Reviewer #1 (Public Review):

In this study the authors first perform global knockout of the gene coding for the polarity protein Crumbs 3 (CRB3) in the mouse and show that this leads to perinatal lethality and anopthalmia. Next, they create a conditional knockout mouse specifically lacking CRB3 in mammary gland epithelial cells and show that this leads to ductal epithelial hyperplasia, impaired branching morphogenesis and tumorigenesis. To study the mechanism by which CRB3 affects mammary epithelial development and morphogenesis, the authors turn to MCF10A cells and find that CRB3 shRNA-mediated knockdown in these cells impairs their ability to form properly polarized acini in 3D cultures. Furthermore, they find that MCF10A cells lacking CRB3 display reduced primary ciliation frequency compared to control cells, which is in agreement with previous studies implicating CRB3 in primary cilia biogenesis. Using a combination of biochemical, molecular- and imaging approaches the authors then provid evidence indicating that CRB3 promotes ciliogenesis by mediating Rab11-dependent recruitment of gamma tubulin ring complex component GCP6 to the centrosome/ciliary base, and they also show that CRB3 itself is localized to the base of primary cilia. Finally, to assess the functional consequences of CRB3 loss on ciliary signaling function, the authors analyze the effect of CRB3 loss on Hedgehog and Wnt signaling using cell-based assays or a mouse model.

Overall, the described findings are interesting and in agreement with previous studies showing an involvement of CRB3 in epithelial cell biology, tumorigenesis and ciliogenesis. The results showing a role for CRB3 in mammary epithelial development and morphogenesis in vivo seem convincing. However, a major weakness of this study is that quantitative analysis of several key results is either lacking, not done appropriately, or is incompletely described. In addition, some of the cell-based experiments are lacking appropriate controls, and the claim that CRB3 directly binds to Rab11 is not supported by the data provided.

Reviewer #1 (Public Review):

In "Striatal ensemble activity in an innate behavior", Minkowicz et al. strive to characterize how the striatum, the primary input nucleus of the basal ganglia, represents grooming. Here, grooming is used as a paradigmatic habitual behavior. The pose dynamics of grooming are stereotyped: mice perform it spontaneously and prior work has shown that it is both represented and controlled by the striatum.

The manuscript presents a valuable contribution to the field by shedding light on how ensembles of neurons encode this innate behavior. Additionally, the use of supervised machine learning allowed the authors to collect and precisely align a large number of grooming repetitions, which enabled most of their downstream analysis.

I found the paper to be well-written and the conclusions are mostly well-supported. However, some of the data analysis was a bit opaque, and some more detail and reanalysis could substantially strengthen the authors' claims.

1) The authors identified grooming bouts using empirically defined thresholds and manual tweaking. Next, the boundaries of grooming were used for trial alignment and linear time warping. This is a completely sensible approach; however, in using only the boundaries of grooming episodes, the dynamics of grooming bouts are ignored. I am particularly concerned that pose dynamics of grooming bouts are most stereotyped at the boundaries (e.g. they always begin and end with specific paw movements). To play devil's advocate, if the striatum encodes pose dynamics and not boundaries and pose dynamics are noisy between the beginning and end of these bouts (either due to the dynamics of the behavior or how it was identified), then a "boundary-like" representation may emerge in the average. I strongly recommend re-running a subset of the analysis after accounting for variability in grooming dynamics. A simple thing to try would be to further cluster grooming bouts using 3D keypoint trajectories. Another would be to warp grooming bouts in a manner that accounts for keypoint trajectories (e.g. DTW or other recent time-warping variants).

2) The authors should consider if the correlation to grooming is due to (at least in part) a correlation with another aspect of movement, e.g. overall velocity, acceleration, height, or angular velocity. This should be straightforward to analyze with the current dataset. To start, I would simply take the velocity and acceleration of the mouse's centroid (head and body could be considered separately). Next, look at the correlation with DLS spiking. If a clear relationship emerges, then check to see how velocity (or another variable) maps onto grooming. It may be that DLS neurons appear to encode the boundaries of grooming when they (at least partially) encode other variables.

3) The ensemble analysis is potentially critical to our understanding of SPNs. Figure 4A suggests that ensembles encode grooming with a probabilistic code - ensembles appear to be engaged for a small number of grooming bouts in the session. First, a basic question is what is the probability a given ensemble is activated during grooming? Second, the more complex question is whether there is an explanation for why one ensemble is engaged for some trials and not others? Related to point 2, I wonder if another aspect of behavior - e.g. vigor, duration, or speed - determines this. I suggest some analysis to at least rule out some simple explanations.

Reviewer #1 (Public Review):

In this manuscript by Douglas et al, the investigative team seeks to identify Staphylococcus aureus genes (and associated polymorphisms) that confer altered susceptibility to human serum, with the hypothesis that such genes might contribute to the propensity of a strain to cause bacteremia, invasive disease, and/or death. Using an innovative GWAS-like approach applied to a bank of over 300 well-characterized clinical S. aureus isolates, the authors discover SNPs in seven different staphylococcal genes that confer increased survival in the setting of serum exposure. The authors then mainly focus on one gene, tcaA, and illustrate a potential mechanism whereby modification of peptidoglycan structure and WTA display leads to altered susceptibility to serum, serum-derived antimicrobial compounds, and antibiotics. One particularly significant finding is that the identified tcaA SNP is significantly associated with patient mortality, in that patients infected with the SNP bearing isolate are less likely to die from infection. It is therefore hypothesized that this SNP represents an adaptive mutation that promotes serum survival while decreasing virulence and host mortality. In a murine model of infection, the strain bearing the WT allele of tcaA is significantly more virulent than the tcaA mutant, suggesting that the role of tcaA in bacteremia is infection-phase dependent.

This manuscript has many strengths. The triangulation of genomic analysis, patient outcomes data, and in vitro and in vivo mechanistic testing adds to the significance of the findings in terms of human disease. Testing the impact of mutating tcaA in multiple staphylococcal lineages and backgrounds also increases the rigor of the study. The identification of bacterial loci that impact susceptibility to both host antimicrobial compounds and commonly used antibiotics is also a strength of this work, given the evolutionary and treatment implications for such genes.

One moderate weakness is that the impact of the identified SNP in tcaA is only tested in some of the assays, whereas the majority of the testing is performed with a whole gene knockout. Additionally, for some experiments, rigor is lacking in that statistical measures are not deployed to support the conclusions of biologically meaningful changes based on data with very modest differences between groups. In some cases this results in more speculative conclusions that will require further testing to validate. Finally, there are instances of inter-experiment variability that require further explanation. All in all, this is an exciting manuscript that will be of interest to the broader research communities focused on staphylococcal pathogenesis, bacterial evolution, and host-pathogen interactions, as well as to clinicians who care for patients with invasive staphylococcal infection.

Reviewer #1 (Public Review):

The manuscript by Hussein et al. uses cryoEM structure, microscale thermophoresis (MST), and molecular dynamics simulations (conventional and CpHMD) to unravel the Zn2+ and proton role in the function of the Cation Diffusion Facilitator YiiP. First, they generate mutants that abolish each of the three Zn2+ models to study the role of each of them separately, both structurally and functionally. Next, they used a Monte Carlo approach refining the CpHMD data with the MST points to establish the Zn2+ or proton binding state depending on the pH. That predicted a stoichiometry of one Zn2+ to 2 or 3 protons (1:3 under lower pH values). Finally, they proposed a mechanism that involves first the binding of Zn2+ to one low-affinity site and then, after the Zn2+ migrates to the highest affinity site in the transmembrane portion of the protein. The lack of Zn2+ in the low-affinity site might induce occlusion of the transporter.

The manuscript is well-written it is of interest to the field of Cation Facilitator Transporters. It is also an excellent example of a combination of different techniques to obtain relevant information on the mechanism of action of a transporter.

I have only a few comments that might need clarification from the authors:

- If the unbinding of Zn2+ to site B triggers the occlusion (and maybe the OF state) and the external pH does not affect that binding, how is it prevented from being always bound to Zn2+ and thus occluded also while it should be transporting protons (B to C panels in Figure 5)? Are there some other factors that I am missing?<br /> - I am not an expert on experiments, but the results for mutants that abolish site C are difficult to understand. For D287A/H263A, the SEC columns data suggest a population of higher oligomers. Still, for the D70A/D287A/H263A and D51A/D287A/H263A, they showed a native dimer. I understand your suggestion that the Fab induces the domain swap, but how do you explain the double mutant SEC column result? Please elaborate.<br /> - Since in the D287A mutant, you are disrupting the preferred tetrahedral coordination of Zn2+, but it still binds, do you observe any waters that compensate for the missing aspartate? Maybe in the MD simulations?

Reviewer #1 (Public Review):

Tomasi et al. performed a combination of bioinformatic, next-generation tRNA sequencing experiments to predict the set of tRNA modifications and their corresponding genes in the tRNAs of the pathogenic bacteria Mycobacterium tuberculosis. Long known to be important for translation accuracy and efficiency, tRNA modifications are now emerging as having regulatory roles. However, the basic knowledge of the position and nature of the modifications present in a given organism is very sparse beyond a handful of model organisms. Studies that can generate the tRNA modification maps in different organisms along the tree of life are good starting points for further studies. The focus here on a major human pathogen that is studied by a large community raises the general interest of the study. Finally, deletion of the gene mnmA responsible for the insertion of s2U at position 34 revealed defects in in growth in macrophage but in test tubes suggesting regulatory roles that will warrant further studies. The conclusions of the paper are mostly supported by the data but the partial nature of the bioinformatic analysis and absence of Mass-Spectrometry data make it incomplete. The authors do not take advantage of the Mass spec data that is published for Mycobacterium bovis (PMID: 27834374) to discuss what they find.

Important points to be considered:

1) The authors say they took a list of proteins involved in tRNA modifications from Modomics and added manually a few but we do not know the exact set of proteins that were used to search the M. mycobacterium genome.

2) The absence of mnmGE genes in TB suggested that the xcm5U derivatives are absent. These are present in M. bovis (PMID: 27834374). Are the MnmEG gene found in M. bovis? If yes, then the authors should perform a phylogenetic distribution analysis in the Mycobacterial clade to see when they disappeared. If they are not present in M. bovis then maybe a non-orthologous set of enzymes do the same reaction and then the authors really do not know what modification is present or not at U34 without LC-MS. The exact same argument can be given for the xmo5U derivatives that are also found in M.bovis but not predicted by the authors in M. tuberculosis.

3) Why is the Psi32 predicted by the authors because of the presence of the Rv3300c/Psu9 gene not detected by CMC-treated tRNA seq while the other Psi residues are? Members of this family can modify both rRNA and tRNA. So the presence of the gene does not guarantee the presence of the modification in tRNAs

4) What are tsaBED not essential but tsaC (called sua5 by the authors) essential?

Reviewer #1 (Public Review):

Muller glia function as retinal stem cells in the adult zebrafish retina. Following retinal injury, Muller glia are reprogramned (reactive Muller glia), and then divide to produce a progenitor that amplifies and differentiates into retinal neurons. Previous scRNAseq analysis used total retinal RNA from uninjured and injured retinas isolated at time points when Muller glia are quiescent, being reprogrammed, and proliferating to reveal genes and gene regulatory networks underlying these events (Hoang et al., 2020). The manuscript by Celotto et al., used double transgenic zebrafish that allow them to purify by FACS quiescent and reactive Muller glia, Muller glia-derived progenitors, and their differentiating progeny at different times post retinal damage. RNA from these cell populations was used in scRNAseq studies to identify the transcriptomes associated with these cell populations. Importantly, they report two quiescent and two reactive Muller glia populations. These results raise the interesting possibility that Muller glia are a heterogenous population whose members may exhibit different regenerative responses to retinal injury. However, without further experimentation, the validity and significance of this result remain unclear. In addition to putative Muller cell heterogeneity, Celotto et al., identified multiple progenitor classes, some of which are specified to regenerate specific retinal neuron types. Because of its focus on Muller glia and Muller glia-derived progenitors at mid to late stages of retina regeneration, this new scRNAseq data will be a useful resource to the research community for further interrogation of gene expression changes underlying retina regeneration.

Major concerns:

1) The identification of multiple populations of Muller glia, reactive Muller glia, and progenitors is interesting, but beyond a few in situ hybridization studies to validate injury-dependent gene inductions, there are no experiments that confirm that multiple cell populations exist in vivo, and no experiments examining the significance of these different populations in the regenerative process. It would be helpful to discuss how the peripheral to the central gradient of Muller cell maturation influences the scRNAseq-based cell clustering results.

2) While the reliance on transient GFP and mCherry expression may be sufficient, the final population used for the scRNAseq analysis is only partial in nature. Permanently marking the MG through a Cre-Lox system is more ideal. The authors mention the possibility of missing highly proliferative populations of MG/RPC through the dilution of fluorescent proteins; a transgenic system that allows for true lineage tracing may then capture more appropriate MG/RPC populations. The lack of gating for a pure GFP population also confounds this problem which the authors do mention in the discussions; this oversight was not explained.

3) Much time was taken to identify each cell cluster and to list the differentially expressed genes, but no functional significance for these genes was probed. While a lot of work has gone into the analysis shown, altering some of the MG/RPC trajectories through differentially expressed genes would go a long way to making this study more impactful.

4) The data presented in this paper has significant overlap with scRNAseq data presented by Hoang et al., 2020 in Science where Muller glia, reactive Muller glia, and Muller glia-derived progenitors were carefully analyzed. How does their data fit with the data presented here? The authors could have used that paper as a jumping-off point and offered more time points for comparison, especially as progenitors differentiate.

5) A major conclusion of the paper is that neurogenic progenitors in the injured retina differentiate into neurons with a similar order as that taking place during development. This analysis is based on two time points, and while the trends stay true to the authors' model, two time points are too few to make such a conclusion. In addition, because of the time points chosen for this analysis, many mature neuronal markers are lacking. Including additional time points so mature neuronal markers are detected in the dataset would enhance the trajectory proposed.

Reviewer #1 (Public Review):

Ciampa et al. investigated the role of the hypoxia-inducible factor 1 (HIF-1) pathway in placental aging. They performed transcriptomic analysis of prior data of placental gene expression over serial timepoints throughout gestation in a mouse model and identified increased expression of senescence and HIF-1 pathways and decreased expression of cell cycle and mitochondrial transcripts with advancing gestational age. These findings were confirmed by RT-PCR, Western blot, and mitochondrial assessment from mouse placental tissues from late gestation time points. Studies of human placental samples at similar late gestational ages showed similar trends in increased HIF-1 targets and decreased mitochondrial abundance with increasing gestation, but were not significantly significant due to the limited availability of uncomplicated preterm placenta samples. The authors demonstrated that stabilization of HIF-1 in vitro using primary trophoblasts and choriocarcinoma cell lines recapitulated the gene and mitochondrial dysfunction seen in the placental tissues and were consistent with senescence. Interestingly, cell-conditioned media from HIF-1 stabilized placenta cell lines induced myometrial cell contractions in vitro and correspondingly, induction of HIF-1 in pregnant mice was associated with preterm labor in vivo. These data support the role of the HIF-1 pathway in the process of placental senescence with increasing gestational age and highlight this pathway as a potentially important contributor to gestational length and a potential target for therapeutics to reduce preterm birth.

Overall, the conclusions of this study are mostly well supported by the data. The concept of placental aging has been controversial, with several prior studies with conflicting viewpoints on whether placental aging occurs at all, is a normal process during gestation, or rather only a pathologic phenomenon in abnormal pregnancies. This has been rather difficult to study given the difficulty of obtaining serial placental samples in late gestation. The authors used both a mouse model of serial placental sampling and human placental samples obtained at preterm, but non-pathologic deliveries, which is an impressive accomplishment as it provides insight into a previously poorly understood timepoint of pregnancy. The data clearly demonstrate changes in the HIF-1 pathway and cellular senescence at increasing gestational ages in the third trimester, which is consistent with the process of aging in other tissues.

