicles affected, e.g. CD3 positive?

Reviewer #1 (Public Review):

Summary:

The authors report evidence for a microprotein of AtHB2-miP. The authors came across HB2 in a screen for alternative transcription start sites in Arabidopsis in response to white light or a white light followed by a far red light representative of shade. Out of 337 potential microproteins, authors selected AtHB2. At the beginning of the manuscript, it is investigated that an alternative transcription start site of HB2 gene can be used in response to far red light. The resulting shorter protein form seems to interact with HB2 protein forms, altering the localization of HB2 in transient expression assays. The functionality of HB2-miP overexpression has been addressed in transgenic Arabidopsis lines using a 35S promoter. The responses and phenotypes were compared with either WT or various types of athb2 mutant lines with disrupted HB2 gene. Such mutants and the 35S promoter-driven AtHB2-miP line showed various types of phenotypes versus each other that can be classified as mild or none, e.g. small effects on root growth, iron homeostasis gene expression, and iron contents.

Strengths:

The authors performed an interesting screen for alternative transcription start sites which resulted in 337 candidates (Figure 1A). Principally, it can be interesting to find that plants may use alternative start sites for HB2 in response to shading light. The authors provide evidence that alternative transcription start sites of HB2 can be present and used in response to FR. The possibility that potentially resulting small protein may have effects under FR light, causing alteration of root growth and physiology, is an interesting idea.

Weaknesses:

In the present manuscript, there are several signs of incomplete analysis.

(1) The transient expression experiments are not conducted with much detail to demonstrate that indeed HB2 miP is produced and can interact with regular protein. The localization of HB2 was found to be linked with condensates, but perhaps not in the presence of HB2 miP. Clearly, the lack of quantitative and qualitative analysis hampers a clear assessment of this point.

(2) The authors, unfortunately, did not provide the data of the screen to demonstrate which concrete candidates may have miPs and whether there is enrichment of certain functions. There is no supplemental table accompanying Figure 1A.

(3) One of the major unclear points that is also not addressed in the discussion is that the function of miR is studied in overexpression plants (35S promoter::miP). The effects are only compared to wild type and various lines of HB2 knockouts or knockdowns, partly with fairly uncharacterized phenotypes. It can now not be clearly determined whether the miP effects are due to a regular function of miP or due to overexpression of it. A needed control would be a 35S::AtHB2 line, or better at least two different lines (only a single miP overexpression line investigated). Since it has not been assessed by deletion mutant analysis to determine which protein parts of miP are involved in the protein regulation, it cannot be ruled out that the observed miP effects are not naturally occurring but the result of ectopic expression of a protein. Clearly, the effect of miP would be ideally studied in an environment where the levels can be controlled and the resulting phenotypes and protein levels quantified.

(4) It is not shown that the microprotein is generated in Arabidopsis in response to shade, e.g. through Western or fluorescence protein detection. The main idea that authors want to claim, namely that miP binds with regular protein and thereby controls its localization or activity has not been addressed in Arabidopsis. There are no localization experiments of HB2 protein data in the presence of miP in Arabidopsis.

(5) The plants with altered HB2 forms seem to grow well and the recorded phenotypes are rather minor. Photos are not shown. At some point, the authors discuss that there could be redundancy or that HB miP might interact with other HB proteins. However, such protein interactions have not been experimentally investigated.

Reviewer #1 (Public Review):

Summary:

In their manuscript entitled 'The domesticated transposon protein L1TD1 associates with its ancestor L1 ORF1p to promote LINE-1 retrotransposition', Kavaklıoğlu and colleagues delve into the role of L1TD1, an RNA binding protein (RBP) derived from a LINE1 transposon. L1TD1 proves crucial for maintaining pluripotency in embryonic stem cells and is linked to cancer progression in germ cell tumors, yet its precise molecular function remains elusive. Here, the authors uncover an intriguing interaction between L1TD1 and its ancestral LINE-1 retrotransposon.

The authors delete the DNA methyltransferase DNMT1 in a haploid human cell line (HAP1), inducing widespread DNA hypo-methylation. This hypomethylation prompts abnormal expression of L1TD1. To scrutinize L1TD1's function in a DNMT1 knock-out setting, the authors create DNMT1/L1TD1 double knock-out cell lines (DKO). Curiously, while the loss of global DNA methylation doesn't impede proliferation, additional depletion of L1TD1 leads to DNA damage and apoptosis.

To unravel the molecular mechanism underpinning L1TD1's protective role in the absence of DNA methylation, the authors dissect L1TD1 complexes in terms of protein and RNA composition. They unveil an association with the LINE-1 transposon protein L1-ORF1 and LINE-1 transcripts, among others.

Surprisingly, the authors note fewer LINE-1 retro-transposition events in DKO cells than in DNMT1 KO alone.

Strengths:

The authors present compelling data suggesting the interplay of a transposon-derived human RNA binding protein with its ancestral transposable element. Their findings spur interesting questions for cancer types, where LINE1 and L1TD1 are aberrantly expressed.

Weaknesses:

Suggestions for refinement:

The initial experiment, inducing global hypo-methylation by eliminating DNMT1 in HAP1 cells, is intriguing and warrants a more detailed description. How many genes experience misregulation or aberrant expression? What phenotypic changes occur in these cells? Why did the authors focus on L1TD1? Providing some of this data would be helpful to understand the rationale behind the thorough analysis of L1TD1.

The finding that L1TD1/DNMT1 DKO cells exhibit increased apoptosis and DNA damage but decreased L1 retro-transposition is unexpected. Considering the DNA damage associated with retro-transposition and the DNA damage and apoptosis observed in L1TD1/DNMT1 DKO cells, one would anticipate the opposite outcome. Could it be that the observation of fewer transposition-positive colonies stems from the demise of the most transposition-positive colonies? Further exploration of this phenomenon would be intriguing.

Reviewer #1 (Public Review):

Summary:

In this manuscript by Thronlow Lamson et al., the authors develop a "beads-on-a-string" or BOAS strategy to link diverse hemagglutinin head domains, to elicit broadly protective antibody responses. The authors are able to generate varying formulations and lengths of the BOAS and immunization of mice shows induction of antibodies against a broad range of influenza subtypes. However, several major concerns are raised, including the stability of the BOAS, that only 3 mice were used for most immunization experiments, and that important controls and analyses related to how the BOAS alone, and not the inclusion of diverse heads, impacts humoral immunity.

Strengths:

Vaccine strategy is new and exciting.

Analyses were performed to support conclusions and improve paper quality.

Weaknesses:

Controls for how different hemagglutinin heads impact immunity versus the multivalency of the BOAS.

Only 3 mice were used for most experiments.

There were limited details on size exclusion data.

Reviewer #1 (Public Review):

Summary:

Rößling et al., report in this study that the perception of RALF1 by the FER receptor is mediated by the association of RALF1 with deesterified pectin, contributing to the regulation of the cell wall matrix and plasma membrane dynamics. In addition, they report that this mode of action is independent from the previously reported cell wall sensing mechanism mediated by the FER-LRX complex.

This manuscript reproduces and aligns with the results from a recently published study (Liu et al., Cell) where they also report that RALF1 can interact with deesterified pectin, forming coacervates and promoting the recruitment of LLG-FER at the membrane.

Reviewer #1 (Public Review):

Summary:

The authors showed that autophagy-related genes are involved in plant immunity by regulating the protein level of the salicylic acid receptor, NPR1.

Strengths:

The experiments are carefully designed and the data is convincing. The authors did a good job of understanding the relationship between ATG6 and NRP1.

Weaknesses:<br /> - The authors can do a few additional experiments to test the role of ATG6 in plant immunity.<br /> I recommend the authors to test the interaction between ATGs and other NPR1 homologs (such as NPR2).

-The concentration of SA used in the experiment (0.5-1 mM) seems pretty high. Does a lower concentration of SA induce ATG6 accumulation in the nucleus?

-Does the silencing of ATG6 affect the cell death (or HR) triggered by AvrRPS4?

-SA and NPR1 are also required for immunity and are activated by other NLRs (such as RPS2 and RPM1). Is ATG6 also involved in immunity activated by these NLRs?