Weaknesses of this study are that although the authors attribute alterations in HIF-1 pathways in advanced gestation to hypoxia, there are no experiments directly assessing whether the changes in HIF-1 pathways are due to hypoxia in either in vitro or in vivo experiments. HIF-1 has both oxygen-dependent and oxygen-independent regulation, so it is unclear which pathways contribute to placental HIF-1 activity during late gestation, especially since the third-trimester placenta is exposed to significantly higher oxygen levels compared to the early pregnancy environment. Additionally, the placenta is in close proximity to the maternal decidua, which consists of immune and stromal cells, which are also significantly affected by HIF-1. Although the in vitro experimental data in this study demonstrate that HIF-1 induction leads to a placenta senescence phenotype, it is unclear whether the in vivo treatment with HIF-1 induction acts directly on the placenta or rather on uterine myometrium or decidua, which could also contribute to the initiation of preterm labor.

Reviewer #1 (Public Review):

Summary

Favate et al. measure the relative levels of metabolites in 12 Escherichia coli strains isolated from different replicate populations after 50,000 generations of the Lenski long-term laboratory evolution experiment. They use untargeted LC/MS methods that include standards and report both positive and negative ionization mode measurements. They initially use principal component analysis (PCA) to broadly compare how the metabolomes of these strains are similar and different. Then, they describe several instances where the changes in metabolite abundance they see in specific pathways correlate with mutations that lead to changes in the expression of genes that encode enzymes in those pathways.

Strengths

The statistical analyses and presentation of the high-throughput data are excellent. The most compelling results are communicated in wonderful figures that integrate their measurements of metabolite levels in this study with results from a prior study they conducted looking at changes in gene expression levels in the same bacterial strains. These sections include the ones describing large increases in NAD(P) pools due to mutations in nadR, changes in the levels of arginine and related compounds due to mutations in argR, and changes in metabolites from glycolysis and the TCA cycle related to iclR and arcB.

Weaknesses

Showing that A-2 and especially A-3 are outliers in the PCA analysis is useful, but it may be hiding other interesting signals in the data. The other strains are remarkably colinear on these plots, hinting that if the outliers were removed, one main component would emerge along which they are situated. It also seems possible that this additional analysis step would allow the second dimension to better differentiate them in a way that is interesting with respect to their mutator status or mutations in key metabolic or regulatory genes.

There is a missed opportunity to connect some key results to what is known about LTEE mutations that reduce the activity of pykF (pyruvate kinase I). This gene is mutated in all 12 LTEE populations, and often these mutations are frameshifts or transposon insertions that should completely knock out its activity. At first glance, inactivating an enzyme for a step in glycolysis does not make sense when the nutrient source in the growth medium is glucose, even though PykF is only one of two isozymes E. coli encodes for this reaction. There has been speculation that inactivating pykF increases the concentration of phosphoenolpyruvate (PEP) in cells and that this can lead to increased rates of glucose import because PEP is used by the phosphotransferase system of E. coli to import glucose (see https://doi.org/10.1002/bies.20629). The current study has confirmed the higher PEP levels, which is consistent with this model.

In the introduction, the papers cited to show the importance of changes in metabolism for adaptation do not seem to fit the focus of this study very well. They stress production of toxins and secondary metabolites, which do not seem to be mechanisms that are at work in the LTEE. I can think of two areas of background that would be more relevant: (1) studies of how bacterial metabolism evolves in adaptive laboratory evolution (ALE) experiments to optimize metabolic fluxes toward biomass production (for example, https://doi.org/10.1038/nature01149 ), and (2) discussions of how cross-feeding, metabolic niche specialization, and metabolic interdependence evolve in microbial communities, including in other evolution experiments (for example, https://doi.org/10.1073/pnas.0708504105 and https://doi.org/10.1128/mBio.00036-12).

Impact and Significance

While there has been past speculation about the effects of LTEE mutations on metabolism, this study measures changes in the levels of metabolites in related metabolic pathways for the first time. Therefore, it provides useful information about how metabolism evolves, in general, and will also be a useful resource for those studying other aspects of the LTEE related to metabolism, such as contingency in the evolution of citrate utilization.

Reviewer #1 (Public Review):

The study examines how hemocytes control whole-body responses to oxidative stress. Using single cell sequencing they identify several transcriptionally distinct populations of hemocytes, including one subset that show altered immune and stress gene expression. They also find that knockdown of DNA Damage Response (DDR) genes in hemocytes increases expression of the immune cytokine, upd3, and that both upd3 overexpression in hemocytes and hemocyte knockdown of DDR genes leads to increased lethality upon oxidative stress.

Strengths

1, The single cell analyses provide a clear description of how oxidative stress can cause distinct transcriptional changes in different populations of hemocytes. These results add to the emerging them in the field that there functionally different subpopulations of hemocytes that can control organismal responses to stress.<br /> 2, The discovery that DDR genes are required upon oxidative stress to limit cytokine production and lethality provides interesting new insight into the DDR may play non-canonical roles in controlling organismal responses to stress.

Weaknesses

1, In some ways the authors interpretation of the data - as indicated, for example, in the title, summary and model figure - don't quite match their data. From the title and model figure, it seems that the authors suggest that the DDR pathway induces JNK and Upd3 and that the upd3 leads to tissue wasting. However, the data suggest that the DDR actually limits upd3 production and susceptibility to death as suggested by several results:<br /> a) PQ normally doesn't induce upd3 but does lead to glycogen and TAG loss, suggesting that upd3 isn't connected to the PQ-induced wasting.<br /> b) knockdown of DDR upregulates upd3 and leads to increased PQ-induced death. This would suggest that activation of DDR is normally required to limit, rather than serve as the trigger for upd3 production and death.<br /> c) hemocyte knockdown of either JNK activity or upd3 doesn't affect PQ-induced death, suggesting that they don't contribute to oxidative stress-induced death. Its only when DDR is impaired (with DDR gene knockdown) that an increase in upd3 is seen (although no experiments addressed whether JNK was activated or involved in this induction of upd3), suggesting that DDR activation prevents upd3 induction upon oxidative stress.

2, The connections between DDR, JNK and upd3 aren't fully developed. The experiments show that susceptibility to oxidative stress-induced death can be caused by a) knockdown of DDR genes, b) genetic overexpression of upd3, c) genetic activation of JNK. But whether these effects are all related and reflect a linear pathway requires a little more work. For example, one prediction of the proposed model is that the increased susceptibility to oxidative stress-induced death in the hemocyte DDR gene knockdowns would be suppressed (perhaps partially) by simultaneous knockdown of upd3 and/or JNK. These types of epistasis experiments would strengthen the model and the paper.

3, The (potential) connections between DDR/JNK/UPD3 and the oxidative stress effects on depletion of nutrient (lipids and glycogen) stores was also not fully developed. However, it may be the case that, in this paper, the authors just want to speculate that the effects of hemocyte DDR/upd3 manipulation on viability upon oxidative stress involve changes in nutrient stores.

Reviewer #1 (Public Review):

In this study, Muronova et al., demonstrate the physiological importance of a centriole and microtubule-associated protein, CCDC146, in sperm flagellar formation and male reproduction. In a previous study, the authors identified two loss-of-function mutations in CCDC146 from the sterile males with multiple morphological abnormalities in flagellar (MMAF) phenotype. To further test physiological significance of the CCDC146, the authors generate its knockout mouse model. The knockout males share the MMAF phenotypes with severely impaired flagellar morphology due to the defective sperm generation in testes. Using CCDC146 knock-in mouse model and expansion microscopy techniques, the authors observed CCDC146 localizes at human and mouse sperm flagella, which is different from the somatic cells. The authors also observed impaired manchette and head-tail coupling apparatus in developing spermatid lacking CCDC146 and address CCDC146 loss-of-function induces molecular and structural defects at axoneme in developing male germ cells, which finally causes MMAF phenotype and male infertility.

This reviewer agrees that identifying and analyzing new pathogenic molecules and variants is hugely valuable to establish male infertility in genetic level. As the authors have done, this study also enlarges the genetic causality underlying MMAF and male infertility. In addition, this study applies new techniques, expansion microscopy, which is also an innovative approach. Although many approaches are used, unfortunately, this study misses the molecular mechanisms to explain pathogenicity to cause MMAF by the CCDC146. Only intracellular localization of the molecule is heavily examined. Although the authors show defective intracellular localization of the centriole and manchette, how CCDC146 loss-of-function and the developmental defects are linked is not examined. These limits provide the impression that this study could be simply another identification of the MMAF-causing gene, which were heavily performed by the authors. Also, in many parts, the results do not clearly support the authors claim. Therefore, this reviewer thinks the current manuscript requires additional results to clearly explain molecular mechanisms underlying the pathogenicity by CCDC146 loss-of-function.

Reviewer #1 (Public Review):

This research article by Watabe T and colleagues characterizes PKA waves triggered by prostaglandin E2 (PGE2). What the author discovered is that waves of PKA occur both in vitro, in MDCK epithelial monolayers, and in vivo, in the ear epidermis in mice. The PKA waves are the consequence PGE2 discharge, that in turn is triggered by Calcium bursts. Calcium level and ERK activity intensity control that mechanism by acting at different levels.

This article is a technological tour de force using different biosensors and optogenetic actuators. What makes this article interesting is the combination of these tools together to dissect a complex, highly dynamic signaling pathway at the single-cell level. For this reason, this paper represents the essence of modern cell biology and paves the way for the cell biology of the future. However, we think that the paper in this stage is still partly descriptive in its nature, and more measurements are needed to increase the strength of the mechanistic insights. Also, the work is not conclusive, some results are over-interpreted, and more work has to be done if the authors want to support all their claims.

Reviewer #1 (Public Review):

The manuscript by Huang et al. examines the potential "self-policing" of Bacillus cells within a biofilm. The authors first discover the co-regulation of lethal extracellular toxins (BAs) and the self-immunity mechanisms; the global regulator spoA controls both. The authors further show that a subpopulation of cells co-express these genes and speculate that these cells engage in preferential cooperation for biofilm formation (over cells that produce neither). Based on previous literature, the authors then evaluate the relative fitness of the wild-type strain compared to mutants locked into either constantly exporting the toxins or permanently immune to these poisons. The wild-type exhibited increased fitness (compared to the mutants) for the tested biofilm conditions. The manuscript raises interesting ideas and provides a potential model to probe questions of cooperatively in Bacillus biofilms.

Strengths:<br /> - The authors use fluorescence-producing reporter strains to discern the spatial expression patterns within biofilms. This real-time imaging provides striking confirmation of their conclusions about shared co-regulation.<br /> - The authors also nicely deploy genetic constructs in microbiological assays to show how toxin production and immunity can influence biofilm phenotypes, including resilience to stress.

Concerns:<br /> - My biggest concern is that the claim of policing on a single-cell level needs more quantitive microscopy, particularly of the xylose-induced strain. The data support a more tempered consideration of self-policing via BAs and self-resistance in this Bacillus species. It seems sufficient that this manuscript opens the door for a novel and readily examinable system for examining potential cooperation and its molecular controls (without making broader claims).<br /> - The discussion is more speculative than the presented data warrants. For example, the speculation in lines 289 - 310 is not anchored in the results. It is hard for this reviewer to imagine how one would use the genetic framework and tools developed in this manuscript to address the ideas proposed in lines 289 - 310.<br /> - Some conclusions (in the results section) are more decisive than the data supports. For example, the microscopy of the PI staining (as presented in Figure 2 and the supplemental movies) does not prove that only non-expressing cells die. Yet the conclusion in line 143 states that "ECM and BAs producers selectively punish the nonproducing siblings." Also, the presented data shows many non-labeled cells without PI; why do some nearby non-gfp-expressing cells remain alive?

Reviewer #1 (Public Review):

Zacharopoulos et al. present a multi-modal investigation into the developmental trajectories of cognitive processing, decision-making processing, and visuomotor processing in children and young adults, and attempt to relate them to neuroimaging measures of functional brain connectivity and neurotransmitter concentrations in two distinct brain regions.

Results suggest specific interactions between neurotransmitter concentrations and visuomotor task performance. Interestingly, GABA and Glu levels appear to have different relationships with task performance if the participant group is trichotomized into older, 'mean-age', and younger participants. These findings appear consistent across three different visuomotor processing tasks and replicate well between two time points at which task performance and MRS measures were established for each participant (1.5 years apart). Visuomotor connectivity (assessed with resting-state-fMRI) also showed age-group-specific relationships with neurotransmitter levels. Finally, the authors present evidence that visuomotor processing mediates the relationship between neurochemical levels and scores of fluid intelligence, but only for older participants.

STRENGTHS

The study has an astonishing sample size in the context of MRS research, a field that has historically struggled to aggregate large datasets because of a severe lack of methodological standardization. Longitudinal MRS data from close to 300 participants means that this is one of the largest MRS datasets to date, enabling the group to add another exciting piece of work to their six previously published manuscripts on relationships between cognitive performance and neurochemical measures from this powerful resource. MRS data quality appears excellent, owed to state-of-the-art acquisition and raw data processing. The authors are further to be commended for making the raw MRS data publicly available - they will serve as a fantastic resource for method developers and applied researchers in the field.

WEAKNESSES

There is generally little to no consideration or discussion concerning age trajectories of MRS-derived metabolite estimates during childhood and early adulthood, which are not clearly established at all. There is evidence for increasing GABA+macromolecules during childhood (Porges et al, eLife 2021, https://elifesciences.org/articles/62575), although it may be ascribed to macromolecules rather than GABA itself (Bell et al, Sci Rep 2021, https://pubmed.ncbi.nlm.nih.gov/33436899/). The findings should at least be discussed in the context of this literature, but I suggest going a step further. The authors have all the data to make a major contribution to the scarce body of evidence on metabolite changes between 6 and 18 years by examining whether GABA and Glu estimates actually appear to change systematically across the age range of their dataset (especially exciting since they have longitudinal data)! It would be immensely valuable to see an analysis like this.

With that said, a methodological weakness concerns the computation of neurochemical concentrations presented here. Firstly, the authors can provide more detail about the acquisition and data processing/modeling decisions. Secondly, and more importantly, MRS-derived estimates of concentration can never be absolute, and always require several assumptions about the relative contributions of tissue classes (GM, WM, CSF) to the measurement volume, tissue water content, water and metabolite MR relaxation times, MR visibility, etc. Quantitative MRS estimates therefore need to be interpreted with caution, especially when these confounding factors are likely to vary between observed groups, or with age, pathology, etc. - there is plenty of reason to assume that cortical maturation, iron accumulation, etc. contribute to changes in relative GM/WM/CSF fractions or relaxation time changes. The authors present two different correction methods to account for some of these aspects, but only present the results of one, stating that "The results showed the same general pattern across all quantification methods.", which is insufficient to assess what changed and what didn't. Interestingly, the authors have presented no less than *four* different quantification methods in a similar manuscript using the same dataset (Zacharopoulos et al, Human Brain Mapp 2021; https://onlinelibrary.wiley.com/doi/10.1002/hbm.25396), but they do not mention normalization to the internal creatine signal in this present work, or whether it yielded different results (which might indicate that their method of tissue correction introduces a confounder rather than correcting for it). There is no mention of whether any further analysis of the water T2 relaxation time estimates was performed, but it would be vital to understand whether they themselves change with age, since this would establish that they are likely to confound GABA and Glu estimation. Generally, the choice to perform additional subject-specific acquisitions to allow corrections for water T2 relaxation is understandable, but not clearly motivated or explained in the experimental section. The authors should further clarify whether the relative tissue volume fractions of GM, WM, and CSF are stable across the age range, or whether there is a systematic tissue composition change with age that may also confound the Glu and GABA estimation.

Finally, I am surprised to find no discussion of limitations at all. It is important to point out the methodological limitations of MRS, which are widely discussed in the MRS literature, but probably less obvious to those readers less intimately familiar with it. This concerns not only the confounding factors for quantification that I described above but also the challenges of the comparably low spectral resolution at 3 Tesla. Even with high-quality data as presented here, it remains unclear whether the small GABA signal can be reliably separated from glutamate, glutamine, and glutathione, all of which exhibit substantial spectral overlap with each other and other strong signals as well as the underlying macromolecular background. The limitations (and how they impact interpretation) ought to be mentioned and discussed in the context of the vast amount of literature. They should provide the reader with the appropriate context and the awareness that all MRS measures are extremely sensitive to many different experimental factors and modeling decisions.

Reviewer #1 (Public Review):

The authors show that TrafE, which is one of the five Dictyostelium discoideum TRAF proteins, is recruited to the Mycobacterium-containing vacuoles (MCVs) and is required for membrane damage repair and xenophagy. They propose that the TrafE-Ub-ALIX axis is important for the regulation of Vps4, and, thereby, for the normal function of ESCRT. They also suggest that TrafE is involved in phagophore sealing.

Overall, the parts of membrane damage repair and xenophagy induction are convincing. Although mammalian TRAF6 was already reported to be involved in the ubiquitination of Chlamydia and Toxoplasma-containing vacuoles (Haldar et al. PNAS, 2015, https://www.pnas.org/doi/epdf/10.1073/pnas.1515966112), how TRAF6 is recruited to pathogen-containing vacuoles remained unknown. This study reveals that the recruitment of TrafE to MCVs is dependent on membrane damage or reduced membrane tension. This is novel. However, the part of phagophore closure is too preliminary. The evidence that TrafE is involved in the phagophore closure is mostly indirect and weak.

Reviewer #1 (Public Review):

This manuscript presents evidence for Kv3 subunits being involved in shaping fast action potentials (APs) within the high-precision circuitry of the zebra finch song circuitry. The authors compare and contrast the morphology of Robustus Arcopallialis (RA) neurons with those in the adjacent intermediate arcopallium (AId) and compare their passive properties, action potential waveforms, and voltage-gated outward currents. Data using pharmacological agents known to interact with Kv3 channels reinforce their other observations.

Strengths:<br /> 1. Interesting avian model of cortical molecular mechanisms.<br /> 2. Comparative study at the level of cortical motoneurons showing those involved in fine motor control for vocalizations express high levels of Kv3.1.<br /> 3. Makes a case for convergent evolutionary utilization of Kv3.1 supporting fast spiking.<br /> 4. Clearly shows other Kv3 subunits are present in the nuclei under study.<br /> 5. Employs well-characterised pharmacological tools to support the physiology.

Weaknesses:<br /> 1. Comparison with Betz Cells comes across as of secondary importance and is perhaps a discussion point rather than the first introductory paragraph.<br /> 2. Fails to adequately quantify the absolute levels of Kv3 mRNA or protein in the zebra finch brain nuclei.<br /> 3. The comparison of % or fold differences between the two avian nuclei (RA and AId neuron) masks important quantitative evidence and the contribution of multiple subunits to functional channels is not well developed.<br /> 4. The voltage-clamp data suggests that the large TEA-sensitive current is too slow to dominantly contribute to the repolarization of a single AP (but would require sustained or cumulative depolarization to be activated), while the fast transient current which could contribute to single APs, is not sufficiently characterised.<br /> 5. It is not possible to conclude that the pharmacology is specific for Kv3.1, it is at best indicative, and the absence of more precise molecular tools (e.g. knockout or gene-edited animals) undermines the authors' justification of the zebra finch as an accessible model.<br /> 6. Although the authors acknowledge the presence of other Kv3 subunits, the report fails to explain whether they are functional, but focuses on Kv3.1 as being dominant, without sufficiently addressing how other subunits contribute (perhaps as heteromeric assemblies of subunits).

Reviewer #1 (Public Review):

This manuscript reports on a rapid and precise CRISPR/Cas9-mediated knock-in approach in the African turquoise killifish, an emerging vertebrate animal and gerontology model. More specifically, it describes an easily adoptable method to efficiently insert fluorescent reporters of different sizes at various genomic loci and to drive cell-type- and tissue-specific expression. This methodology will allow the development of humanized disease models and of cell-type specific molecular probes to study complex vertebrate biology, including aging biology, in the killifish. While this knock-in methodology is already widely used in common vertebrate animal models, the efficient generation of stable lines with germline transmission has been missing in killifish. As killifish have the shortest generation time of vertebrate animal models in laboratory conditions, show a rapid sexual maturity, and a short lifespan, the established method enables the generation of stable lines of homozygous transgenic vertebrate animals in 2-3 months. Overall, we believe this first report on efficient long (1.8kb) construct knock-in using CRISPR/Cas9 in the killifish establishes the killifish as a system for precise genetic engineering at scale, which has been challenging so far in vertebrates.

The establishment of this methodology will have a major impact in the field and be of extreme use within the scientific community. It will allow the development of scalable human disease models and integrate both genetics and age as risk factors, thus having the potential to identify future therapeutic targets for age-related diseases. It also has a generic character as the generated protocol can serve as a template for knock-in approaches in other emerging model organisms.

Although the reported data are of major interest and relevance to the scientific field, they are, as yet not sufficiently shown in convincing figures. The methodology is state-of-the-art and entails an extensive set of molecular, biochemical, and morphological/imaging technologies. While most of the data are nicely presented and accompanied by illustrative figures, the manuscript would benefit from the inclusion of a more detailed material and methods section, and a little more elaboration on morphometrical expression data in the results section, e.g, expression shown for all the studied genes in the larval fish, and a more critical discussion, that also highlights a few of the limitations, e.g., those related to the fast generation of homozygous F1 fish.

Reviewer #1 (Public Review):

The underlying principle of the experimental system described here is to test potential candidate genes that intersect with the proteotoxic-induced UPR by screening an siRNA pool that diminishes the UPR transcription reporter activated by sec-11 RNAi-mediated ER proteotoxic stress. The authors specifically focused on genes reported to play roles in LD biology, instead of general lipid synthesis genes. Systematic evaluation of the LD genes with respect to the induction of the UPR provides important insights into the overall functions and mechanisms of the UPR.

Using this set-up, the authors identified the hydroxysteroid dehydrogenase gene let-767/HSD17B12. Subsequent analyses revealed that let-767-mediated signaling is a key component that establishes the orchestration of both ER lipid and protein homeostasis and ER organismal functions, including ER lipid storage and ER structural changes. In addition, the authors found that acs-1i, knockdown of a gene involved in metabolism of lipids such as LCFA and mmBCFA, also diminished UPRE-GFP levels induced by sec-11i, albeit to a lesser extent than let-767i. Supplementation of lipid metabolites such as LCFA and mmBCFA recovered not only the sec-11-induced UPRE-GFP reporter phenotypes in acs-1i worms, but also the ER size and morphology and the LD and body sizes.

In contrast, the UPRE-GFP reporter phenotype in let-767i worms was not recovered by exogenously added LCFA or mmBCFA, although it was recovered by spb-1 RNAi, knockdown of a major lipogenic enzyme/pathway. The system established by the authors allowed them to quantitatively dissect the involvement of the Ire1-Xbp1 splicing UPR signaling branch. Finally, the authors demonstrated similar effects in mammalian tissue culture cells, suggesting conservation of the mechanisms.

The conclusions of this manuscript are generally in agreement with the data and the authors' interpretations are reasonable. However, at this point, the work remains descriptive and does not provide a mechanistic understanding. Overall contributions/advances towards providing new insights into how the UPR pathway is wired with respect to lipid-associated perturbations remain somewhat limited.

Reviewer #1 (Public Review):

The work in this study builds on previous studies by some of the same authors and aims to test whether the heartbeat evoked response was modulated by the local/global auditory regularities and whether this differed in post-comatose patients with different contagiousness diagnosis. The authors report that during the global effect there were differences between the MCS and UWS patients.

The study is well constructed and analysed and has data from 148 participants (although the maximum in anyone group was 59). The reporting of the results is excellent and the conclusions are supported by the results presented. This study and the results presented are discussed as evidence that EEG based techniques maybe a low cost diagnostic tool for consciousness in post-comatose patients, although it should be stressed that here no classification of diagnostics was performed on the EEG data.

One potential weakness was the relationship between the design of the experiment and the analysis pathway for the results. If I have understood correctly the experimental design the auditory regularity changed on whether the local/global regularity was standard/deviant. In the analysis the differences between all conditions in which the local or global regularity were compared between the standard and deviant trials. This difference was then compared between MCS and UWS patient groups. For these analyses the results for the health and emerging MCS were not included. If this is correct it would be interesting to understand the motivation for this. Relatedly, it would be good to clarify if the effects reported were corrected for the multiple planned contrasts and if not why they should not be corrected.

Reviewer #1 (Public Review):

Kazrin appears to be implicated in many diverse cellular functions, and accordingly, localizes to many subcellular sites. Exactly what it does is unclear. The authors perform a fairly detailed analysis of Kazrin in-cell function, and find that it is important for the perinuclear localization of TfN, and that it binds to members of the AP-1 complex (e.g., gamma-adaptin). The authors note that the C-terminus of Kazrin (which is predicted to be intrinsically disordered) forms punctate structures in the cytoplasm that colocalize with components of the endosomal machinery. Finally, the authors employ co-immunoprecipitation assays to show that both N and C-termini of Kazrin interacts with dynactin, and the dynein light-intermediate chain.

Much of the data presented in the manuscript are of fairly high quality and describe a potentially novel function for Kazrin C. However, I had a few issues with some of the language used throughout, the manner of data presentation, and some of their interpretations. Most notably, I think in its current form, the manuscript does not strongly support the authors' main conclusion: that Kazrin is a dynein-dynactin adaptor, as stated in their title. Without more direct support for this function, the authors need to soften their language. Specific points are listed below.

Major comments:<br /> 1) I agree with the authors that the data provided in the manuscript suggest that Kazrin may indeed be an endosomal adaptor for dynein-dynactin. However, without more direct evidence to support this notion, the authors need to soften their language stating as much. For example, the title as stated would need to be changed, as would much of the language in the first paragraph of the discussion. Alternatively, the manuscript could be significantly strengthened if the authors performed a more direct assay to test this idea. For example, the authors could use methods employed previously (e.g., McKenney et al., Science 2014) to this end. In brief, the authors can simply use their recombinant Kazrin C (with a GFP) to pull out dynein-dynactin from cell extracts and perform single molecule assays as previously described.<br /> 2) I'm not sure I agree with the use of the term 'condensates' used throughout the manuscript to describe the cytoplasmic Kazrin foci. 'Condensates' is a very specific term that is used to describe membraneless organelles. Given the presumed association of Kazrin with membrane-bound compartments, I think it's more reasonable to assume these foci are quite distinct from condensates.<br /> 3) The authors note the localization of Tfn as perinuclear. Although I agree the localization pattern in the kazKO cells is indeed distinct, it does not appear perinuclear to me. It might be useful to stain for a centrosomal marker (such as pericentrin, used in Figure 5B) to assess Tfn/EEA1 with respect to MT minus ends.<br /> 4) "Treatment with the microtubule depolymerizing drug nocodazole disrupted the perinuclear localization of GFP-kazrin C, as well as the concomitant perinuclear accumulation of EE (Fig. 5C & D), indicating that EEs and GFP-kazrin C localization at the pericentrosomal region required minus end-directed microtubule-dependent transport, mostly affected by the dynactin/dynein complex (Flores-Rodriguez et al., 2011)."<br /> - I don't agree that the nocodazole experiment indicates that minus end-directed motility is required for this perinuclear localization. In the absence of other experiments, it simply indicates that microtubules are required. It might, however, "suggest" the involvement of dynein. The same is true for the subsequent sentence ("Our observations indicated that kazrin C can be transported in and out of the pericentriolar region along microtubule tracks...").<br /> 5) Although I see a few examples of directed motion of Tfn foci in the supplemental movies, it would be more useful to see the kymographs used for quantitation (and noted by the authors on line 272). Also related to this analysis, by "centripetal trajectories", I assume the authors are referring to those moving in a retrograde manner. If so, it would be more consistent with common vernacular (and thus more clear to readers) to use 'retrograde' transport.<br /> 6) The error bars on most of the plots appear to be extremely small, especially in light of the accompanying data used for quantitation. The authors state that they used SEM instead of SD, but their reasoning is not stated. All the former does is lead to an artificial reduction in the real deviation (by dividing SD by the square root of whatever they define as 'n', which isn't clear to me) of the data which I find to be misleading and very non-representative of biological data. For example, the error bars for cell migration speed in Figure 2B suggest that the speeds for WT cells ranged from ~1.7-1.9 µm/sec, which I'm assuming is largely underrepresenting the range of values. Although I'm not a statistician, as someone that studies biochemical and biological processes, I strongly urge the authors to use plots and error bars that more accurately describe the data to your readers (e.g., scatter plots with standard deviation are the most transparent way to display data).

Reviewer #1 (Public Review):

Nicotine preference is highly variable between individuals. The paper by Mondoloni et al. provided some insight into the potential link between IPN nAchR heterogeneity with male nicotine preference behavior. They scored mice using the amount of nicotine consumption, as well as the rats' preference of the drug using a two-bottle choice experiment. An interesting heterogeneity in nicotine-drinking profiles was observed in adult male mice, with about half of the mice ceasing nicotine consumption at high concentrations. They observed a negative association of nicotine intake with nicotine-evoked currents in the antiparticle nucleus (IPN). They also identified beta4-containing nicotine acetylcholine receptors, which exhibit an association with nicotine aversion. The behavioral differentiation of av vs. n-avs and identification of IPN variability, both in behavioral and electrophysiological aspects, add an important candidate for analyzing individual behavior in addiction.

The native existence of beta4-nAchR heterogeneity is an important premise that supports the molecules to be the candidate substrate of variabilities. However, only knockout and re-expression models were used, which is insufficient to mimic the physiological state that leads to variability in nicotine preference.

Reviewer #1 (Public Review):

Cerebellar parallel fiber to Purkinje cell synapses display multiple forms of long-term plasticity, expressed in both presynaptic and postsynaptic compartments. At this synapse, a prominent form of presynaptic LTP was once thought to operate through cAMP-dependent activation of PKA, and subsequent phosphorylation of RIM1a. However, recent studies have questioned this hypothesis. LTP is not blocked by selective inhibitors of PKA, or by mutations in Rim1a designed to block PKA-dependent serine phosphorylation. In this study, Wang and colleagues use a wide array of pharmacology and genetics to elucidate a potential signaling cascade for presynaptic LTP in parallel fibers, where cAMP activates EPAC, leading to PKCε-dependent phosphorylation of RIM1α. Presynaptic ablation of either EPAC or PKCε leads to loss of presynaptic LTP and forskolin-induced potentiation. The experiments are generally well conceived and executed. The findings provide a new framework for understanding how presynaptic cAMP elevations can alter vesicle release machinery and drive synaptic plasticity, and open new avenues for exploration at synapses throughout the CNS. The manuscript could be improved by better a more transparent citation of previous studies and a more open discussion of the unknown steps in the newly-elucidated signaling cascade.