Reviewer #1 (Public Review):

In this study, the authors reported that disruption of the X-linked ciliary protein OFD in the cranial neural crest-derived cells (CNCCs) leads to a migration defect in the CNCCs and that aberrant CNCCs abnormally differentiate into osteoblasts due to a lack of Hh signal. Furthermore, CNCC defects lead to the failure of mesoderm-derived cells to differentiate into myoblasts and instead result in abnormal differentiation of mesoderm-derived cells into adipocytes. The Ofd cko mouse model has a very striking phenotype and nicely mimics the phenotype of human patients, making it a very valuable model to understand human disease.

Reviewer #1 (Public Review):

Recent work reported that the AP2-associated kinase 1 (AAK1) downregulates Wnt signaling by phosphorylating, thus activating, the µ-subunit of the AP2 complex (AP2M1), which recognizes an endocytic signal on the intracellular domain of the Wnt co-receptor LRP6 leading to its internalization (Agajanian, et al., 2018). It has also long been known that DPY-23/AP2M1 and the retromer complex, which controls trafficking between endosomes and the trans-golgi network and recycling from endosomes to the plasma membrane, regulate Wnt signaling in C. elegans, at least in part by modulating trafficking of the Wnt-secretion factor MIG-14/WLS (Pan, et al., 2008; Yan et al., 2008).

Here the authors first set out to ask whether SEL-5/AAK1 plays a conserved role in Wnt signaling via phosphorylation of DPY-23/AP2M1 by assessing the function of SEL-5 in Wnt-regulated morphogenetic events; specifically, the well-characterized migration and polarization of several neurons and the less-understood process of excretory canal cell outgrowth.

The authors found that the simultaneous removal of sel-5 and the retromer complex gene vps-29 resulted in synthetic neuronal and excretory canal outgrowth phenotypes, indicating that sel-5 and the retromer complex function in parallel in these processes. Genetic interactions between sel-5 and Wnt pathway components were also examined, and for QL neuroblast migration, loss of sel-5 exacerbated phenotypes caused by loss of the Wnt receptor LIN-17/FZD, but not those caused by loss of a different receptor, MIG-1/FZD. The authors assessed the site of sel-5 function in neuronal migration defects via tissue-specific rescue and identified the hypodermis, a known source of Wnt ligands, and muscles as sites where sel-5/AAK1 activity is required.

The novelty in this work comes from the discovery of a function for sel-5/AAK1 and the retromer complex in excretory canal outgrowth, identified by phenotypes caused by simultaneous loss of sel-5 and retromer components. This synthetic phenotype is rescued by restoring sel-5 to either the excretory canal cell or the hypodermis, suggesting autonomous and non-autonomous functions for sel-5 in canal outgrowth. The authors also confirmed previous results showing that loss of LIN-17/FZD results in excretory canal overgrowth, and by carrying out an extensive survey of Wnt-pathway mutants they discovered that LIN-44/Wnt is likely the ligand that functions via LIN-17 as a "stop" signal in canal outgrowth. They also implicate a CWN-1/Wnt-CFZ-2/FZD pathway as required for canal outgrowth and find genetic interactions between sel-5/AAK1 and the lamellipodin ortholog mig-10, suggesting that these genes function in parallel to promote excretory canal outgrowth.

The most intriguing claim in this work is the suggestion that neither DPY-23 phosphorylation nor SEL-5 kinase activity is required for their function in Wnt signaling. However, the tools used to support these conclusions are not well-characterized. First, a new dpy-23 phosphorylation site-mutant is not genetically characterized, thus it is difficult to interpret the negative results obtained with this allele. Second, although the mutations introduced into SEL-5 are expected to abolish kinase activity, this is not demonstrated biochemically, nor are the effects, if any, of mutations on protein stability/localization assessed. Finally, experiments testing the function of SEL-5 kinase mutants are reported using only one multi-copy extrachromosomal array per construct. Because these types of transgenes vastly overexpress proteins, it is likely that even proteins with reduced function will rescue, raising concerns regarding the conclusion that kinase activity is not necessary for SEL-5 function.

In conclusion, it is not clear that the findings presented here will be of great general interest, as they mostly support previously-known functions for SEL-5/AAK1, DPY-23/AP2M1, and the retromer complex in Wnt-mediated signaling. Thus, this work will mainly be of interest to researchers studying Wnt-mediated cell outgrowth, and more specifically to those studying the C. elegans excretory canal. Moreover, the study lacks coherence: initially, there is a clear hypothesis testing a role for SEL-5/AAK1 in DPY-23/AP2M1 phosphorylation and how this impinges on Wnt signaling. This model appears to be refuted (although, as noted above the tools used to do this need to be better validated), but the authors do not explore alternative targets or functions for SEL-5/AAK1, nor do they directly assess how SEL-5 or the retromer complex impinge on Wnt signaling in excretory canal outgrowth. Thus, there is little mechanistic insight provided by this work.

Reviewer #1 (Public Review):

Salt-inhibited germination and growth in Arabidopsis and other plant species. Here the authors demonstrated that part of that inhibitory effect is caused by the arginine-derived urea hydrolysis, a novel mechanism. They also postulated that urea transport is involved in germination inhibition, but they do not link urea transport from cotyledons to pH changes in roots. At last, they generalized the mechanisms to other glycophytic crops and halophytic plants, but the salt concentration used is the same for the four groups, which are supposed to have very different salt tolerance ranges, questioning the validity of this generalization.<br /> Overall, the authors have provided well-organized genetic and pharmacological evidence to support most of their conclusions.

Reviewer #1 (Public Review):

HMCV encodes various immunoevasins to inhibit being presented by MHC class I molecules to the cytotoxic cells of the immune system. Here, the authors studied the role and specificity of US10, a relatively uncharacterized immunoevasin from HCMV. They found that US10 differentially affects antigen presentation by different MHC class I allotypes. HLA-A and certain HLA-B and C alleles (so-called "tapasin-independent") were unaffected, while other HLA-B and C alleles (so-called "tapasin-dependent") as well as HLA-G were negatively affected. US10 can bind to different MHC class I allotypes, which inhibits their incorporation into peptide loading complex and slowers maturation. By comparing US10 to the other well-studied immunoevasins from HCMV, US2, US3, and US11, the authors demonstrated only partial overlap between them suggesting the cumulative action of immunoevasins in inhibiting MHC class I antigen presentation of HMCV epitopes. This work contributes to our understanding of the complex immune evasion mechanism by HCMV.

The strengths include using a broad use of available techniques, including overexpression of US10 and US10 siRNA in the infection context that allowed comparison of its net and cumulative effects. Bioinformatic analysis of US10 and US11 to describe how transcription and expression of these two gene products contribute to the control of immunoevasion by HCMV. The conclusions are mostly supported by the experiments.

2 comments on the same text

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors delineate the crucial role of the SIRT2-ACSS2 axis in ACSS2 degradation. They demonstrate that SIRT2 acts as an ACSS2 deacetylase specifically under nutrient stress conditions, notably during amino acid deficiency. The SIRT2-mediated deacetylation of ACSS2 at K271 consequently triggers its proteasomal degradation. Additionally, they illustrate that acetylation of ACSS2 at K271 enhances ACSS2 protein levels, thereby promoting De Novo lipogenesis.

Strengths:

The findings presented in this manuscript are clearly interesting.

Weaknesses:

Further support is required for the model put forward by the authors.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Bell et al. provide an exhaustive and clear description of the diversity of a new class of predicted type IV restriction systems that the authors denote as CoCoNuTs, for their characteristic presence of coiled-coil segments and nuclease tandems. Along with a comprehensive analysis that includes phylogenetics, protein structure prediction, extensive protein domain annotations, and in-depth investigation of encoding genomic contexts, they also provide detailed hypothesis about the biological activity and molecular functions of the members of this class of predicted systems. This work is highly relevant, it underscores the wide diversity of defence systems that are used by prokaryotes and demonstrates that there are still many systems to be discovered. The work is sound and backed up by a clear and reasonable bioinformatics approach.