Reviewer #1 (Public Review):

This paper uses light field microscopy to measure calcium signals across the fly brain while it is walking and turning, and also while the fly is externally driven to walk and turn, using a treadmill. The authors drive calcium indicator expression using pan-neuronal drivers, as well as drivers specific to individual neurotransmitters and neuromodulators. From their experiments, the authors show that inhibitory and excitatory neurons in the brain are activated in similar patterns by walking and that neurons expressing machinery for different neuromodulatory amines tend to show differentially strong calcium signals during walking. By examining spontaneous and forced walking and turning, the authors identify brain regions that activate before spontaneous turning and that activate asymmetrically in concert with spontaneous or forced turning.

Strengths: Overall, the strength of this paper is in its careful descriptions and analyses of whole brain activation patterns that correlate with spontaneous and forced behaviors. Showing how the pattern of activity relates to broad classes of cells is also useful for understanding brain activation. Especially in brain regions identified as preceding spontaneous walking and in being asymmetrically involved in spontaneous and forced turning, it provides a wealth of potential hypotheses for new experiments. Overall, it contributes to a coarse-grained understanding of broad changes in brain activity during behavior.

Weaknesses: The primary weakness of this paper is that it presents some speculative interpretations and conclusions too strongly. Most importantly, average activity in a neuropil can represent the calcium activity of hundreds or thousands of neurons, and it is hard to know what fraction is active, for instance, or how expression pattern differences might play into calcium signals. Calcium signals also do not reliably indicate hyperpolarization, so a net increase in the average Ca++ indicator signal does not necessarily reflect that the average neuron is becoming more active, just that some labeled neurons are becoming more active, while others may be inactive or hyperpolarized. The conclusions about regions triggering walk (rather than just preceding it) are too strong for the manipulations in this paper, as are some of the links with individual neuron types. Thus, more presenting substantial caveats is required for the conclusions being drawn from the data presented here.

DOI: 10.1016/j.celrep.2023.112365

Resource: RRID:ZFIN_ZDB-ALT-071016-1

Curator: @scibot

SciCrunch record: RRID:ZFIN_ZDB-ALT-071016-1

What is this?

The space and the dual space is having the same dimension.

Linear functonal maps from a vector space to the real space.

If a vector is bounded, and we put this vector into the linear operator, then the output vector from the space would be bounded by the norm too.

Joint Public Review:

This paper presents two new tools for investigating GLP-1 signaling. The genetically encoded sensor GLPLight1 follows the plan for other GPCR-based fluorescent sensors, inserting a circularly permuted GFP into an intracellular loop of the GPCR. The light-uncaged agonist peptide, photo-GLP1, has no detectable agonist activity (as judged by the GLPLight1 sensor) until it is activated by light. However, based on the current characterization, it is unclear how useful either of these tools will be for investigating native GLP-1 signaling.

The GLPLight1 sensor has a strong fluorescent response to GLP-1 with an EC50 of ~10 nM, and its specificity is high, as shown by lack of response to ligands of related class B GPCRs. However, the native GLP1R enables biological responses to concentrations that are ~1000-fold lower than this (as shown, for instance, in a supplemental figure of this paper). This makes it difficult to see how the sensor will be useful for in vivo detection of GLP-1 release, as claimed; although there may be biological situations where the concentration is adequate to stimulate the sensor, this is not established. Data using a GLP-1 secreting cell line suggest that the sensor has bound some of the released GLP-1, but it is difficult to have confidence without seeing an actual fluorescence response to stimulated release.

Alternatively, the sensor might be used for drug screening, but it is unclear that this would be an improvement over existing high-throughput methods using the cAMP response to GLP1R activation (since those are much more sensitive and also allow detection of signaling through different downstream pathways).

The utility of the caged agonist PhotoGLP1 is similarly unclear. The data demonstrate a substantial antagonism of GLP-1 binding by the still-caged compound, and it is therefore unclear whether the kinetics of the response to PhotoGLP1 itself would mimic the normal activation by GLP-1 in the absence of the caged compound. A further concern is that the light-dependence of the agonist effect of PhotoGLP1 was evaluated only with the GLPLight1 sensor and not with GLP1R signaling itself, which is 1000x more sensitive and which would be the presumed target of the tool. In addition, PhotoGLP1 is based upon native GLP-1, which is rapidly truncated and inactivated by the peptidase DPPIV, expressed in most cell types, and expressed at very high levels in the plasma. The utility of PhotoGLP1 is therefore limited to acute (minutes) in vitro experiments.

Reviewer #1 (Public Review):

Using a combination of structural biology methods, this report aims at describing the auto-inhibited architecture of kinesin 1 either as homodimers or hetero-tetramers. Hence, the multiple contacts between the protein domains and their folding pattern is addressed using cross-linking mass spectrometry (XL-MS), negative stain electron microscopy and Alpha Fold based structure prediction. Based on the existing literature, the key domains and amino acids responsible for kinesin 1 inhibited state were not clearly deciphered. The synergetic use of different methods now seems to describe in detail the molecular cues which could induce kinesin-1 refolding and opening. Multiple interactions between the different domains seem to induce the folded conformation.

The combination of methodologies is an efficient way to unravel details that could not be addressed previously. The paper is well written. However, the methodology is sometimes not sufficiently detailed and the paper would benefit from additional explanations and demonstrations. The methods for generating the electron microscopy data and its relevance and quality, for instance, are barely described. In addition, the conclusions drawn would be more convincing if similar investigations would be carried out similarly for all isoforms (KIF5B and FIF5C) in parallel.

This article raises the potential strength and power of deep learning structure prediction methods combined simultaneously with other structural biology methods to answer specific questions. In the present context, this study will certainly be helpful to reveal and understand the activation mechanism of kinesin motor proteins.

Reviewer #1 (Public Review):

This work is a follow-up of the work from the same group where the authors showed that Lactiplantibacillus plantarum can enhance juvenile growth by activating the expression of an intestinal protease. They previously showed that this process was mediated by the dlt operon which is involved in the D-Alanylation of teichoic acid.

In the present study, the authors characterized the structure and enzymatic activity of the first protein encoded by this operon and show that the first gene of this operon encodes for an esterase releasing D-Ala from D-Ala lipoteichoic acids (LTA) and renamed it here DltE. The gene encoding this protein was previously uncharacterized and annotated as a peptidoglycan-binding protein putatively involved in peptidoglycan maturation. With the structure and enzymatic characterization of this protein, this study revealed that this protein does not act as peptidoglycan, but instead releases D-Ala from D-alanylated-LTA.

The authors use a Drosophila mutant impaired in response to mDAP-Peptidoglycan fragments (affected in the IMD pathway) to show that this mutant still responds to D-Ala-LTAs. This result is important to show that D-Ala-LTAs act as additional cues sensed by Drosophila independent of m-DAP-peptidoglycan by a still unknown sensory pathway. The study convincingly shows that D-Ala-LTA from the gut microbe L. plantarum leads to increase intestinal peptidase expression (intestinal activity) and enhance juvenile larva growth.

Reviewer #1 (Public Review):

This manuscript by Walker et. al. explores the interplay between the global regulators HapR (the QS master high cell density (HDC) regulator) and CRP. Using ChIP-Seq, the authors find that at several sites, the HapR and CRP binding sites overlap. A detailed exploration of the murPQ promoter finds that CRP binding promotes HapR binding, which leads to repression of murPQ. The authors have a comprehensive set of experiments that paints a nice story providing a mechanistic explanation for converging global regulation. I did feel there are some weak points though, in particular the lack of integration of previously identified transcription start sites, the lack of replication (at least replication presented in the manuscript) for many figures, some oddities in the growth curve, and not reexamining their HapR/CRP cooperative binding model in vivo using ChIP-Seq.

Reviewer #1 (Public Review):

Germe and colleagues have investigated the mode of action of bacterial DNA gyrase, a tetrameric GyrA2GyrB2 complex that catalyses ATP-dependent DNA supercoiling. The accepted mechanism is that the enzyme passes a DNA segment through a reversible double-stranded DNA break formed by two catalytic Tyr residues-one from each GyrA subunit. The present study sought to understand an intriguing earlier observation that gyrase with a single catalytic tyrosine that cleaves a single strand of DNA, nonetheless has DNA supercoiling activity, a finding that led to the suggestion that gyrase acts via a nicking closing mechanism. Germe et al used bacterial co-expression to make the wild-type and mutant heterodimeric BA(fused). A complexes with only one catalytic tyrosine. Whether the Tyr mutation was on the A side or BA fusion side, both complexes plus GyrB reconstituted fluoroquinolone-stabilised double-stranded DNA cleavage and DNA supercoiling. This indicates that the preparations of these complexes sustain double strand DNA passage. Of possible explanations, contamination of heterodimeric complexes or GyrB with GyrA dimers was ruled out by the meticulous prior analysis of the proteins on native Page gels, by analytical gel filtration and by mass photometry. Involvement of an alternative nucleophile on the Tyr-mutated protein was ruled unlikely by mutagenesis studies focused on the catalytic ArgTyrThr triad of residues. Instead, results of the present study favour a third explanation wherein double-strand DNA breakage arises as a consequence of subunit (or interface/domain) exchange. The authors showed that although subunits in the GyrA dimer were thought to be tightly associated, addition of GyrB to heterodimers with one catalytic tyrosine stimulates rapid DNA-dependent subunit or interface exchange to generate complexes with two catalytic tyrosines capable of double-stranded DNA breakage. Subunit exchange between complexes is facilitated by DNA bending and wrapping by gyrase, by the ability of both GyrA and GyrB to form higher order aggregates and by dense packing of gyrase complexes on DNA. By addressing a puzzling paradox, this study provides support for the accepted double strand break (strand passage) mechanism of gyrase and opens new insights on subunit exchange that may have biological significance in promoting DNA recombination and genome evolution.

The conclusions of the work are mostly well supported by the experimental data.

Strengths:

The study examines a fundamental biological question, namely the mechanism of DNA gyrase, an essential and ubiquitous enzyme in bacteria, and the target of fluoroquinolone antimicrobial agents.

The experiments have been carefully done and the analysis of their outcomes is comprehensive, thoughtful and considered.

The work uses an array of complementary techniques to characterize preparations of GyrA, GyrB and various gyrase complexes. In this regard, mass photometry seems particularly useful. Analysis reveals that purified GyrA and GyrB can each form multimeric complexes and highlights the complexities involved in investigating the gyrase system.

The various possible explanations for the double-strand DNA breakage by gyrase heterodimers with a single catalytic tyrosine are considered and addressed by appropriate experiments.

The study highlights the potential biological importance of interactions between gyrase complexes through domain-or subunit-exchange

Weaknesses:

The mutagenesis experiments described do not fully eliminate the perhaps unlikely participation of an alternative nucleophile.

Joint Public Review

The molecular composition of synaptic vesicles (SVs) has been defined in substantial detail, but the function of many SV-resident proteins are still unknown. The present study focused on one such protein, the 'orphan' SV-resident transporter SLC6A17. By utilizing sophisticated and extensive mouse genetics and behavioral experiments, the authors provide convincing support for the notion that certain SLC6A17 variants cause intellectual disability (ID) in humans carrying such genetic variations. This is an important and novel finding. Furthermore, the authors propose, based on LC-MS analyses of isolated SVs, that SLC6A17 is responsible for glutamine (Gln) transport into SVs, leading to the provocative idea that Gln functions as a neurotransmitter and that deficits in Gln transport into SVs by SLC6A17 represents a key pathogenetic mechanism in human ID patients carrying variants of the SLC6A17 gene.

This latter aspect of the present paper is not adequately supported by the experimental evidence so that the main conceptual claims of the study appear insufficiently justified at this juncture. Key weaknesses are as follows:

A. Detection of Gln, along with classical neurotransmitters such as glutamate, GABA, or ACh, in isolated SV fractions does not prove that Gln is transported into SVs by active transport. Gln is quite abundant in extracellular compartments. Its appearance in SV samples can therefore also be explained by trapping in SVs during endocytosis, presence in other - contaminating - organelles, binding to membrane surfaces, and other processes. Direct assays of Gln uptake into SVs, which have the potential to stringently test key postulates of the authors, are lacking.

B. The authors generated multiple potentially very useful genetic tools and models. However, the validation of these models is incomplete. Most importantly, it remains unclear whether the different mutations affect SLC6A17 expression levels, subcellular localization, or the expression and trafficking of other SV and synapse components.

C. Apart from the caveats mentioned above regarding Gln uptake into SVs, the data interpretation provided by the authors lacks stringency with respect to the biophysics of plasma membrane and SV transporters.

Reviewer #1 (Public Review):

Abdellahi et al. used targeted memory reactivation (TMR) and machine learning tools to look for evidence that waking neural activity is reinstated during subsequent REM sleep. Prior work has demonstrated that learning content is successfully decoded following TMR cues during NREM sleep, but a direct link between patterns of brain activity recorded during wakefulness and subsequent REM sleep in humans has never been reported. In this paper, the authors report that an LDA classifier detects wake-like neural activity (specifically, neural activity recorded while imaging performing a trained serial reaction time task) approximately one second after TMR cues are presented during REM sleep. Decoding performance is better when the classifier is trained on sleep trials with high theta compared to low theta power, and classifier performance was correlated with overnight improvement on the task.

Finding evidence of reinstated waking neural activity during REM sleep is an exciting result, and the authors present a promising method that holds implications for advancing our understanding of how memories are reprocessed during REM sleep. I think it is a particular strength of the paper that the authors trained on sleep data and tested in wake data, which is analogous to prior rodent studies that found evidence of replay during REM. I also thought playing sounds during the adaptation night, prior to SRTT training, provided a nice control.

The conclusions of this paper are mostly supported by the results presented, but it is not always clear how those results were obtained. Some aspects of the experimental and data analytic methods need to be clarified and expanded, both for a better understanding of how the results of this study were obtained, as well as for future reproducibility.

Reviewer #1 (Public Review):

Most previous studies investigating the phenomenon of crowding in depth use small stereoscopic differences in depth. Taken together their results suggest that a depth difference between target and flankers reduces crowding. A potential problem is that stereo displays can reduce depth perception. The studies that have used a real-depth display have provided some inconsistent findings. The present study investigated larger differences, representative of those among many objects in the real world. These larger differences increased crowding, even in the absence of diplopia (double vision).

This study is likely to be impactful in the field as it shows that crowding occurs in-depth and strengthens the importance of crowding in natural 3D environments. All existing models of crowding would need to be modified to explain this experimental finding.

The novel multi-depth plane display that the authors used enables measurements of depth differences that are more likely to correspond to differences in the real world, and could be used by others to further investigate crowding in-depth or other perceptual processes (e.g., visual search).

In general, there are some interactions that were reported and others that were not reported, but it would be important to know if they are significant. (pages 15-16) For example, when the target is at fixation and the target is at a variable flanker depth: In Experiment 1, was there a significant interaction between (a) target-fixation depth and flanker depth (in front versus behind) and (b) target-fixation depth and target-flanker spacing? In Experiment 3, it is reported that perceptual error was higher when the target was in from or behind the flanker ring and fixation and that the greatest perceptual error occurred when the target was behind, but it is not reported if this interaction was significant. Its presence is important to know whether the data should be independently analyzed for 'in front' and 'behind'. In Experiment 5, was the interaction between target-flanker spacing and depth significant?

The findings are clear but the explanation(s) for the findings is not. The authors state that large interocular disparity differences likely induce diplopia, which could increase perceptual error by increasing the number of features. The authors should explain what they mean by features and how an increased perceived number of features would increase crowding. Moreover, the authors acknowledge that only a few observers reported experiencing diplopia; however, they speculate that observers may have experienced diplopia but not noticed it consciously given the short stimulus presentation time.