Strengths:

The analysis provided by the authors is extensive and covers the three most important aspects that can be covered computationally when analysing a new family/superfamily: phylogenetics, genomic context analysis, and protein-structure-based domain content annotation. With this, one can directly have an idea about the superfamily of the predicted system and infer about their biological role. The bioinformatics approach is sound and makes use of the most current advances in the fields of protein evolution and structural bioinformatics.

Weaknesses:

It is not clear how coiled-coil segments were assigned if only based on AF2-predicted models or also backed by sequence analysis, as no description is provided in the methods. The structure prediction quality assessment is based solely on the average pLDDT of the obtained models (with a threshold of 80 or better). However, this is not enough, particularly when multimeric models were used. The PAE matrix should be used to evaluate relative orientations, particularly in the case where there is a prediction that parts from 2 proteins are interacting. In the case of multimers, interface quality scores, as the ipTM or pDockQ, should also be considered and, at minimum, reported.

These weaknesses were addressed during revision, and the results provided by the authors support their conclusions. The data resulting from this work will be useful for the general life sciences community, particularly the prokaryotic defense and microbiology communities. It also underscores the high range of functionally unknowns in sequenced genomes that are now much easier to find and interpret due to the success of deep-learning based methods and automated robust bioinformatics pipelines.

Reviewer #1 (Public Review):

Summary:

Campbell et al investigated the effects of light on the human brain, in particular the subcortical part of the hypothalamus during auditory cognitive tasks. The mechanisms and neuronal circuits underlying light effects in non-image forming responses are so far mostly studied in rodents but are not easily translated in humans. Therefore, this is a fundamental study aiming to establish the impact light illuminance has on the subcortical structures using the high-resolution 7T fMRI. The authors found that parts of the hypothalamus are differently responding to illuminance. In particular, they found that the activity of the posterior hypothalamus increases while the activity of the anterior and ventral parts of the hypothalamus decreases under high illuminance. The authors also report that the performance of the 2-back executive task was significantly better in higher illuminance conditions. However, it seems that the activity of the posterior hypothalamus subpart is negatively related to the performance of the executive task, implying that it is unlikely that this part of the hypothalamus is directly involved in the positive impact of light on performance observed. Interestingly, the activity of the posterior hypothalamus was, however, associated with an increased behavioural response to emotional stimuli. This suggests that the role of this posterior part of the hypothalamus is not as simple regarding light effects on cognitive and emotional responses. This study is a fundamental step towards our better understanding of the mechanisms underlying light effects on cognition and consequently optimising lighting standards.

Strengths:

While it is still impossible to distinguish individual hypothalamic nuclei, even with the high-resolution fMRI, the authors split the hypothalamus into five areas encompassing five groups of hypothalamic nuclei. This allowed them to reveal that different parts of the hypothalamus respond differently to an increase in illuminance. They found that higher illuminance increased the activity of the posterior part of the hypothalamus encompassing the MB and parts of the LH and TMN, while decreasing the activity of the anterior parts encompassing the SCN and another part of TMN. These findings are somewhat in line with studies in animals. It was shown that parts of the hypothalamus such as SCN, LH, and PVN receive direct retinal input in particular from ipRGCs. Also, acute chemogenetic activation of ipRGCs was shown to induce activation of LH and also increased arousal in mice.

Weaknesses:

While the light characteristics are well documented and EDI calculated for all of the photoreceptors, it is not very clear why these irradiances and spectra were chosen. It would be helpful if the authors explained the logic behind the four chosen light conditions tested. Also, the lights chosen have cone-opic EDI values in a high correlation with the melanopic EDI, therefore we can't distinguish if the effects seen here are driven by melanopsin and/or other photoreceptors. In order to provide a more mechanistic insight into the light-driven effects on cognition ideally one would use a silent substitution approach to distinguish between different photoreceptors. This may be something to consider when designing the follow-up studies.

Reviewer #1 (Public Review):

Summary:

Major findings or outcomes include a genome for the wasp, characterization of the venom constituents and teratocyte and ovipositor expression profiles, as well as information about Trichopria ecology and parasitism strategies. It was found that Trichopria cannot discriminate among hosts by age, but can identify previously parasitized hosts. The authors also investigated whether superparasitism by Trichopria wasps improved parasitism outcomes (it did), presumably by increasing venom and teratocyte concentrations/densities. Elegant use of Drosophila ectopic expression tools allowed for functional characterization of venom components (Timps), and showed that these proteins are responsible for parasitoid-induced delays in host development. After finding that teratocytes produce a large number of proteases, experiments showed that these contribute to digestion of host tissues for parasite consumption.<br /> The discussion ties these elements together by suggesting that genes used for aiding in parasitism via different parts of the parasitism arsenal arise from gene duplication and shifts in tissue of expression (to venom glands or teratocytes).

Strengths:

The strength of this manuscript is that it describes the parasitism strategies used by Trichopria wasps at a molecular and behavioral level with broad strokes. It represents a large amount of work that in previous decades might have been published in several different papers. Including all of these data in a manuscript together makes for a comprehensive and interesting study.

Weaknesses:

The weakness is that the breadth of the study results in fairly shallow mechanistic or functional results for any given facet of Trichopria's biology. Although none of the findings are especially novel given results from other parasitoid species in previous publications, integrating results together provides significant information about Trichopria biology.

Reviewer #1 (Public Review):

Summary:

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease that does not respond to immunotherapy. This work represents an extension of the authors' prior observation that PI3Ka deletion in an orthotopic KPC pancreatic tumor model confers susceptibility to immune-mediated elimination. The authors' major claims in the present manuscript are as follows:

(1) PI3Ka (Pik3ca) knockout in KPC pancreatic tumor cells induces clonal T cell expansion.

(2) Genome-wide LOF screen in aKPC cells to identify tumor-intrinsic determinants of PI3Ka-KO-enhanced T cell response identified Pccb.

(3) When Pccb is knocked out in the context of Pi3ka knockout KPC, anti-tumor T cell response is reduced as measured by<br /> a. Increased tumor progression<br /> b. Decreased survival<br /> c. T cells are still clonally expanded but less functional

(4) ICB is able to "reactivate" clonally expanded T cells.

(5) Conclusion: Pccb modulates the activity of T cells in PDAC.

Overall, the experiments were appropriately executed and technically sound, albeit underpowered for single-cell analyses. Upon careful consideration of the data, the biggest weakness of the paper is the authors' interpretations of results, particularly for claims 1 and 4 (see below for details). Much of the data is correlative and does not delve into causation, leaving this reviewer wishing for experiments that would clearly demonstrate that Pccb in tumor cells directly impacts T cell anti-tumor activity.

Strengths:

(1) Tumor intrinsic determinants of intratumoral T cell infiltration in PDAC are less commonly evaluated as combination therapies for ICB. This is a point of conceptual innovation and importance.

(2) A sensitized CRISPR screen to identify mutations that rescue KPC/PI3Ka-KO tumors from immune-mediated killing is an elegant method to better understand the molecular mechanisms contributing to KPC immunosurveillance. Further, one screen candidate (Pccb) was experimentally validated.

(3) Single-cell clonotype analyses hold promise for identifying tumor-reactive T cells (though authors never demonstrated that specific clones were tumor antigen specific).

Weaknesses:

(1) "Clonal expansion of cytotoxic T cells infiltrating the pancreatic αKO tumors"<br /> a. Only two tumor-bearing hosts were evaluated by single-cell TCR sequencing, thus limiting conclusions that may be drawn regarding repertoire diversity and expansion.<br /> b. High abundance clones in the TME do not necessarily have tumor specificity, nor are they necessarily clonally expanded. They may be clones which are tissue-resident or highly chemokine-responsive and accumulate in larger numbers independent of clonal expansion. Please consider softening language to clonal enrichment or refer to clone size as clonal abundance throughout the paper.<br /> c. The whole story would be greatly strengthened by cytotoxicity assays of abundant TCR clones to show tumor antigen specificity.

(2) "A genome-wide CRISPR gene-deletion screen to identify molecules contributing to Pik3ca-mediated pancreatic tumor immune evasion"<br /> a. CRISPR mutagenesis yielded outgrowth of only 2/8 tumors. A more complete screen with an increased total number of tumors would yield much stronger gene candidates with better statistical power. It is unsurprising that candidates were observed in only one of the two tumors. Nevertheless, the authors moved forward successfully with Pccb.

(3) T cells infiltrate p-αKO tumors with increased expression of immune checkpoints<br /> a. In Figure 4D, cell counts are not normalized to totalCD8+ T cell counts making it difficult to directly compare aKO to p-aKO tumors. Based on quantifications from Figure 4D, I suspect normalization will strengthen the conclusion that CD8+ infiltrate is more exhausted in p-aKO tumors.<br /> b. Flow cytometric analysis to further characterize the myeloid compartment is incomplete (single replicate) and does not strengthen the argument that p-aKO TME is more immunosuppressive.<br /> c. It could, however, strengthen the argument that TIL has less anti-tumor potential if effector molecule expression in CD8+ infiltrating cells were quantified.

(4) Inhibition of PD1/PD-L1 checkpoint leads to elimination of most p-αKO tumors<br /> a. It is reasonable to conclude that p-aKO tumors are responsive to immune checkpoint blockade. However, there is no data presented to support the statement that checkpoint blockade reactivates an existing anti-tumor CD8+ T cell response and does not instead induce a de novo response.<br /> b. The discussion of these data implies that anti-PD-1 would not improve aKO tumor control, but these data are not included. As such, it is difficult to compare the therapeutic response in aKO versus p-aKO. Further, these data are at best an indirect comparison of the T cell responsiveness against tumor, as the only direct comparison is infiltrating cell count in Figure 4 and there are no public TCR clones with confirmed anti-tumor specificity to follow in the aKO versus p-aKO response.

Reviewer #1 (Public Review):

Summary:

This study has as its goal to determine how the structure and function of the circuit that stabilizes gaze in the larval zebrafish depends on the presence of the output cells, the motor neurons. A major model of neural circuit development posits that the wiring of neurons is instructed by their postsynaptic cells, transmitting signals retrogradely on which cells to contact and, by extension, where to project their axons. Goldblatt et al. remove the motor neurons from the circuit by generating null mutants for the phox2a gene. The study then shows that, in this mutant that lacks the isl1-labelled extraocular motor neurons, the central projection neurons have 1) largely normal responses to vestibular input; 2) normal gross morphology; 3) minimally changed transcriptional profiles. From this, the authors conclude that the wiring of the circuit is not instructed by the output neurons, refuting the major model.

Strengths:

I found the manuscript to be exceptionally well-written and presented, with clear and concise writing and effective figures that highlight key concepts. The topic of neural circuit wiring is central to neuroscience, and the paper's findings will interest researchers across the field, and especially those focused on motor systems.

The experiments conducted are clever and of a very high standard, and I liked the systematic progression of methods to assess the different potential effects of removing phox2a on circuit structure and function. Analyses (including statistics) are comprehensive and appropriate and show the authors are meticulous and balanced in most of the conclusions that they draw. Overall, the findings are interesting, and with a few tweaks, should leave little doubt about the paper's main conclusions.

Weaknesses:

The main point is the incomplete characterisation of the effects of removing phox2a on the extra-ocular motor neurons. Are these cells no longer there, or are they there but no longer labelled by isl1:GFP? If they are indeed removed, might they have developed early on, and subsequently lost? These questions matter as the central focus of the manuscript is whether the presence of these cells influences the connectivity and function of their presynaptic projection neurons. Therefore, for the main conclusions to be fully supported by the data, the authors would need to test whether 1) the motor neurons that otherwise would have been labelled by the isl1:GFP line are physically no longer there; 2) that this removal (if, indeed, it is that) is developmental. If these experiments are not feasible, then the text should be adjusted to take this into account. A further point to address is the context of the manipulation. If the phox2a removal does indeed take out the extra-ocular motor neurons, what percentage of postsynaptic neurons to the projection neurons are still present? In other words, how does the postsynaptic nMLF output relate to the motor neurons? If, for instance, the nMLF (which, as the authors state, are likely still innervated by the projection neurons) are the main output of the projections neurons, then this would affect the interpretation of the results.

Reviewer #1 (Public Review):

Though the Norrin protein is structurally unrelated to the Wnt ligands, it can activate the Wnt/β-catenin pathway by binding to the canonical Wnt receptors Fzd4 and Lrp5/6, as well as the tetraspanin Tspan12 co-receptor. Understanding the biochemical mechanisms by which Norrin engages Tspan12 to initiate signaling is important, as this pathway plays an important role in regulating retinal angiogenesis and maintaining the blood-retina-barrier. Numerous mutations in this signaling pathway have also been found in human patients with ocular diseases. The overarching goal of the study is to define the biochemical mechanisms by which Tspan12 mediates Norrin signaling. Using purified Tspan12 reconstituted in lipid nanodiscs, the authors conducted detailed binding experiments to document the direct, high-affinity interactions between purified Tspan12 and Norrin. To further model this binding event, they used AlphaFold to dock Norrin and Tspan12 and identified four putative binding sites. They went on to validate these sites through mutagenesis experiments. Using the information obtained from the AlphaFold modeling and through additional binding competition experiments, it was further demonstrated that Tspan12 and Fzd4 can bind Norrin simultaneously, but Tspan12 binding to Norrin is competitive with other known co-receptors, such as HSPGs and Lrp5/6. Collectively, the authors proposed that the main function of Tspan12 is to capture low concentrations of Norrin at the early stage of signaling, and then "hand over" Norrin to Fzd4 and Lrp5/6 for further signal propagation. Overall, the study is comprehensive and compelling, and the conclusions are well supported by the experimental and modeling data.

Strengths:

• Biochemical reconstitution of Tspan12 and Fzd4 in lipid nanodiscs is an elegant approach for testing the direct binding interaction between Norrin and its co-receptors. The proteins used for the study seem to be of high purity and quality.

• The various binding experiments presented throughout the study were carried out rigorously. In particular, BLI allows accurate measurement of equilibrium binding constants as well as on and off rates.

• It is nice to see that the authors followed up on their AlphaFold modeling with an extensive series of mutagenesis studies to experimentally validate the potential binding sites. This adds credence to the AlphaFold models.

• Table S1 is a further testament to the rigor of the study.

• Overall, the study is comprehensive and compelling, and the conclusions are well supported by the experimental and modeling data.

Suggestions for improvement:

• It would be helpful to show Coomassie-stained gels of the key mutant Norrin and Tspan12 proteins presented in Figures 2E and 2F.

• Many Norrin and Tspan12 mutations have been identified in human patients with FEVR. It would be interesting to comment on whether any of the mutations might affect the Norrin-Tspan12 binding sites described in this study.

• Some of the negative conclusions (e.g. the lack of involvement of Tspan12 in the formation of the Norrin-Lrp5/6-Fzd4-Dvl signaling complex) can be difficult to interpret. There are many possible reasons as to why certain biological effects are not recapitulated in a reconstitution experiment. For instance, the recombinant proteins used in the experiment may not be presented in the correct configurations, and certain biochemical modifications, such as phosphorylation, may also be missing.

Reviewer #1 (Public Review):

Summary:<br /> Bendzunas, Byrne et al. explore two highly topical areas of protein kinase regulation in this manuscript. Firstly, the idea that Cys modification could regulate kinase activity. The senior authors have published some standout papers exploring this idea of late, and the current work adds to the picture of how active site Cys might have been favoured in evolution to serve critical regulatory functions. Second, BRSK1/2 are understudied kinases listed as part of the "dark kinome" so any knowledge of their underlying regulation is of critical importance to advancing the field.

Strengths:<br /> In this study, the author pinpoints highly-conserved, but BRSK-specific, Cys residues as key players in kinase regulation. There is a delicate balance between equating what happens in vitro with recombinant proteins relative to what the functional consequence of Cys mutation might be in cells or organisms, but the authors are very clear with the caveats relating to these connections in their descriptions and discussion. Accordingly, by extension, they present a very sound biochemical case for how Cys modification might influence kinase activity in cellular environs.

Comments on revised version:

The authors have satisfactorily addressed my concerns.