Reviewer #1 (Public Review):

Pelentritou and colleagues investigated the brain's ability to infer temporal regularities in sleep. To do so, they measured the effect on brain and cardiac activity to the omission of an expected sound. Participants were presented with three different categories of sounds: fixed sound-to-sound intervals (isochronous), fixed heartbeat-to-sound intervals (synchronous), and a control condition without any regularity (asynchronous). When omitting a sound, they observed a difference in the isochronous and synchronous conditions compared to the control condition, in both wakefulness and sleep (NREM stage 2). Furthermore, in the synchronous condition, sounds were temporally associated with sleep slow waves suggesting that temporal predictions could influence ongoing brain dynamics in sleep. Finally, at the level of cardiac activity, the synchronous condition was associated with a deceleration of cardiac frequency across vigilance states. Overall, this work suggests that the sleeping brain can learn temporal expectations and responds to their violation.

Major strengths and weaknesses:<br /> The paradigm is elegant and robust. It represents a clever way to investigate an important question: whether the sleeping brain can form and maintain predictions during sleep. Previous studies have so far highlighted the lack of evidence for predictive processes during sleep (e.g. (Makov et al., 2017; Strauss et al., 2015; Wilf et al., 2016)). This work shows that at least a certain type of prediction still takes place during sleep.

However, there are some important aspects of the methodology and interpretations that appear problematic.<br /> (1) The methodology and how it compares to previous articles would need to be clarified. For example, the Methods section indicates that the authors used a right earlobe electrode as a reference. This is quite different from the nose reference used by SanMiguel et al. (2013) or in Dercksen et al. (2022). This could affect the polarity and topographies of the OEP or AEP and thus represents a very significant difference. Likewise, SOs are typically detected in a montage reference to the mastoids. Perhaps the left/right asymmetries present in many plots (e.g. Figure 3) could be due to the right earlobe reference used. Also, the authors did not use the same filters in wakefulness and sleep, which could introduce an important bias when comparing sleep and wake results or sleep results with previous wake papers.<br /> (2) The ERP to sound omission shows significant differences between the isochronous and asynchronous conditions in wakefulness (Figure 3A and Supp. Fig.) but this difference is very different from previous reports in wakefulness. Topographies are also markedly different, which questions whether the same phenomenon is observed. For example, SanMiguel and colleagues observed an N1 in response to omitted but expected sounds. The authors argue that they observe a similar phenomenon in the iso vs baseline contrast, but the timing and topography of their effect are very different from the typical N1. The authors also mention that, within their study, wake and N2 OEPs were "largely similar" but they differ in terms of latencies and topographies (Figure 3A-B). It would be better to have a more objective way to explore differences and similarities across the different analyses of the paper or with the literature.<br /> (3) The authors applied a cluster permutation to identify clusters of significant time points. However, some aspects of this analysis are puzzling. Indeed, the authors restricted the cluster permutation to a temporal window of 0 to 350ms in wake (vs. -100 to 500ms in sleep). This can be misleading since the graphs show a larger temporal window (-100 to 500ms). Consequently, portions of this time window could show no cluster because the analysis revealed an absence of significant clusters but because the cluster permutation was not applied there. Besides, some of the reported clusters are extremely brief (e.g. l. 195, cluster's duration: 62ms), which could question their physiological relevance or raise the possibility that some of these clusters could be false positives (there was no correction for multiple comparisons across the many cluster permutations performed). Finally, there seems to be a duplication of the bar graphs showing the number of significant electrodes in the positive and first negative cluster for Figure 2 Supp. Fig. 1.<br /> (4) More generally, regarding statistics, the absence of exact p-values can render the interpretation of statistical outputs difficult. For example, the authors report a significant modulation of the sound-to-SO latency across conditions (p<0.05) but no significant effect of heartbeat peak-to-SO latency (p>0.05). They interpret this pattern of results rather strongly as evidence that the "readjustment of SOs was specific to auditory regularities and not to cardiac input". Yet, examining the reported chi-square values show very close values between the two analyses (7.9 vs. 7.4). It seems thus difficult to argue for a real dissociation between the two effects. Providing exact p-values for all statistical tests could help avoid this pitfall.

Reviewer #1 (Public Review):

Kozol et al adapt an important tool, in the form of the atlas, to the Astyanax research community. While broadly the atlas appears to correctly identify large brain regions, it is unclear what is the significance of the finer divisions. The external confirmations are restricted to just a few large brain regions (by independent human observer: e.g., optic tectum, hypothalamus. By molecular marker: hypothalamus only.). As such, interpretations of results from as many as 180 small subregions should be interpreted sceptically.<br /> The authors also suggest that some brain regions have increased in size during cavefish evolution (e.g., hypothalamus, subpallium). The analysis of progeny from a genetic cross of cave and surface morphs suggest a complex genetic program has evolved to control this variant set of brain structures. With the development of genetic manipulation tools in this species, an exciting series of experiments may link causal variants with brain development differences.

MAJOR ISSUES<br /> Line 85+. Segmentation accuracy is not well established by the authors.<br /> For example, Figure S2 states that the pixel correlation is high between Astyanax populations. But the details of how this cross-correlation was done are sparse. Is the Y-axis here showing the fraction of pixels that are shared in the morphs? While the annotation appears to function similarly across morphs, the 80% machine:human correlation is difficult to put into context. On the one hand, this seems low. For what values should one strive? Are there common "mistakes" or differences in human & machine annotations that lead to certain regions being excluded? A discussion of these is warranted and will be useful to others who wish to use this approach.

Line 87. "such as" is misleading since these were the only two antibodies used to confirm molecular definitions of regions.<br /> But more to the point, additional markers should be used to confirm more than just the ISL+ hypothalamic divisions.<br /> This is particularly warranted, as Fig 1d is not convincing. I believe that the yellow label is ISL; this is difficult to see in the figures. ISL is not ideal since this is widespread in the hypothalamus. There are no ISL-negative regions depicted, which would be necessary to demonstrate that the resolution of this subregion labeling tool is high. A complementary approach would be to find molecular markers that are more restricted than ISL which label only subsets of hypothalamic regions.<br /> Finally, do the mid/hindbrain ISL labeled regions correspond to known ISL+ subregions?

The molecular and human-observed confirmations of brain regions suggests that the annotated borders of gross anatomical regions are correctly identified by the algorithm. However, data is not presented that indicates whether the smaller regions correspond to biologically meaningful compartments.

Parameters used in CobraZ to perform the segmentation are not defined. More transparency is required here for others to replicate.

Reviewer #1 (Public Review):

The authors of this manuscript aimed to systematically evaluate the pleiotropic effects of MCR-1-mediated colistin resistance. They evaluated the effect of MCR-1 and MCR-3 carried on different plasmids on antimicrobial peptides (AMPs) and assessed their ultimate effect on virulence. The authors find that MCR-1-mediated colistin resistance correlates with increased resistance against some host AMPs, but also increased sensitivity to others. The authors also find that MCR-1 alone is associated with resistance to human serum and to elements of the complement system. This highlights a potential selective advantage for MCR-1-mediated resistance to host immune factors and a potential for enhanced virulence.

The methods have been well established before and adequately support their main findings. While determining the role of MCR-1 in a single genetic background is important to better understand its potential pleiotropic effects against a diversity of AMPs and in a variety of scenarios, the impact and significance of the results are partially ameliorated because different genetic backgrounds, particularly those most relevant to a clinical (or agricultural) context were not considered. The results depicted here are still a necessary and important step towards a more comprehensive understanding of the pleiotropic effects of MCR-1. But, interactions between plasmids and host genomes and their co-evolution can have important effects more generally. The authors do mention this in the discussion and suggest it to be an important avenue for future work. However, given the objective of the study and the clinical and agricultural context in which the authors have framed their work, it seems more relevant to include those distinct genetic backgrounds already here.

The conclusions stemming from the results found in Figure 3, and Figures 4c and d seem too overreaching to me. The associated resistance to AMPs from pigs seems to be only strong enough against one of the five tested AMPs and hence concluding that these impose a strong selective pressure in the pig's gut seems unsubstantiated. Similarly, the difference in survival probability within their in vivo system, though statistically significant, seems to be very ild between their MCR-1 and empty vector control.

Reviewer #1 (Public Review):

The adhesion of Leishmania promastigotes to the stomodeal valve in the anterior region of the sandfly vector midgut is thought to be important to facilitate the transmission of the parasites by bite. The promastigote form found in attachment is termed a 'haptomonad', although its adhesion mechanism and role in facilitating transmission have not been well studied. Using 3D EM techniques, the paper provides detailed new information pertaining to the adhesion mechanism. Electron tomography was especially useful to reveal the ultrastructure of the attachment plaque and the extensive remodelling of the flagellum that occurs. A few of the attached haptomonads were found to be in division, which is a novel observation. The attachment of cultured promastigotes to plastic and glass surfaces in vitro was found to involve a similar remodeling of the flagellum and was exploited to image the sequential steps in attachment, flagellar remodeling, and haptomonad differentiation. The in vitro attachment was found to be calcium2+ dependent. Based mainly on the in vitro observations, a sound model of the haptomonad attachment plaque and differentiation process is provided.

Reviewer #1 (Public Review):

In this manuscript, Sampaio et al. tackle the role of fluid flow during left-right axis symmetry breaking. The left-right axis is broken in the left-right organiser (LRO) where cilia motility generates a directional flow that permit to dictate the left from the right embryonic side. By manipulating the fluid moved by cilia in zebrafish, the authors conclude that key symmetry breaking event occurs within 1 hour through a mechanosensory process.

Overall, while the study undeniably represents a huge amount of work, the conclusions are not sufficiently backed up by the experiments. Furthermore, the results provided present a limited advance to the field: the transient activity of the LRO is well established, and narrowing down this activity to 1 hour (even though unclear from the presented data that it is a valid conclusion) does not help to understand better the mechanism of symmetry breaking. Importantly, the authors do not provide any convincing experiments to back up the mechanosensory hypothesis because the fluid extraction experiments affect both the chemical and physical features of the LRO, so it is impossible to disentangle the two with this approach.

Reviewer #1 (Public Review):

In this manuscript, Lee and colleagues address the participation of NBR1 in chloroplast clearance after treatment with high light intensity. Authors use NBR1 fused to reporter proteins (GFP, mCherry), with the aid of nbr1, atg7, and nbr1-atg7 mutants, in combination with immunogold labelling to show localization of NBR1 to surface and interior of photodamaged chloroplasts, which follows with their engulfment in the vacuole, a process which is independent of ATG7. The combined use of ATG8 fused to GFP further shows that NBR1 and ATG8 are recruited independently to photodamaged chloroplasts. In addition, the use of mutant versions of NBR1 in combination with mutants lacking E3 ligases PUB4 and SP1 and mutant toc132-2 and tic40-4 lacking members of the TIC-TOC complex of protein translocation to the chloroplast, authors show that chloroplast localization of NBR1 requires the ubiquitin ligase domain (UBA2) of the protein, whereas, the PB1 domain exerts a negative effect on NBR1 chloroplast association, yet neither the PUB4 and SP1 E3 ligases nor the TOC-TIC are required for NBR1 association to photodamaged chloroplasts. All these approaches are well described and strongly support the authors' conclusions that the loss of chloroplast envelope integrity allows the entrance of cytosolic ubiquitin ligases and the participation of NBR1 in photodamaged chloroplast clearance by a process of microautophagy. All these findings add valuable information to our knowledge of chloroplast homeostasis in response to light stress.

To further support these conclusions, authors perform a chloroplast proteomic analysis of the WT, nbr1, atg7, and nbr1-atg7 mutants. However, in contrast with the above results, the description of the proteomic data is rather confusing. The paragraph on Page 17 (lines 393-406) is hard to follow. The term "over-representation of less abundant chloroplast protein" is also quite confusing, like the data in Fig. 6 and supplementary to this figure (what does show the PCA analysis in Fig. 6-suppl. 1?). I wonder whether it would be possible to show all these data as supplementary and try to present the data supporting the major conclusion of these analyses (if I understood correctly, that nbr1, atg7, and the double mutant have lower contents of chloroplast proteins), in a more simple and clear format.

Reviewer #1 (Public Review):

The manuscript entitled "Pooled genome-wide CRISPRa screening for rapamycin resistance gene in Drosophila cells" by Xia et al. is a well-structured piece of work with clear objectives and experiments. The authors successfully demonstrated genome-wide gene activation using CRISPRa using a novel sgRNA design, which overcame previous failed attempts to replicate gene activation that worked well in mammalian systems. The study is detailed and highly relevant for the application of CRISPRa in understanding the molecular mechanism of gene candidates.

Reviewer #1 (Public Review):

The model put forward by the authors in this manuscript is a simple and exciting one, explaining the function of AGS3 as a negative regulator of LGN, acting as a 'dominant-negative' version of LGN. Overall, the results support the model very well, and the results shown in Fig 6, which clearly reveal the functional relevance of AGS3, add strength to the paper.

In Figures 3A and B, the authors claim that AGS3 overexpression leads to depolarization of LGN in epidermal stem cells. However, in the example provided in Figure 3A, the LGN signal appears to be stronger than the control, with more LGN still on the apical side (many would categorize this as 'apically polarized'). In the scoring shown in Figure 3B, I am not sure if 'eyeballing' is the right way to decide whether it is polarized/depolarized/absent. The authors should come up with a bit more quantitative method to quantify the localization/amount of LGN and explain the method well in the manuscript. A similar concern regarding the determination of the LGN localization pattern applies to the rest of figure 3 as well.

Reviewer #1 (Public Review):

Previously the authors showed that ERK3 plays a critical role in the production of IL-8, immune cell chemotaxis, and metastasis (Bogucka et al, eLife 2020). This is a follow-up study on these observations in which they uncover a critical role for ERK3 in the activation of RhoGTPases, formation of actin-rich protrusions, and actin polymerization. Previous publications have reported a critical role of ERK3 in regulating cell morphology and migration. However, the molecular mechanisms responsible for these phenotypes remain elusive. The polarized phenotype of motile cells involves complex actin cytoskeleton re-arrangements, and in this study, the authors demonstrate a direct role for MAPK6 kinase in regulating actin dynamics.

First, the authors confirm that loss of ERK3 negatively affects MDA-MB231 cell motility and migration, both in vitro and in vivo. Interestingly loss of ERK3 reduced F-actin content in primary breast mammary epithelial cells. The authors used a multi-disciplinary approach to elucidate the underlying mechanisms. Using biochemical methods, they elegantly show the direct link between ERK3 and RhoGTPases as well as the ARP2/3 complex. Furthermore, direct binding of ERK3 to Rac1, Cdc42, and Arp2/3 complex is shown by biochemical assays, and these observations are validated by monitoring the interaction between ERK3 and the Cdc42/ARP2/3 complex in cells at endogenous levels. The finding that ERK3 acts as a GEF for Cdc42 and not Rac1 is interesting and further links this kinase to PAKs. PAK kinases have been shown to phosphorylate ERK3 at Ser 189 in the SEG motif to activate ERK3 (Deleris et al JBC,2011). Overall, this study generated a lot of interesting data and the work has been well-executed and properly interpreted. The main findings are novel and important, and they are of particular interest to readers in the fields of cell migration and actin dynamics. This manuscript is also likely to stimulate additional investigations using biophysical and structural methods to further decipher GEF activity controls ERK3.

Reviewer #1 (Public Review):

This paper by Zhuang and colleagues seeks to answer an important clinical question by trying to come up with novel predictive biomarkers to predict high-risk T1 colorectal cancers that are at risk for nodal involvement. The current clinical features may both miss patients who underwent local therapy and who should have gone on to have surgery and patients for whom surgery was done based on risk features but perhaps unnecessarily. Using a training and validation set, they developed a protein-based classifier with an AUC of 0.825 based on mass spec analyses and proteomic analyses of patients with and without LN importantly linking biological rationale to the proteomic discoveries.