Reviewer #1 (Public Review):

Summary:

The authors perform a multidisciplinary approach to describe the conformational plasticity of P-Rex1 in various states (autoinhibited, IP4 bound and PIP3 bound). Hydrogen-deuterium exchange (HDX) is used to reveal how IP4 and PIP3 binding affect intramolecular interactions. While IP4 is found to stabilize autoinhibitory interactions, PIP3 does the opposite, leading to deprotection of autoinhibitory sites. Cryo-EM of IP4 bound P-Rex1 reveals a structure in the autoinhibited conformation, very similar to the unliganded structure reported previously (Chang et al. 2022). Mutations at observed autoinhibitory interfaces result in a more open structure (as shown by SAXS), reduced thermal stability and increased GEF activity in biochemical and cellular assays. Together their work portrays a dynamic enzyme that undergoes long-range conformational changes upon activation on PIP3 membranes. The results are technically sound and the conclusions are justified. The main drawback is the limited novelty due to the recently published structure of unliganded P-Rex1, which is virtually identical to the IP4 bound structure presented here. Novel aspects suggest a regulatory role for IP4, but the exact significance and mechanism of this regulation has not been explored.

Strengths:

The authors use a multitude of techniques to describe the dynamic nature and conformational changes of P-Rex1 upon binding to IP4 and PIP3 membranes. The different approaches together fit well with the overall conclusion that IP4 binding negatively regulates P-Rex1, while binding to PIP3 membranes leads to conformational opening and catalytic activation. The experiments are performed very thoroughly and are technically sound. The results are clear and support the conclusions.

Weaknesses:

(1) The novelty of the study is compromised due to the recently published structure of unliganded P-Rex1 (Chang et al. 2022). The unliganded and IP4 bound structure of P-Rex1 appear virtually identical, however, no clear comparison is presented in the manuscript. In the same paper a very similar model of P-Rex1 activation upon binding to PIP3 membranes and Gbeta-gamma is presented.

(2) The authors demonstrate that IP4 binding to P-Rex1 results in catalytic inhibition and increased protection of autoinhibitory interfaces, as judged by HDX. The relevance of this in a cellular setting is not clear and is not experimentally demonstrated. Further, mechanistically, it is not clear whether the biochemical inhibition by IP4 of PIP3 activated P-Rex1 is due to competition of IP4 with activating PIP3 binding to the PH domain of P-Rex1, or due to stabilizing the autoinhibited conformation, or both.

(3) Fig.1B-C: To give a standard deviation from 2 data points has no statistical significance. In this case it would be better to define as range/difference of the 2 data points.

Reviewer #1 (Public Review):

Summary:

Tuberculous meningitis (TBM) is one of the most severe form of extrapulmonary TB. TBM is especially prevalent in people who are immunocompromised (e.g. HIV-positive). Delays in diagnosis and treatment could lead to severe disease or mortality. In this study, the authors performed the largest ever host whole blood transcriptomics analysis on a cohort of 606 Vietnamese participants. The results indicated that TBM mortality is associated with increased neutrophil activation and decreased T and B cell activation pathways. Furthermore, increased angiogenesis was also observed in HIV-positive patients who died from TBM, whereas activated TNF signaling and down-regulated extracellular matrix organisation were seen in the HIV-negative group. Despite similarities in transcriptional profiles between PTB and TBM compared to healthy controls, inflammatory genes were more active in HIV-positive TBM. Finally, 4 hub genes (MCEMP1, NELL2, ZNF354C and CD4) were identified as strong predictors of death from TBM.

Strengths:

This is a really impressive piece of work, both in terms of the size of the cohort which took years of effort to recruit, sample and analyse and also the meticulous bioinformatics performed. The biggest advantage of obtaining a whole blood signature is that it allows an easier translational development into test that can be used in the clinical with a minimally invasive sample. Furthermore, the data from this study has also revealed important insights in the mechanisms associated with mortality and the differences in pathogenesis between HIV-positive and HIV-negative patients, which would have diagnostic and therapeutic implications.

Weaknesses:

The authors have addressed all the weaknesses in the revised version.

Reviewer #2 (Public Review):

Summary:

This study from Bamgbose et al. identifies a new and important interaction between H4K20me and Parp1 that regulates inducible genes during development and heat stress. The authors present convincing experiments that form a mostly complete manuscript that significantly contributes to our understanding of how Parp1 associates with target genes to regulate their expression.

Strengths:

The authors present 3 compelling experiments to support the interaction between Parp1 and H4K20me, including:

(1) PR-Set7 mutants remove all K4K20me and phenocopy Parp mutant developmental arrest and defective heat shock protein induction.

(2) PR-Set7 mutants have dramatically reduced Parp1 association with chromatin and reduced poly-ADP ribosylation.

(3) Parp1 directly binds H4K20me in vitro.

Reviewer #1 (Public Review):

Summary:

The endocannabinoid system (ECS) components are dysregulated within the lesion microenvironment and systemic circulation of endometriosis patients. Using endometriosis mouse models and genetic loss of function approaches, Lingegowda et al. report that canonical ECS receptors, CNR1 and CNR2, are required for disease initiation, progression, and T-cell dysfunction.

Strengths:

The approach uses genetic approaches to establish in vivo causal relationships between dysregulated ECS and endometriosis pathogenesis. The experimental design incorporates bulk RNAseq approaches, as well as imaging mass spectrometry to characterize the mouse lesions. The identification of immune-related and T-cell-specific changes in the lesion microenvironment of CNR1 and CNR2 knockout (KO) mice represents a significant advance

Weaknesses:

Although the mouse phenotypic analyses involve a detailed molecular characterization of the lesion microenvironment using genomic approaches, detailed measurements of lesion size/burden and histopathology would provide a better understanding of how CNR1 or CNR2 loss contributes to endometriosis initiation and progression. The cell or tissue-specific effects of the CNR1 and CNR2 are not incorporated into the experimental design of the studies. Although this aspect of the approach is recognized as a major limitation, global CNR1 and CNR2 KO may affect normal female reproductive tract function, ovarian steroid hormone levels, decidualization response, or lead to preexisting alterations in host or donor tissues, which could affect lesion establishment and development in the surgically induced, syngeneic mouse model of endometriosis.

Reviewer #1 (Public Review):

Strengths

The paper has shown the expression of RGS10 is related to the molecular subtype, distant metastasis, and survival status of breast cancer. The study utilizes bioinformatic analyses, human tissue samples, and in vitro and in vivo experiments which strengthen the data. RGS10 was validated to inhibit EMT through a novel mechanism dependent on LCN2 and miR-539-5p, thereby reducing cancer cell proliferation, colony formation, invasion, and migration. The study elaborated the function of RGS10 in influencing the prognosis and biological behavior which could be considered as a potential drug target in breast cancer.

Weakness<br /> The mechanism by which the miR-539-5p/RGS10/LCN2 axis may be related to the prognosis of cancer patients still needs to be elucidated. In addition, the sample size used is relatively limited. Especially, if further exploration of the related pathways and mechanisms of LCN2 can be carried out by using organoid models, as well as the potential of RGS10 as a biomarker for further clinical translation to verify its therapeutic target effect, which will make the data more convincing.

Reviewer #1 (Public Review):

Summary:

The authors want to explore how much two known minibinder protein domains against the Spike protein of SARS-CoV-2 can function as a binding domain of 2 sets of synthetic receptors (SNIPR and CAR); the authors also want to know how some modifications of the linkers of these new receptors affect their activation profile.

Major strengths and weaknesses of the methods and results:

- Strengths include: analysis of synthetic receptor function for 2 classes of synthetic receptors, with robust and appropriate assays for both kinds of receptors. The modifications of the linkers are also interesting and the types of modifications that are often used in the field.

- Weaknesses include: none of the data analysis provides statistical interpretation of the results (that I could find). One dataset is confusing: Figures 5A and C, are said to be the same assay with the same constructs, but the results are 30% in A, and 70% in C.

An appraisal of whether the authors achieved their aims, and whether the results support their conclusions:

Given the open-ended nature of the goal (implicit in it being an exploration), it is hard to say if the authors have reached their aims; they have done an exploration for sure; is it big enough an exploration? This reviewer is not sure.

The results are extremely clearly presented, both in the figures and in the text, both for the methods and the results. The claims put forward (with limited exceptions see below) are very solidly supported by the presented data.