In the training cohort, they took 105 candidate proteins reduced to 55, and did a validation in the training cohort first and then in two validation cohorts (one of which was prospective). They also looked at a 9 protein classifier which also performed well and furthermore looked at IHC for clinical ease.

Reviewer #1 (Public Review):

This paper presents a systematic and novel examination of how pupil size relates to BOLD fMRI signal in a set of subcortical nuclei. It provides some important novel findings that should help advance understanding of how pupil size relates to activity in subcortical nuclei as well as providing important advances in how to measure these relationships.

The authors first tried replicating prior findings of a relationship between pupil size and BOLD signal using the prior methods. They could not (despite replicating pupil-cortical region relationships), and so tested whether the delay in the hemodynamic response function might differ in subcortical and cortical regions. They found that BOLD signal in the subcortical nuclei showed associations with pupil size at short delays. This is a critical finding as typical fMRI analyses assume a longer delay and so likely obscure the ability to see effects in these subcortical regions. The authors provide a number of helpful 'control' analyses that help strengthen confidence in their findings. For instance, it buttresses their findings that the pons control region did not show any significant effect to time-to-peak on correlations with pupil size or derivative measures. It also is helpful to know that pupil size fluctuations were associated with cortical activity in the regions expected from prior studies. The rigor of the study is also supported by the fact that there was a preregistration and that data are publicly shared.

Reviewer #1 (Public Review):

The manuscript by Mansur et al examines the roles of KLHL40, mutations which lead to the development of skeletal muscle disease (nemaline myopathy, NM). The authors use CRISPR-based gene editing in a model organism (zebrafish) to disrupt the two fish isoforms of KLHL40 (a and b) and examine the resulting phenotypes. The authors find that disease-like phenotypes develop in adulthood selectively with the deletion of the KLHL40a isoform. Phenotypes include reduced body size, reduced endurance, and reduced life span with cellular effects that include perturbations to sarcomere organization, perturbed morphology of secretory organelles and mitochondria, and defects in collagen secretion and ECM deposition. The system provided the advantage of following both development and pre-disease state (onset) allowing the authors to look at changes in translation but mainly in the proteome with a focus on ubiquitylation (followed by mass spectrometry). Selective changes to the proteomthe e in KLHL40a deletion mutant are evident in the pre-symptomatic stage. Pathway analysis suggests that mutant cells show selective increases in glycolytic and biosynthetic enzymes/pathways, perhaps, akin to a Warburg effect. Monitoring the correlation between loss of KLHL40a-dependent ubiquitylation and increased protein levels defined the small GTPase Sar1a as a direct target for KLHL40a-directed degradation. Sar1a interacts with KLHL40a and is ubiquitylated by Cul3-KLHL40 in cell-free and over-expression assays in mammalian cells. Overexpression of Sar1a in muscle leads to endoplasmic reticulum (ER) membrane tubulation and thickening of the Z-lines similar to ones showing in KHLH40a deletion and NM patients. Markedly elevated levels of Sar1a and defects in collagen secretion are also recorded in patients with KLHL40 mutations. These observations suggest that selective control of COPII coat protein Sar1a levels (and thus the activity of the COPII coat, which mediates biosynthetic secretion from the ER), perturbs collagen secretion and ECM deposition. Overall this comprehensive work delineates the roles of Cul3-KLHL40a in the development of NM and specifically in regulating secretion by controlling the levels of one component of the COPII coat. The work is very interesting yet requires additional experimental clarifications and analysis.

Strength

This is a very interesting study showing global developmental and disease onset-related changes to the proteome focusing on changes derived from KLHL40a deletion. The work demonstrates a key role of ubiquitylation and selective protein degradation in the development and muscle disease onset. The global proteome view identified changes to energy production modes and defined direct regulation of Sar1a levels by Cul3-KHLH40a ubiquitylation which regulates ECM secretion, providing a mechanistic explanation for the development of NM in patients with KLHL40 mutations. Furthermore, the study highlights an interesting mechanism in which the levels of an individual component of the COPII coat are controlled by degradation to regulate biosynthetic secretion from the ER.

Weaknesses

There are weaknesses in the analysis that would markedly benefit from added clarifications. The differential outcome with the deletion of klhl40 a and b requires explanation. Morphological observations, which are key to understanding the overall phenotypes of KLHL40a deletion should be developed to provide a better definition of effects on organelle morphology and in particular ones involved in secretion. Some of the transcriptome-proteome data are left unexplored, in particular a view of the unfolded protein response (UPR) within the data, which will complement the documented defects in protein secretion and provide intrinsic controls to the work. The findings on Sar1a and the role of controlled degradation in regulating COPII activities are highly interesting yet a more complete analysis of COPII components is missing. Information on Sar1b, previously implicated in selective effects on secretion, Sec23-Sec24 and ratio (where levels are regulated by ubiquitylation and de ubiquitylation), and outer layer COPII proteins Sec13 and in particular Sec31, which is by itself a target for Cul3-KLHL12 regulation during development and modifies selective biosynthetic secretion, is lacking. Added analysis can provide new perspectives on the potential broader implications and significance of this study.

Reviewer #1 (Public Review):

The authors aimed to study the contribution of bacterial factors to poor treatment outcomes in drug-susceptible TB, an important issue that has not been well studied. The authors performed GWAS on a very large population-based (3 sites in China) dataset of 3416 Mtb WGS data of pre-treatment isolates linked with clinical data to predict treatment outcomes. Logistic regression was used to assess the association between predictors and outcomes and ROC curves were generated to assess the value of the genomic signatures to predict poor TB treatment outcomes. The authors were successful in identifying 14 Mtb variants in 13 genes and reactive oxygen species that were more likely to occur in patients with poor treatment outcomes.

The investigators were very thorough, in investigating both fixed and unfixed mutations, and analyzing the changes in gene expression under stress (exposure to first-line drugs and hypoxic conditions) for the 13 genes identified, which further strengthened the evidence generated by GWAS. The authors attempted to perform an external validation of their findings but could not identify a suitable existing dataset.

These data can be used by others to guide their analyses, and confirm if these 13 genes are also found in other settings. If confirmed, then the results could open the possibility for individualised tailoring of treatment of drug-susceptible TB, especially to prevent the risk of relapse.

Reviewer #1 (Public Review):

This study sought to establish a model of targeted lung endothelial ablation and subsequently study the regeneration process post-ablation using single-cell RNA-sequencing in order to identify key subpopulations and underlying mechanisms of regeneration.

Strengths of the study include:

1. The elegance of the DT endothelial ablation model which leverages local lung instillation of DT to locally ablate the endothelium and cause significant lung vascular leakiness while keeping the endothelium of other organs intact, as is convincingly demonstrated in Fig 1 and Fig 2.

2. The temporal analyses using scRNA-seq demonstrate key shifts in endothelial and non-endothelial cell populations following endothelial injury. These experiments identify a highly proliferative subpopulation of endothelial cells that expresses the transcription factor FoxM1 during the regeneration phase.

3. The authors discover that the traditionally designated "gCap" lung endothelial population contains additional subpopulations that have regenerative potential and that there is a transient expression of apelin in the regenerative population. Pharmacological inhibition of the apelin receptor increase mortality.

Potential weaknesses include:

1. The description of the "stem-like" nature of endothelial cells is not experimentally proven. "Stem-like" is a vague term and the usage of this term is primarily based on the expression of Procr. However, that itself does not justify the usage of "stem-like" unless there is more clear evidence of what "stem-like" properties these cells have, such as multipotency.

2. The intriguing finding of the proliferative EC population raises the question as to how these cells emerge. Do they have a specific subpopulation/cluster origin in the baseline lung endothelium, and was Apelin expression both necessary as well as sufficient to induce the switch to the proliferative state? Such mechanistic analyses would be very helpful in understanding the coordination of the lung endothelial regeneration program.

3. The authors mention that endothelial ablation also induces shifts in the numbers of other cell types such as epithelial cells, alveolar macrophages, and immune cells but there is no analysis beyond the quantification of the cells. Are these cells involved in the regeneration of the endothelium by providing ligands such as growth factors?

Reviewer #1 (Public Review):

Lammer et al. examined the effects of social loneliness, and longitudinal change in social loneliness, on cognitive and brain aging. In a large sample longitudinal dataset, the authors found that both baseline loneliness and an increase in loneliness at follow-up were significantly associated with smaller hippocampal volume, reduced cortical thickness, and worse cognition in healthy older adults. In addition, those older adults with high loneliness at baseline showed even smaller hippocampal volume at follow-up. These results are interesting in identifying the importance of social support to cognitive and brain health in old age. With a longitudinal design, they were able to show that increased loneliness was related to reduced brain structural measures. Such results could help guide clinicians and policymakers in designing social support systems that would benefit the growing aging population.

The strength of the current study lies in the large sample size and longitudinal follow-up design. The multilevel models used to separate within and between subject effects are well constructed. Combining neuroimaging data with behavioral changes provided further evidence that social loneliness may be related to accelerated brain aging. Stringent FDR correction, Bayes factor comparison, and the additional analyses for sensitivity showed the robustness and credibility of the results.

Weaknesses of the study were related to the interpretation and discussion of their findings.

Social loneliness is a relatively little-studied factor in cognitive ageing, and the authors should consider expanding the discussion, with some additional analyses, as to how their results could be used by clinicians and older adults to monitor social behaviors.

The authors examined the interaction between baseline and age change to see if higher baseline loneliness was associated with accelerated decline. The interaction was significant, but the authors did not further explore the interaction effect, which may have clinical significance. The authors should consider identifying a cut-off point in LSNS that suggests persons scoring less than this score on the LSNS may be at greater risk of accelerated brain decline than others. Such a cut-off point is important for clinicians, as well as for future researchers to compare their results.

Although it was not directly tested in the paper, LSNS scores did not seem to change with increasing age (Table 1). This general stability of LSNS scores in older adults should be discussed further. The authors should consider how their relatively healthy and high SES sample may be less vulnerable to loss of family or friends in old age, making this sample sub-optimal for the question they have. The significance of the subject effect suggests that some individuals still experience a loss of social connectedness. The authors may want to elaborate on this and give some explanations of such subject differences in the ageing effect on social loneliness. Although stress was not a significant mediating factor, is it related to baseline loneliness or changes in loneliness in the current sample?

The presentation of longitudinal data (Figure 1) lacks dimensionality. The scatter plots presented here are more suitable for cross-sectional studies and could cause confusion regarding the interpretation of the results. The authors should consider individual growth curves or spaghetti plots in visualizing change within subjects.

Reviewer #1 (Public Review):

Muscle is a major insulin-responsive tissue for the disposal of glucose, a process dependent on the translocation of GLUT4 glucose transporter from intracellular compartments to the plasma membrane. Knudsen and co-workers provide an analysis of the impact of microtubule-based movement on GLUT4 biology in muscle cell lines, and rodent and human muscle fibers ex vivo. A role for microtubules in the control of GLUT4 vesicle dynamics in both unstimulated and insulin-stimulated adipocytes (cultured and primary) has been previously reported by a number of groups. Less is known about the requirement for microtubules for GLUT4 translocation in muscle. A strength of this study is that key aspects of the work were performed in muscle fibers rather than muscle cell lines.

Conclusions that are strongly supported by the data presented include:

1. Demonstration of constitutive GLUT4 movement along microtubule tracks in both unstimulated and insulin-stimulated muscle fibers. GLUT4 dynamics in unstimulated fibers were captured by fluorescence recover after photobleaching (FRAP) and by quantifying vesicle movements by live cell microscopy, whereas in insulin-stimulated cells GLUT4 dynamics were captured by following the movements of GLUT4-containing vesicles. These data support a model in which intracellular GLUT4 is dynamic in both unstimulated and insulin-stimulated muscle fibers rather than being static in unstimulated conditions and only mobilized upon insulin-stimulation.

2. Similar microscopy analyses of GLUT4-containing vesicles demonstrate that depolymerization of microtubules reduced GLUT4 vesicle movement and impacted insulin-stimulated glucose uptake. Short term depolymerization of microtubules (5 min) did not affect insulin-stimulated glucose uptake, whereas insulin-stimulated glucose uptake was blocked after prolonged depolymerization (2 hrs). The use of a muscle on a chip method to monitor glucose uptake in real time was critical for these experiments.

The changes in glucose uptake were accompanied by changes in the morphologies of intracellular GLUT4-containing structures. The differences between short and long term depolymerization of microtubules support a model in which GLUT4 can be translocated to the plasma membrane by insulin stimulation in the absence of microtubules but an intact microtubule cytoskeleton is required to maintain GLUT4 in a "compartment" that can be recruited by insulin. Stated another way, the microtubule-dependent dynamics of GLUT4-containing vesicles in unstimulated cells is permissive for insulin-stimulated GLUT4 translocation.

3. Knockdown of the microtubule motor protein, Kif5b, blunts insulin-stimulated translocation of GLUT4 to the plasma membrane of cultured muscle cells. These findings agree with previously demonstrated role for Kif5b in adipocytes.

4. In an in vitro model of insulin resistance (incubation of muscle fibers with short chain C2 ceramide) unstimulated and insulin-stimulated GLUT4-containing vesicle movement was blunted and unstimulated and insulin-stimulated microtubule polymerization was reduced.

Weakness of the study include:

1. There are no data supporting a role for insulin regulation of microtubule-dependent GLUT4-containg vesicle movement. The data in Fig.2B do not support a differences in the number of "moving" GLUT4 vesicles between basal and insulin-stimulated fibers. The statement on line 103 that they "observed a ~16% but insignificant increase" to be confusing. These data do not support an effect of insulin on the number of moving GLUT4 vesicles that can be detected in an individual experiment. There is also effect of insulin on GLUT4 vesicles in the data reported in Fig.S2D, Fig.S5B, and Fig.S5F. However, the data in Fig. 2C suggest there was a consistent increase in "moving" vesicles in insulin-stimulated conditions in 4 independent experiments (how are these data normalized?). Because the basis of insulin-regulation of glucose uptake is the control of GLUT4 translocation to the plasma membrane, the authors need to clarify their thinking on why they do not detect insulin robust effects on GLUT4 dynamics in the individual experiments. Is it that they are not measuring the correct parameter? That the assay is not sensitive to the changes?

The small (or no effect) of insulin distracts a bit from the findings that there is microtubule-dependent GLUT4 movement in basal and stimulated muscle fibers, and that disruption of this movement by depolymerization of microtubules or Kif5b knockdown blunts GLUT4 translocation. As noted above, the data strongly support microtubule-dependent GLUT4 dynamics as permissive for insulin-stimulated GLUT4 translocation even if this dynamics might not be a target of insulin action.

2. The analyses of GLUT4-containing structures are not particularly informative. Co-localization with other markers (beyond syntaxin6) are needed to understand these structures. Defining structures as small, medium or large is incomplete. In particular, it is important to probe the microtubule nucleation site clusters for other membrane markers. Transferrin receptor? IRAP?

3. The Kinesore data do not support the authors hypothesis. The data show that Kinesore increases the amount of GLUT4 in the plasma membrane of basal cells and that insulin further increases plasma membrane GLUT4 to the same extent as it does in control cells. How does that provide insight into the role microtubules (or kif5b) in GLUT4 biology? Why does Kinesore increase plasma membrane GLUT4? Is it an effect of Kinesin 1 on GLUT4 vesicles? Kinesore is reported to remodel the microtubule cytoskeleton by a mechanism dependent on Kinesin 1. Is that the reason for the change in GLUT4?