A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community:

The work may stimulate others to consider minibinders as potential binding domains for synthetic receptors. The modifications that are presented although not novel, do provide a starting point for larger-scale analysis.

It is not clear how much this is generalizable to other binders (the authors don't make such claims though). The claims are very focused on the tested modifications, and the 2 receptors and minibinder used, a scope that I would define as narrow; the take-home message if one wants to try it with other minibinders or other receptors seems to be: test a few things, and your results may surprise you.

Any additional context you think would help readers interpret or understand the significance of the work:

We are at the infancy stage of synthetic receptors optimization and next-generation derivation; there is a dearth of systematic studies, as most focus is on developing a few ones that work. This work is an interesting attempt to catalyze more research with these new minibinders. Will it be picked up based on this? Not sure.

Reviewer #1 (Public Review):

In this study, the authors explored how the reduced growth fitness, resulting from genome reduction, can be compensated through evolution. They conducted an evolution experiment with a strain of Escherichia coli that carried a reduced genome, over approximately 1,000 generations. The authors carried out sequencing, and found no clear genetic signatures of evolution across replicate populations. They carry out transcriptomics and a series of analyses that lead them to conclude that there are divergent mechanisms at play in individual evolutionary lineages. The authors used gene network reconstruction to identify three gene modules functionally differentiated, correlating with changes in growth fitness, genome mutation, and gene expression, respectively, due to evolutionary changes in the reduced genome.

I think that this study addresses an interesting question. Many microbial evolution experiments evolve by loss of function mutations, but presumably a cell that has already lost so much of its genome needs to find other mechanisms to adapt. Experiments like this have the potential to study "constructive" rather than "destructive" evolution.

Comments on revised version:

I think the authors have carefully gone through the manuscript and addressed all of my concerns.

Reviewer #1 (Public Review):

⍺-synuclein (syn) is a critical protein involved in many aspects of human health and disease. Previous studies have demonstrated that post-translational modifications (PTMs) play an important role in regulating the structural dynamics of syn. However, how post-translational modifications regulate syn function remains unclear. In this manuscript, Wang et al. reported an exciting discovery that N-acetylation of syn enhances the clustering of synaptic vesicles (SVs) through its interaction with lysophosphatidylcholine (LPC). Using an array of biochemical reconstitution, single vesicle imaging, and structural approaches, the authors uncovered that N-acetylation caused distinct oligomerization of syn in the presence of LPC, which is directly related to the level of SV clustering. This work provides novel insights into the regulation of synaptic transmission by syn and might also shed light on new ways to control neurological disorders caused by syn mutations.

Reviewer #1 (Public Review):

Summary:

The mammalian Shieldin complex consisting of REV7 (aka MAD2L2, MAD2B) and SHLD1-3 affects pathway usage in DSB repair favoring non-homologous endjoining (NHEJ) at the expense of homologous recombination (HR) by blocking resection and/or priming fill-in DNA synthesis to maintain or generate near blunt ends suitable for NHEJ. While the budding yeast Saccharomyces cerevisiae does not have homologs to SHLD1-3, it does have Rev7, which was identified to function in conjunction with Rev3 in the translesion DNA polymerase zeta. Testing the hypothesis that Rev7 also affects DSB resection in budding yeast, the work identified a direct interaction between Rev7 and the Rad50-Mre11-Xrs2 complex by two-hybrid and direct protein interaction experiments. Deletion analysis identified that the 42 amino acid C-terminal region was necessary and sufficient for the 2-hybrid interaction. Direct biochemical analysis of the 42 aa peptide was not possible. Rev7 deficient cells were found to be sensitive to HU only in synergy with G2 tetraplex forming DNA. Importantly, the 42 aa peptide alone suppressed this phenotype. Biochemical analysis with full-length Rev7 and a C-terminal truncation lacking the 42 aa region shows G4-specific DNA binding that is abolished in the C-terminal truncation and with a substrate containing mutations to prevent G4 formation. Rev7 lacks nuclease activity but inhibits the dsDNA exonuclease activity of Mre11. The C-terminal truncation protein lacking the 42 aa region also showed some inhibition suggesting the involvement of additional binding sites besides the 42 aa region. Also, the Mre11 ssDNA endonuclease activity is inhibited by Rev7 but not the degradation of linear ssDNA. Rev7 does not affect ATP binding by Rad50 but inhibits in a concentration-dependent manner the Rad50 ATPase activity. The C-terminal truncation protein lacking the 42 aa region also showed some inhibition but significantly less than the full-length protein.

Using an established plasmid-based NHEJ assay, the authors provide strong evidence that Rev7 affects NEHJ, showing a four-fold reduction in this assay. The mutations in the other Pol zeta subunits, Rev3 and Rev1, show a significantly smaller effect (~25% reduction). A strain expressing only the Rev7 C-terminal 42 aa peptide showed no NHEJ defect, while the truncation protein lacking this region exhibited a smaller defect than the deletion of REV7. The conclusion that Rev7 supports NHEJ mainly through the 42 aa region was validated using a chromosomal NHEJ assay. The effect on HR was assessed using a plasmid:chromosome system containing G4 forming DNA. The rev7 deletion strain showed an increase in HR in this system in the presence and absence of HU. Cells expressing the 42 aa peptide were indistinguishable from the wild type as were cells expressing the Rev7 truncation lacking the 42 aa region. The authors conclude that Rev7 suppresses HR, but the context appears to be system-specific and the conclusion that Rev7 abolished HR repair of DSBs is unwarranted and overly broad.

Strength:

This is a well-written manuscript with many well-executed experiments that suggest that Rev7 inhibits MRX-mediated resection to favor NEHJ during DSB repair. This finding is novel and provides insight into the potential mechanism of how the human Shieldin complex might antagonize resection.

Weaknesses:

The nuclease experiments were conducted using manganese as a divalent cation, and it is unclear whether there is an effect with the more physiological magnesium cation. Additional controls for the ATPase and nuclease experiments to eliminate non-specific effects would be helpful. Evidence for an effect on resection in cells is lacking. The major conclusion about the role of Rev7 in regulating the choice between HR and NHEJ is not justified, as only a highly specialized assay is used that does not warrant the broad conclusion drawn. Specifically, the results that the Rev7 C-terminal truncation lacking the 42 aa region still suppresses HR is unexpected and unexplained. The effect of Rev7 on G4 metabolism is underdeveloped and distracts from the main results that Rev7 modulated MRX activity. The authors should consider removing this part and develop a more complete story on this later.

Reviewer #1 (Public Review):

Summary:

Extracellular ATP represents a danger-associated molecular pattern associated to tissue damage and can act also in an autocrine fashion in macrophages to promote proinflammatory responses, as observed in a previous paper by the authors in abdominal sepsis. The present study addresses an important aspect possibly conditioning the outcome of sepsis that is the release of ATP by bacteria. The authors show that sepsis-associated bacteria do in fact release ATP in a growth dependent and strain-specific manner. However, whether this bacterial derived ATP play a role in the pathogenesis of abdominal sepsis has not been determined. To address this question, a number of mutant strains of E. coli has been used first to correlate bacterial ATP release with growth and then, with outer membrane integrity and bacterial death. By using E. coli transformants expressing the ATP-degrading enzyme apyrase in the periplasmic space, the paper nicely shows that abdominal sepsis by these transformants results in significantly improved survival. This effect was associated with a reduction of peritoneal macrophages and CX3CR1+ monocytes, and an increase in neutrophils. To extrapolate the function of bacterial ATP from the systemic response to microorganisms, the authors exploited bacterial OMVs either loaded or not with ATP to investigate the systemic effects devoid of living microorganisms. This approach showed that ATP-loaded OMVs induced degranulation of neutrophils after lysosomal uptake, suggesting that this mechanism could contribute to sepsis severity.

Strengths:

A strong part of the study is the analysis of E. coli mutants to address different aspects of bacterial release of ATP that could be relevant during systemic dissemination of bacteria in the host.

Weaknesses:

As pointed out in the limitations of the study whether ATP-loaded OMVs provide a mechanistic proof of the pathogenetic role of bacteria-derived ATP independently of live microorganisms in sepsis is interesting but not definitively convincing. It could be useful to see whether degranulation of neutrophils is differentially induced by apyrase-expressing vs control E. coli transformants. Also, the increase of neutrophils in bacterial ATP-depleted abdominal sepsis, which has better outcomes than "ATP-proficient" sepsis, seems difficult to correlate to the hypothesized tissue damage induced by ATP delivered via non-infectious OMVs. Are the neutrophils counts affected by ATP delivered via OMVs? A comparison of cytokine profiles in the abdominal fluids of E. coli and OMV treated animals could be helpful in defining the different responses induced by OMV-delivered vs bacterial-released ATP. The analyses performed on OMV treated versus E. coli infected mice are not closely related and difficult to combine when trying to draw a hypothesis for bacterial ATP in sepsis. Also it was not clear why lung neutrophils were used for the RNAseq data generation and analysis.

Joint Public Review

This work investigates the evolutionary conservation and functional significance of FoxO transcription factors in the response of airway epithelia to diverse stressors, ranging from hypoxia to temperature fluctuations and oxidative stress. Utilizing a comprehensive approach encompassing Drosophila, murine models, and human samples, the study investigates FoxO's role across species. The authors demonstrate that hypoxia triggers a dFOXO-dependent immune response in Drosophila airways, with subsequent nuclear localization of dFOXO in response to various stressors. Transcriptomic analysis reveals differential regulation of crucial gene categories in respiratory tissues, highlighting FoxO's involvement in metabolic pathways, DNA replication, and stress resistance mechanisms.

The study underscores FoxO's importance in maintaining homeostasis by revealing reduced stress resistance in dFOXO Drosophila mutants, shedding light on its protective role against stressors. In mammalian airway cells, FoxO exhibits nuclear translocation in response to hypoxia, accompanied by upregulation of cytokines with antimicrobial activities. Intriguingly, mouse models of asthma show FoxO downregulation, which is also observed in sputum samples from human asthma patients, implicating FoxO dysregulation in respiratory pathologies.

Overall, the manuscript suggests that FoxO signaling plays a critical role in preserving airway epithelial cell homeostasis under stress conditions, with implications for understanding and potentially treating respiratory diseases like asthma. By providing compelling evidence of FoxO's involvement across species and its correlation with disease states, the study underscores the importance of further exploration into FoxO-mediated mechanisms in respiratory health.

Strengths

(1) This study shows that FoxO transcription factors are critical for regulating immune and inflammatory responses across species, and for orchestrating responses to various stressors encountered by airway epithelial cells, including hypoxia, temperature changes, and oxidative stress. Understanding the intricate regulation of FoxO transcription factors provides insights into modulating immune and inflammatory pathways, offering potential avenues for therapeutic interventions against respiratory diseases and other illnesses.

(2) The work employs diverse model systems, including Drosophila, murine models, and human samples, thereby establishing a conserved role for FoxOs in airway epithelium and aiding translational relevance to human health.

(3) The manuscript establishes a strong correlation between FoxO expression levels and respiratory diseases such as asthma. Through analyses of both murine models of asthma and asthmatic human samples, the study demonstrates a consistent reduction in FoxO expression, indicating its potential involvement in the pathogenesis of respiratory disorders. This correlation underscores the clinical relevance of FoxO dysregulation and opens avenues for developing treatments for respiratory conditions like asthma, COPD, and pulmonary fibrosis, addressing significant unmet clinical needs.

(4) The study unveils intriguing mechanistic details regarding FoxO regulation and function. Particularly noteworthy is the observation of distinct regulatory mechanisms governing dFOXO translocation in response to different stressors. The independence of hypoxia-induced dFOXO translocation from JNK signaling adds complexity to our understanding of FoxO-mediated stress responses. Such mechanistic insights deepen our understanding of FoxO biology and pave the way for future investigations into the intricacies of FoxO signaling pathways in airway epithelial cells.

Weaknesses

(1) The manuscript does not distinguish between FoxO expression levels and FoxO activation status. While FoxO nuclear localization is observed in Drosophila and murine models, it remains unclear whether this reflects active FoxO signaling or merely FoxO expression, limiting the mechanistic understanding of FoxO regulation.

(2) The manuscript utilizes various stressors across different experiments without providing a clear rationale for their selection. This lack of coherence in stressor choice complicates the interpretation of results and diminishes the ability to draw meaningful comparisons across experiments.

(3) The manuscript frequently refers to "FoxO signaling" without providing specific signaling readouts. This ambiguity undermines the clarity of the conclusions drawn from the data and hinders the establishment of clear cause-and-effect relationships between FoxO activation and cellular responses to stress.

(4) Many conclusions drawn in the manuscript rely heavily on the quantification of immunostaining images for FoxO nuclear localization. While this is an important observation, it does not provide a sufficient mechanistic understanding of FoxO expression or activation regulation.

(5) The primary weakness in the Drosophila experiments is the analysis of dFoxO in homozygous dFoxO mutant animals, which precludes determining the specific role of dFoxO in airway cells. Despite available tools for tissue-specific gene manipulation, such as tissue-specific RNAi and CRISPR techniques, these approaches were not employed, limiting the precision of the findings.

(6) In mammalian experiments, the results are primarily correlative, lacking causal evidence. While changes in FoxO expression are observed under pathological conditions, the absence of experiments on FoxO-deficient cells or tissues precludes establishing a causal relationship between FoxO dysregulation and respiratory pathologies.

(7) Although the evidence suggests a critical role for FoxO in airway tissues, the precise nature of this role remains unclear. With gene expression changes analyzed only in Drosophila, the extent of conservation in downstream FoxO-mediated pathways between mammals and Drosophila remains uncertain. Additionally, the functional consequences of FoxO deficiency in airway cells were not determined, hindering comparisons between species and limiting insights into FoxO's functional roles in different contexts.

Reviewer #1 (Public Review):

In this manuscript, entitled " Merging Multi-OMICs with Proteome Integral Solubility Alteration Unveils Antibiotic Mode of Action", Dr. Maity and colleagues aim to elucidate the mechanisms of action of antibiotics through combined approaches of omics and the PISA tool to discover new targets of five drugs developed against Helicobacter pylori.

Strengths:

Using transcriptomics, proteomic analysis, protein stability (PISA), and integrative analysis, Dr. Maity and colleagues have identified pathways targeted by five compounds initially discovered as inhibitors against H. pylori flavodoxin. This study underscores the necessity of a global approach to comprehensively understanding the mechanisms of drug action. The experiments conducted in this paper are well-designed and the obtained results support the authors' conclusions.

Weaknesses:

This manuscript describes several interesting findings. A few points listed below require further clarification:

(1) Compounds IVk exhibits markedly different behavior compared to the other compounds. The authors are encouraged to discuss these findings in the context of existing literature or chemical principles.

(2) The incubation time for treating H. pylori with the drugs was set at 4 hours for transcriptomic and proteomic analyses, compared to 20 min for PISA analysis. The authors need to explain the reason for these differences in treatment duration.

(3) The PISA method facilitates the identification of proteins stabilized by drug treatment. DnaJ and Trigger factor (tig), well-known molecular chaperones, prevent protein aggregation under stress. Their enrichment in the soluble fraction is expected and does not necessarily indicate direct stabilization by the drugs. The possibility that their stabilization results from binding to other proteins destabilized by the drugs should be considered. To prevent any misunderstanding, the authors should clarify that their methodology does not solely identify direct targets. Instead, the combination of their findings sheds light on various pathways affected by the treatment.

(4) At the end of the manuscript, the authors conclude that four compounds "strongly interact with CagA". However, detailed molecule/protein interaction studies are necessary to definitively support this claim. The authors should exercise caution in their statement. As the authors mentioned, additional research (not mandated in the scope of this current paper) is necessary to determine the drug's binding affinity to the proposed targets.

(5) The authors should clarify the PISA-Express approach over standard PISA. A detailed explanation of the differences between both methods in the main text is important.

Reviewer #1 (Public Review):

Summary:

This manuscript provides an initial characterization of three new missense variants of the PLCG1 gene associated with diverse disease phenotypes, utilizing a Drosophila model to investigate their molecular effects in vivo. Through the meticulous creation of genetic tools, the study assesses the small wing (sl) phenotype - the fly's ortholog of PLCG1 - across an array of phenotypes from longevity to behavior in both sl null mutants and variants. The findings indicate that the Drosophila PLCG1 ortholog displays aberrant functions. Notably, it is demonstrated that overexpression of both human and Drosophila PLCG1 variants in fly tissue leads to toxicity, underscoring their pathogenic potential in vivo.