4. The analysis of Kif5b is a bit cursory. Depolymerization of microtubules in muscle fibers essentially blocks all GLUT4 movement (only the insulin condition is shown in Fig.2B but I assume basal would be equally inhibited), and fully inhibits insulin-stimulated glucose uptake in muscle fibers. What are the effects of nocodazole in L6 cells (cell used for kif5b studies) and is it similar in magnitude to kif5b knockdown? Those data would identify there are non-Kif5b microtubule-dependent effects.

5. The authors need to show that the fibers isolated from the HFD mice remain insulin-resistant ex vivo by measuring glucose uptake. It is possible that once removed from the mice they "revert" to normal insulin-sensitivity, which might contribute to the differences reported in Fig5.

6. Although it is interesting that the authors have included the insulin-resistance models/experiments, they are not well developed and therefore the conclusions are not particularly strong.

7. The data do not support the title.

Reviewer #1 (Public Review):

The authors study the control of the timing of Q neuroblast migration, through the precisely timed expression of the Wnt receptor MIG-1/Frizzled, which halts migration of the QR.pa cell at its intended position. Understanding the underlying mechanism is important, as similar mechanisms might play a role in controlling the timing of biological processes in development much more broadly. The authors use precise measurements of mig-1 mRNA molecules, fitted to mathematical models of different mechanisms to control the timing of mig-1 expression, and couple this with experimental perturbations of mig-1 expression. In this way, the authors convincingly show that mig-1 dynamics is best explained by a model where mig-1 expression is controlled by the accumulation of an activator, rather than the degradation of a repressor, which is an important result. In addition, they show that the asymmetric division of QR.p into the larger QR.pa and smaller QR.pp cells is important for proper mig-1 expression in Qr.pa, likely by asymmetric inheritance of the activator. In the process, the authors identify novel conserved binding motifs that are responsible for different aspects of mig-1 dynamics, which will potentially allow identifying the putative activator in the future.

In its current form, I find the manuscript has two main weak points: First, the connection between the experiments and models is relatively weak. Now, the model is mostly used to aid the interpretation of experiments, by predicting rough trends. However, even though the model is in principle fitted to the experimental data in some cases, a detailed comparison between experimental results and the model is often lacking. For example, there are multiple occasions where the data appears to not fit the model in some aspects, but the potential origin of these mismatches is typically not discussed. Second, the authors present experimental evidence of an earlier model prediction, that positive feedback loops in mig-1 expression reduce variability in timing. Here, the authors speculate that this feedback loop might be due to the activation of mig-1 expression by mig-1-induced Wnt signaling, which in itself is an interesting idea. However, the genetic perturbation used here - manipulation of the Wnt pathway, rather than perturbing specifically the induction of mig-1 expression by Wnt signaling - likely changes the expression of many genes in the cell, making it difficult to establish whether the increased variability in Qr.pa position is indeed due breaking the proposed feedback loop.

Reviewer #1 (Public Review)

This paper utilizes two well-established mathematical models of colorectal cancer (CRC) screening to estimate the impact of disruptions in screening caused by the COVID-19 pandemic on long-term outcomes related to CRC. For screening, the authors use two recommendations from the US Preventive Services Task Force (USPSTF) (which were informed by the results of these models): screening colonoscopy every 10 years at ages 50, 60, and 70, and annual fecal immunochemical tests (FIT) from ages 50-75. Separate model runs were performed for 8 different cohorts at the time of the pandemic based on age, screening history, and adherence to screening. For each cohort, microsimulations were performed for 3 different scenarios--no disruption, delays in screening, or discontinuation from screening. The primary outcome was life-years gained (LYG) from screening.

In general, severe prolonged disruptions in any screening led to the largest loss of benefit from screening - for example, unscreened 50-year-olds forced to wait until age 65 (Medicare eligibility) had the largest absolute and relative loss in screening-associated LYG compared to shorter delays of 18 months or less. Losses were also higher in those who were semi-adherent to screening recommendations. The prolonged disruption had a consistently much greater impact than short-term reductions, changes in regimen, or assumptions about test sensitivity. The results are consistent between the two models. The authors point out that, since pandemic-induced disruptions in insurance coverage had a greater impact on minority populations already at risk for reduced access to screening and other preventive services, the pandemic may lead to further exacerbations in existing disparities in CRC incidence and mortality.

The strengths of this paper include the use of well-validated models, the consistent results between the models, the relatively intuitive nature of the findings, and the use of LYG, a commonly used metric for screening recommendations. As the authors point out, estimates of the population impact of the pandemic given the current age structure of the US would be helpful, these would be inherently speculative given the lack of empirical data on pandemic effects on screening. Although prioritizing screening individuals with long pandemic-induced delays is clearly the optimal policy approach, how this might be achieved is unclear.

Reviewer #1 (Public Review):

In this study, the authors use open-access datasets of Neuropixel recordings to explore the relationship between ripple strength and propagation in the septal/dorsal hippocampal pole. They found that the ripple strength correlates with the direction of propagation and that the duration of the events is dependent on the site of initiation. Medial pole ripples are longer and engage significantly more neurons than lateral ripples. These findings may have theoretical and practical implications for the study of sharp-wave ripples, a main oscillatory event underlying memory consolidation. While the approach is not entirely novel (e.g. Patel et al., JN 2013; Kumar and Deshmukh 2020), the study provides some additional insights. The strength of evidence of propagation dynamics is solid and claims are broadly supported. Some points however may require revision. In particular, issues regarding the definition of the longitudinal and transversal axes, as well as additional analysis on microcircuit interactions and neuronal dynamics per cell types and hippocampal sectors should be more thoroughly addressed in support of mechanisms.

Reviewer #1 (Public Review):

Nikolaos Koutras et al shed light on potential distinct functions of the Src family kinases (SFKs) Lck and Lyn in lymphoid signal transduction. The authors therefore overexpress Lyn and ectopic Lck in the B lymphoid cell line BJAB in an elegant Dox-inducible manner and compare the SFK's ability to trigger and shape B-lymphoid signal transduction. The findings indicate that ectopic expression of Lck is sufficient to phosphorylate the B cell receptor (BCR) ITAMs in BJAB cells. In these cells, constitutive ITAM and ITIM phosphorylation by both overexpressed Lck and Lyn induces BCR signaling, as demonstrated by phosphorylation of Syk and Akt, as well as CD22 inhibitory signaling, as shown by SHP-1 phosphorylation. In direct comparison, the influence of Lyn on said phosphorylation is stronger when it is (over-)expressed in the same amounts as Lck. This outcome was somewhat expected, since ITIM/ITAM phosphorylation is considered to be the principal function of Lyn in B cells.

The study finds Lyn to be degraded more efficiently via the proteasome and to be more tightly controlled by phosphatases when compared to Lck. However, rather than interpreting the findings as distinct kinase-intrinsic properties, one could attribute the slower degradation and stricter PTP control of Lyn to the fact that Lyn is the principal and predominant SFK in B cells and thus a "standard target" of the B-lymphoid molecular machinery, to which it is better adapted to.

Next, the authors present a RNAseq transcriptome analysis of Lck- and Lyn-expressing B cells and validate selected findings via qPCR. The data show Lyn and Lck to regulate pathways and biological functions of critical importance to B lymphocytes. Generally, most of the Lck/Lyn-regulated biological functions and pathways shown here (antigen presentation, cytokine production, migration, apoptosis, autophagy, etc.) are well known to be controlled by BCR signaling, which the overexpression of SFKs are constitutively activating, as shown earlier. While the authors draw a Venn diagram depicting differentially regulated transcripts between Lck- and Lyn-expressing cells, it does not seem like Lck is able to regulate pathways which are not "canonically" regulated by Lyn. There is also the persisting problem of Lck being expressed to a much higher extent and the effect of the endogenously expressed Lyn, since the model systems are not based on a Lyn-deficient cell line.

Lastly, the authors follow up their finding of deregulated transcripts belonging to the ER/UPR ontology cluster. Flow cytometric analysis indeed shows an influence of Lck and Lyn expression on ER homeostasis, which can be reverted with SFK inhibitors. Alas, additional follow-up experiments to functionally investigate the deregulated pathways suggested by the RNAseq analysis are not included in this study.

While there definitely are implications for the role of ectopic expression of Lck in CLL cells, this work however presents no direct comparison of expression strength or signaling outcomes between the study's BJAB (Burkitt lymphoma) cell line-based model and a model of CLL - be it a mouse model, human patient samples or a CLL cell line. Since the B-lymphoid cell line used, the Burkitt lymphoma line BJAB, is not CLL-derived, the conclusions that can be drawn for the pathophysiology of CLL is limited.

In principal, the authors show that the Src kinase Lck - when ectopically expressed - largely fills out the role of the predominant B-lymphoid Src kinase Lyn, namely phosphorylation of the CD79-ITAMs and induction of constitutive antigen receptor signaling. Given that the established role of Lck is the phosphorylation of ITAMs and activation of the T cell receptor in T cells, where it is predominantly expressed, these findings provide limited advancement of our current understanding of antigen receptor signal transduction. As a distinct functional difference between Lck and Lyn is not established in this work, said SFKs' largely exclusive expression in T and B cells remains enigmatic.

Reviewer #1 (Public Review):

Members of the SLC11/NRAMP family of transporters permit the movement of transition metals across cell membranes in all kingdoms of life. The current study builds off previous structural and mechanistic work on the SLC11/NRAMP family of transporters by Manatschal and colleagues reported in eLife; the current study presents a cryo-EM structure of a plant aluminum (Al3+) transporter that combats aluminum toxicity in soil. The structure was not determined in the presence of added metal ions, so the paper also employs a variety of established functional assays to test the effects of mutating suggested binding site residues. One notable result is the identification of a mutation (S68A) that maintains divalent transport but disrupts trivalent binding/transport. Strengths of the manuscript include the extensive legwork required to identify a combination of plant homologue, cameloid nanobody, and amphipol that is required to provide homogenous protein and interpretable cryo-EM data. The cryo-EM maps are reliable with low orientation bias and clear features. In addition, the authors perform a number of biochemical and transport assays with divalent metals to bolster their structural model.

Reviewer #1 (Public Review):

In this paper the authors are estimating the amount of transmission (via the force of infection) of EV-D^8 in England. The strengths of the study are the use of serological data for understanding underlying transmission, and the assessment of the sensitivity of the conclusions to the seropositivity cut off and the model form used. The weaknesses are the data not being annually and the lack of link to HFMD cases,, but these do not detract from the conclusions that can be drawn from the paper. The results do support the conclusions.

Reviewer #1 (Public Review):

In this paper, the authors developed a method that allows one to test a large number of drug combinations in a single cell culture sample. In principle, the experiments rely on the randomness of drug uptake in individual cells as a tool to create and encode drug treatments. They used a single sample containing thousands of cells treated with a combination of fluorescent barcoded drugs, and created transient drug gradients. They also developed segmentation- free image analysis capable of handling optical fields with a substantial number of cells. The major strength of this work is the demonstration of the feasibility of testing drug combinations in a relatively straightforward manner that could be used by many laboratories. As such this paper could have a significant impact on the early drug discovery of combinatorial therapy. One of the weaknesses in this manuscript is the absence of studies beyond just HeLa cells. In addition, the phenotype tested is cell death, which might limit the application to other drug interactions that might look at other phenotypes; e.g inhibition of cell proliferation or changes in differentiation phenotypes. Finally, there is a basic assumption that drug leakage does not occur or is minimal, but secondary uptake of the drug is likely and may not be homogeneous. Notwithstanding, the approach is feasible and likely will be applied in several laboratories.

Reviewer #1 (Public Review):

In this manuscript, Nocka and colleagues reveal a novel layer of regulation of the Btk tyrosine kinase, a key signaling protein in B lymphocyte signaling and an important drug target with 3 recently FDA-approved drugs, by the SH3-SH2 domain-containing adaptor protein Grb2. The authors nicely demonstrate a critical role of the interaction of the Grb2 SH3 domains with the Pro-rich linker C-terminal to the Btk PH-TH domains on membranes for full kinase activation of Btk. Hence this interaction recruits Btk to scaffold-mediated signaling clusters.

This is a technically sound paper with high-quality experiments. The manuscript is easy to follow and excellently written. The findings are novel and of high relevance towards a complete understanding of Btk regulation and signaling in cancer and normal cells.

Reviewer #1 (Public Review):

The study by Oikawa and colleagues demonstrates for the first time that a descending inhibitory pathway for nociception exists in non-mammalian organisms, such as Drosophila. This descending inhibitory pathway is mediated by a Drosophila neuropeptide called Drosulfakinin (DSK), which is homologous to mammalian cholecystokinin (CCK). The study creates and uses several Drosophila mutants to convincingly show that DSK negatively regulates nociception. They then use several sophisticated transgenic manipulations to demonstrate that a descending inhibitory pathway for nociception exists in Drosophila.

Strengths:

This study creates the possibility of using Drosophila to study descending nociceptive systems.

CRISPR/Cas9 is used to generate mutants of dsk, CCKLR-17D1, and CCKLR-17D3. The authors then use these mutants to clearly show that DSK negatively regulates nociception.

Several GAL4s are used to clearly show that these effects are likely mediated by two sets of neurons in the brain, MP1 and Sv.

RNAi and rescue experiments further show that CCKLR-17D1, a DSK receptor, functions in Goro neurons to negatively regulate nociception.

Thermogenetic experiments nicely show that activation of DSK neurons attenuates the nociceptive response.

Weaknesses:

A minor weakness in the study is that it is unclear how DSK negatively regulates nociception. An earlier study at the Drosophila nmj shows that loss of DSK signaling impairs neurotransmission and synaptic growth. In the current study, loss of CCKLR-17D1 in Goro neurons seems to increase intracellular calcium levels in the presence of noxious heat. An interesting future study would be the examination of the underlying mechanisms for this increase in intracellular calcium.

Reviewer #1 (Public Review):

Chan et al. tried identifying the binding sites or pockets for the KCNQ1-KCNE1 activator mefenamic acid. Because the KCNQ1-KCNE1 channel is responsible for cardiac repolarization, genetic impairment of either the KCNQ1 or KCNE1 gene can cause cardiac arrhythmia. Therefore, the development of activators without side effects is highly demanded. Because the binding of mefenamic acid requires both KCNQ1 and KCNE1 subunits, the authors performed drug docking simulation by using KCNQ1-KCNE3 structural model (because this is the only available KCNQ1-KCNE structure) with substitution of the extracellular five amino acids (R53-Y58) into D39-A44 of KCNE1. That could be a limitation of the work because the binding mode of KCNE1 might differ from that of KCNE3. Still, they successfully identified some critical amino acid residues, including W323 of KCNQ1 and K41 and A44 of KCNE1. They subsequently tested these identified amino acid residues by analyzing the point mutants and confirmed that they attenuated the effects of the activator. They also examined another activator, yet structurally different DIDS, and reported that DIDS and mefenamic acid share the binding pocket, and they concluded that the extracellular region composed of S1, S6, and KCNE1 is a generic binding pocket for the IKS activators.

The data are solid and well support their conclusions, although there are a few concerns regarding the choice of mutants for analysis and data presentation.

Other comments:

1. One of the limitations of this work is that they used psKCNE1 (mostly KCNE3), not real KCNE1, as written above. It is also noted that KCNQ1-KCNE3 is in the open state. Unbinding may be facilitated in the closed state, although evaluating that in the current work is difficult.<br /> 2. According to Figure 2-figure supplement 2, some amino acid residues (S298 and A300) of the turret might be involved in the binding of mefenamic acid. On the other hand, Q147 showing a comparable delta G value to S298 and A300 was picked for mutant analysis. What are the criteria for the following electrophysiological study?<br /> 3. It is an interesting speculation that K41C and W323A stabilize the extracellular region of KCNE1 and might increase the binding efficacy of mefenamic acid. Is it also the case for DIDS? K41 may not be critical for DIDS, however.<br /> 4. Same to #2, why was the pore turret (S298-A300) not examined in Figure 7?