Strengths:

The research effectively highlights the physiological significance of sl in Drosophila. In addition, the study establishes the in vivo toxicity of disease-associated variants of both human PLCG1 and Drosophila sl.

Weaknesses:

The study's limitations include the human PLCG1 transgene's inability to compensate for the Drosophila sl null mutant phenotype, suggesting potential functional divergence between the species. This discrepancy signals the need for additional exploration into the mechanistic nuances of PLCG1 variant pathogenesis, especially regarding their gain-of-function effects in vivo.

Overall:

The study offers compelling evidence for the pathogenicity of newly discovered disease-related PLCG1 variants, manifesting as toxicity in a Drosophila in vivo model, which substantiates the main claim by the authors. Nevertheless, a deeper inquiry into the specific in vivo mechanisms driving the toxicity caused by these variants in Drosophila could significantly enhance the study's impact.

Reviewer #1 (Public Review):

Summary:

Casas-Tinto et al. present convincing data that injury of the adult Drosophila CNS triggers transdifferentiation of glial cells and even the generation of neurons from glial cells. This observation opens up the possibility of getting a handle on the molecular basis of neuronal and glial generation in the vertebrate CNS after traumatic injury caused by Stroke or Crush injury. The authors use an array of sophisticated tools to follow the development of glial cells at the injury site in very young and mature adults. The results in mature adults revealing a remarkable plasticity in the fly CNS and dispels the notion that repair after injury may be only possible in nerve cords which are still developing. The observation of so-called VC cells which do not express the glial marker repo could point to the generation of neurons by former glial cells.

Conclusion:

The authors present an interesting story that is technically sound and could form the basis for an in-depth analysis of the molecular mechanism driving repair after brain injury in Drosophila and vertebrates.

Strengths:

The evidence for transdifferentiation of glial cells is convincing. In addition, the injury to the adult CNS shows an inherent plasticity of the mature ventral nerve cord which is unexpected.

Weaknesses:

Traumatic brain injury in Drosophila has been previously reported to trigger mitosis of glial cells and generation of neural stem cells in the larval CNS and the adult brain hemispheres. Therefore this report adds to but does not significantly change our current understanding. The origin and identity of VC cells is unclear.

Good example with capitalization.

Reviewer #1 (Public Review):

In this manuscript, Huang and colleagues explored the role of iron in bacterial therapy for cancer. Using proteomics, they revealed the upregulation of bacterial genes that uptake iron, and reasoned that such regulation is an adaptation to the iron-deficient tumor microenvironment. Logically, they engineered E. Coli strains with enhanced iron-uptake efficiency, and showed that these strains, together with iron scavengers, suppress tumor growth in a mouse model. Lastly, they reported the tumor suppression by IroA-E. Coli provides immunological memory via CD8+ T cells. In general, I find the findings in the manuscript novel and the evidence convincing.

(1) Although the genetic and proteomic data are convincing, would it be possible to directly quantify the iron concentration in (1) E. Coli in different growth environments, and (2) tumor microenvironment? This will provide functional consequence of upregulating genes that import iron into the bacteria.

(2) Related to 1, the experiment to study the synergistic effect of CDG and VLX600 (lines 139-175) is very nice and promising, but one flaw here is a lack of the measurement of iron concentration. Therefore, a possible explanation could be that CDG acts in another manner, unrelated to iron uptake, that synergizes with VLX600's function to deplete iron from cancer cells. Here, a direct measurement of iron concentration will show the effect of CDG on iron uptake, thus complementing the missing link.

(3) Lines 250-268: Although statistically significant, I would recommend the authors characterize the CD8+ T cells a little more, as the mechanism now seems quite elusive. What signals or memories do CD8+ T cells acquire after IroA-E. Coli treatment to confer their long-term immunogenicity?

(4) Perhaps this goes beyond the scope of the current manuscript, but how broadly applicable is the observed iron-transport phenomenon in other tumor models? I would recommend the authors to either experimentally test it in another model, or at least discuss this question.

Reviewer #1 (Public Review):

In this paper, the authors evaluate the utility of brain age derived metrics for predicting cognitive decline by performing a 'commonality' analysis in a downstream regression that enables the different contribution of different predictors to be assessed. The main conclusion is that brain age derived metrics do not explain much additional variation in cognition over and above what is already explained by age. The authors propose to use a regression model trained to predict cognition ('brain cognition') as an alternative suited to applications of cognitive decline. While this is less accurate overall than brain age, it explains more unique variance in the downstream regression.

REVISED VERSION: while the authors have partially addressed my concerns, I do not feel they have addressed them all. I do not feel they have addressed the weight instability and concerns about the stacked regression models satisfactorily. I also must say that I agree with Reviewer 3 about the limitations of the brain age and brain cognition methods conceptually. In particular that the regression model used to predict fluid cognition will by construction explain more variance in cognition than a brain age model that is trained to predict age. This suffers from the same problem the authors raise with brain age and would indeed disappear if the authors had a separate measure of cognition against which to validate and were then to regress this out as they do for age correction. I am aware that these conceptual problems are more widespread than this paper alone (in fact throughout the brain age literature), so I do not believe the authors should be penalised for that. However, I do think they can make these concerns more explicit and further tone down the comments they make about the utility of brain cognition. I have indicated the main considerations about these points in the recommendations section below.

In this paper, the authors evaluate the utility of brain age derived metrics for predicting cognitive decline by performing a 'commonality' analysis in a downstream regression that enables the different contribution of different predictors to be assessed. The main conclusion is that brain age derived metrics do not explain much additional variation in cognition over and above what is already explained by age. The authors propose to use a regression model trained to predict cognition ('brain cognition') as an alternative that explains more unique variance in the downstream regression.

This is a reasonably good paper and the use of a commonality analysis is a nice contribution to understanding variance partitioning across different covariates. I have some comments that I believe the authors ought to address, which mostly relate to clarity and interpretation

First, from a conceptual point of view, the authors focus exclusively on cognition as a downstream outcome. I would suggest the authors nuance their discussion to provide broader considerations of the utility of their method and on the limits of interpretation of brain age models more generally.

Second, from a methods perspective , there is not a sufficient explanation of the methodological procedures in the current manuscript to fully understand how the stacked regression models were constructed. I would request that the authors provide more information to enable the reader to better understand the stacked regression models used to ensure that these models are not overfit.

Please also provide an indication of the different regression strengths that were estimated across the different models and cross-validation splits. Also, how stable were the weights across splits?

Please provide more details about the task designs, MRI processing procedures that were employed on this sample in addition to the regression methods and bias correction methods used. For example, there are several different parameterisations of the elastic net, please provide equations to describe the method used here so that readers can easily determine how the regularisation parameters should be interpreted.

Reviewer #1 (Public Review):

Summary

A new method, tCFS, is introduced to offer richer and more efficient measurement of interocular suppression. It generates a new index, the suppression depth, based on the contrast difference between the up-ramped contrast for the target to breakthrough suppression and the down-ramped contrast for the target to disappear into suppression. A uniform suppression depth regardless of image types (e.g., faces, gratings and scrambles) was discovered in the paper, favoring an early-stage mechanism involving CFS. Discussions about claims of unconscious processing and the related mechanisms.

Strength

The tCFS method adds to the existing bCFS paradigms by providing the (re-)suppression threshold and thereafter the depression depth. Benefiting from adaptive procedures with continuous trials, the tCFS is able to give fast and efficient measurements. It also provides a new opportunity to test theories and models about how information is processed outside visual awareness.

Weakness:

This paper reports the surprising finding of uniform suppression depth over a variety of stimuli. This is novel and interesting. But given the limited samples being tested, the claim of uniformity suppression depth needs to be further examined, with respect to different complexities and semantic meanings.

From an intuitive aspect, the results challenged previous views about "preferential processing" for certain categories, though it invites further research to explore what exactly could suppression depth tell us about unconscious visual processing.