Title: How Alive Is 1.5? Part One – A Small Budget, Shrinking Fast

Author: - Kevin Anderson - Dan Calverley

Key Messages - For a 50:50 chance of staying below 1.5°C, we’re using up the remaining carbon budget at around 1% every month. - Following current national emissions pledges (NDCs) to 2030 puts the temperature commitments within the Paris Agreement beyond reach. - Claims that 1.5°C is now inevitable also assign “well below 2°C” to the scrapheap. - An ‘outside chance’ of not exceeding 1.5°C remains viable, but ongoing fossil fuel use is rapidly undermining it. - The few credible pathways for an outside chance of 1.5°C are not being discussed. This is an active choice by policymakers and experts, who have largely dismissed equity-based social change.

Reviewer #1 (Public Review):

This is a valuable study demonstrating convincingly that PI3K signaling lies downstream of Pdgfra signaling in zebrafish cardiomyocyte progenitors as they undergo latero-medial migration and midline fusion, essential for heart tube formation, likely via chemotaxis. Whereas the authors used both multiple inhibitory drugs and dominant negative transgene expression to interrupt PI3K expression, with findings strongly aligning, the manuscript would have been stronger if genetic approaches were used to complement the above approaches. Nonetheless, the impact of dnPI3K inhibition allowed the authors to suggest that the effects were cell autonomous to migrating cardiomyocytes. The authors used contemporary live imaging techniques allowing quantification of key cell behaviors, and this is a strength of the paper. There are some issues about the inter-study alignment of trajectory data that need to be addressed. Perhaps the most conspicuous weakness is that the authors have not advanced the model for cardiomyocyte migration beyond adding the involvement of PI3K downstream of Pdgfra, which is to a significant degree expected. The recording of cardiomyocyte protrusions biased in their orientation towards the direction of migration, which is lost in the mutants, is an interesting advance, although it was not shown whether protrusions are causally related to migration.

Reviewer #1 (Public Review):

This manuscript addresses the important and understudied issue of circuit-level mechanisms supporting habituation, particularly in pursuit of the possible role of increases in the activity of inhibitory neurons in suppressing behavioral output during long-term habituation. The authors make use of many of the striking advantages of the larval zebrafish to perform whole brain, single neuronal calcium imaging during repeated sensory exposure, and high throughput screening of pharmacological agents in freely moving, habituating larvae. Notably, several blockers/antagonists of GABAA(C) receptors completely suppress habituation of the O-bend escape response to dark flashes, suggesting a key role for GABAergic transmission in this form of habituation. Other substances are identified that strikingly enhance habituation, including melatonin, although here the suggested mechanistic insight is less specific. To add to these findings, a number of functional clusters of neurons are identified in the larval brain that has divergent activity through habituation, with many clusters exhibiting suppression of different degrees, in line with adaptive filtration during habituation, and a single cluster that potentiates during habituation. Further assessment reveals that all of these clusters include GABAergic inhibitory neurons and excitatory neurons, so we cannot take away the simple interpretation that the potentiating cluster of neurons is inhibitory and therefore exerts an influence on the other adapting (depressing) clusters to produce habituation. Rather, a variety of interpretations remain in play.

Overall, there is great potential in the approach that has been used here to gain insight into circuit-level mechanisms of habituation. There are many experiments performed by the authors that cannot be achieved currently in other vertebrate systems, so the manuscript serves as a potential methodological platform that can be used to support a rich array of future work. While there are several key observations that one can take away from this manuscript, a clear interpretation of the role of GABAergic inhibitory neurons in habituation has not been established. This potential feature of habituation is emphasized throughout, particularly in the introduction and discussion sections, meaning that one is obliged as a reader to interrogate whether the results as they currently stand really do demonstrate a role for GABAergic inhibition in habituation. Currently, the key piece of evidence that may support this conclusion is that picrotoxin, which acts to block some classes of GABA receptors, prevents habituation. However, there are interpretations of this finding that do not specifically require a role for modified GABAergic inhibition. For instance, by lowering GABAergic inhibition, an overall increase in neural activity will occur within the brain, in this case below a level that could cause a seizure. That increase in activity may simply prevent learning by massively increasing neural noise and therefore either preventing synaptic plasticity or, more likely, causing indiscriminate synaptic strengthening and weakening that occludes information storage. Sensory processing itself could also be disrupted, for instance by altering the selectivity of receptive fields. Alternatively, it could be that the increase in neural activity produced by the blockade of inhibition simply drives more behavioral output, meaning that more excitatory synaptic adaptation is required to suppress that output. The authors propose two specific working models of the ways in which GABAergic inhibition could be implemented in habituation. An alternative model, in which GABAergic neurons are not themselves modified but act as a key intermediary between Hebbian assemblies of excitatory neurons that are modified to support memory and output neurons, is not explored. As yet, these or other models in which inhibition is not required for habituation, have not been fully tested.

This manuscript describes a really substantial body of work that provides evidence of functional clusters of neurons with divergent responses to repeated sensory input and an array of pharmacological agents that can influence the rate of a fundamentally important form of learning.

Reviewer #1 (Public Review):

Rosas et al studied the mechanism/s that enabled carbapenems resistance of a Klebsiella isolate, FK688, which was isolated from an infected patient. To identify and characterize this mechanism, they used a combination of multiple methods. They started by sequencing the genome of this strain by a combination of short and long read sequencing. They show that Klebsiella FK688 does not encode a carbapenemase, and thus looked for other mechanisms that can explain this resistance. They discover that both DHA-1 (located on the mega-plasmid) and an inactivation of the porin OmpK36, are required for carbapenem resistance in this strain. By using experimental evolution, it was shown that resistance is lost rapidly in the absence of antibiotics selection, by a deletion in pNAR1 that removed blaDHA-1. Moreover, their results suggested that it is likely that exposure to other antibiotics selected for the acquisition of the mega-plasmid that carries DHA-1, which then enabled this strain to gain resistance to carbapenemase by a single deletion.

The major strength of this study is the use of various approaches, to tackle an important and interesting problem.

The conclusions of this paper are mostly well supported by data, but one aspect is not clear enough. The description of the evolutionary experiment is not clear. I could not find a clear description of the names of the evolved populations. However, the authors describe strains B3 and A2, but their source is not clear. The legends of the relevant figure (Figure 5) are confusing. For example, the text describing panel B is not related to the image shown in this panel. Moreover, it is shown in panel C (and written in the main text) that the OmpK36+ evolved populations had only translucent colonies, so what is the source of B3(o)?

Reviewer #1 (Public Review):

The glideosome-associated connector is an essential piece of the machinery used by the apicomplexa parasites as they invade host cells. This GAC makes important interactions with the membrane and with actin during this process. Here, Kumar et al present the first structure of the GAC from T. gondii, showing a complex fold in a closed form. This structure was determined at pH 5, and they show that at more physiological pH values the structure is far more open. However, this is not in the context of actin, membrane, or other binding partners, and so the question remains about how open the structure is in its physiological context. The authors next use molecular dynamics, NMR, and mutagenesis to identify the residues involved in membrane binding and also assess actin binding through modelling which is not validated by experiment. This paper presents an important contribution to our understanding of the molecular machinery involved in host cell invasion but leaves many questions remaining about how this protein links to the cytoskeleton and functions during the invasion process.

• The structure of TgGAC provides the first such structure of this complex and is an important contribution to our understanding. The structure presented in Figure 1A is a composite, containing the crystal structure of the majority of the protein, determined at pH 5, to which has been docked the PH domain structure, determined by NMR. It would be good to see more clarity in the figure about what is experimentally determined and what is modelled.<br /> • SAXS data shows that, at pH 8, a substantial fraction of the protein is in a very extended conformation, which differs significantly from the compact structure seen in crystals at pH 5. I would prefer to see the models in Figure 2d represented as spheres or surfaces, to prevent over-interpretation associated with showing models with low-resolution data. However, the SAXS findings are robust and this is clearly a dynamic molecule in solution. It will be interesting to see what the situation is in the context of binding partners.<br /> • Molecular dynamic simulations next indicate the region which binds to a lipid bilayer, with contact residues forming a consistent interaction surface in three independent simulations. This identified the PH domain and neighbouring residues as the membrane interaction surface.<br /> • Switching to Plasmodium falciparum protein, the authors next use NMR to investigate the binding of the PH domain to membrane nanodiscs, and show that the same protein region identified in the MD simulations was found to bind in the NMR experiments.<br /> • These membrane binding assays were then followed up through liposome pelleting assays, using TgGAP, which showed that the protein only pellets in the presence of PA lipid and that mutation of residues identified through NMR abolished liposome binding. The mutations didn't have the same effect on full-length and PH domains (noting KER for example) suggesting that lipid binding is not entirely mediated by the PH domain in the full-length protein.<br /> • The authors next put the mutants into toxoplasma and assay the effect on apical localisation and on invasion percentage. Interestingly the mutants had little effect, perhaps due to the role of other regions of the GAC on lipid binding, suggesting that abolishing PH domain lipid binding is not sufficient. Unfortunately, as the mutations only partly reduced lipid binding in the context of full-length GAC, as shown in liposome experiments, it is hard to come to a firm conclusion about the importance of lipid binding from this data as the protein used in this experiment will still have partial lipid binding properties.<br /> • The authors next investigate actin binding by TgGAC and show that most of the N-terminal half of the protein is required for this function. The authors propose, using AlphaFold2 and similarities to catenins, how GAC might bind to actin. In the absence of any validation from experimental data, caution is needed here, and I would personally not rely on the accuracy of these models.

Reviewer #1 (Public Review):

During the height of the Covid19-pandemic, there was great and widely spread concern about the lowered protection the screening programs within the cancer area could offer. Not only were programs halted for some periods because of a lack of staff or concern about the spreading of SARS CoV2. When screening activities were upheld, participation decreased, and follow-up of positive test results was delayed. Mariam El-Zein and coworkers have addressed this concern in the context of cervical screening in Canada, one of the rather few countries in the world with well organized, population-based, although regionalized, cervical screening program.

Despite the existence of screening registries, they choose to do this in form of a survey on the internet, to different professional groups within the chain of care in cervical screening and colposcopy. The reason for taking this "soft data" approach is somewhat diffuse. The authors claim they want to "capture modifications". However, the suggestions that come from this study are limited and are submitted for publication 2 years after the survey when the height of the pandemic has passed long since, and its burden on the screening program has largely disappeared. The value of the study had been larger if either the conclusions had been communicated almost directly, or if the survey had been done later, to sum up the total effect of the pandemic on the Canadian cervical screening program.

Another major problem with this study is the coverage. The results of persistent activities to get a large uptake is somewhat depressing although this is not expressed by the authors. 510 professionals filled out the survey partially or in total. 10 professions were targeted. The authors make no attempt to assess the coverage or the validity of the sample. They state the method used does not make that possible. But the number of family practicians, colposcopists, cytotechnicians, etc. involved in the program should roughly be known and the proportion of those who answered the survey could have been calculated. My guess is that it is far below 10%. Also, the national distribution seems shewed despite the authors boosting its pan-Canadian character. I am just faintly familiar with the Canadian regions, but, as an example, only 2 replies from Quebec must question the national validity of this survey.

The result section is dominated by quantitative data from the responses to the 61 questions. All questions and their answers are tabulated. As there is no way to assess the selection bias of the answers these quantitative results have no real value from an epidemiological standpoint. The replies to the open-ended questions are summarized in a table and in the text. The main conclusion of the content analysis of the answers to the direct questions, and one of the main conclusions of the study, is that the majority favors HPV self-sampling in light of the pandemic. However, this not-surprising view is taken by only 80 responders while almost as many (n=60) had no knowledge about HPV self-sampling.

The authors conclude that their study identified the need for recommendations and strategies and building resilience in the screening system. No one would dispute the need, but the additional weight this study adds, unfortunately, is low, from a scientific standpoint.

The conclusion I draw from this study is that the authors have done a good job in identifying some possible areas within the Canadian screening programs where the SARS-Cov2 pandemic had negative effects and received some support for that in a survey. Furthermore, they listed a few actions that could be taken to alleviate the vulnerability of the program in a future similar situation, and received limited support for that. No more, no less.

Reviewer #1 (Public Review):

OTOP ion channels are proton-activated, proton-permeable proteins that participate in sour tasting but for which other physiological roles are just beginning to be elucidated. The authors of this manuscript noticed that the isoform OTOP3 shows activation by protons that are potentiated in the presence of Zn2+ and other divalent ions, while other isoforms are not weakly or not at all potentiated. This allowed them to apply a chimeric approach to define which regions of the protein are responsible for the Zn2+ effect. The authors found that a single extracellular loop and a single histidine residue located in it are sufficient to explain the potentiation and propose that this histidine is part of a binding site that allosterically couples to yet undefined proton binding sites(s) responsible for proton gating.

The authors have performed very high-quality experiments and carried out a careful analysis of the data. This characterization of gating behavior of OTOP channels should be a step in elucidating physiological roles and in understanding the dynamics of these proteins. For these reasons, it should be of interest to researchers working in molecular biophysics and the physiological roles of ion channels.

Reviewer #1 (Public Review):

In this study, the authors set out to determine the degree to which early language experience affects neural representations of concepts. To do so, they use fMRI to measure responses to 90 words in adults who are deaf. One group of deaf adults (n=16) were native signers (and thus had early language exposure); a second group (n=21) was exposed to sign language later on. The groups were relatively well-matched in other respects. The primary finding was that the high dimensional representations of concepts in the left lateral anterior temporal lobe (ATL) differed between native and delayed signers, suggesting a role for early language experience in concept representation.

The analyses are carefully conducted and reflect a number of thoughtful choices. These include the "inverted MDS" method for constructing semantic RDMs, a normal hearing comparison group for both behavioral and fMRI data, and care taken to avoid bias in defining functional ROIs. And, comparing early and delayed signing groups is a clever way to study the role of early language experience on adult language representations.

One interesting result that I struggled to put in a broader context relates to the disconnect between behavioral and neural results. Specifically, the behavioral semantic RDMs (Figure 1a) did not differ between any of the groups of participants. This suggests that the representations of the 90 concepts are represented similarly in all of the participants. However, the similarity of the neural RDMs in left lateral ATL differs between the native and delayed signing groups (but not in other regions). Given the similarity of the behavioral semantic RDMs, it is unclear how to interpret the difference in left lateral ATL representations. In other words, the neural differences in left ATL do not affect behavior (semantic representation). The importance of the differences in neural RDMs is therefore questionable.

An important point is that, if I understand correctly, the semantic space is defined by the 90 experimental items. That is, behavioral RDMs were created by having normal hearing participants arrange 90 items spatially, and neural RDMs were created by comparing patterns of responses to these 90 experimental items. This 90-dimensional space is thus both (a) lower dimensional than many semantic space models that include hundreds of directions and (b) constrained by the specific 90 experimental items chosen. On the one hand, this seems to limit the generalizability of the findings for semantic representations more broadly.

The logic behind using a categorical semantic RDM (e.g., Figure 2a) was not clear. The behavioral semantic RDMs (Figure 1a) clearly show gradations in dissimilarity, particularly for the abstract categories. It would seem that using the behavioral semantic RDM would capture a more accurate representation of the semantic space than the categorical one.

Reviewer #1 (Public Review):

In this paper, the authors present a method for discovering response properties of neurons, which often have complex relationships with other experimentally measured variables, like stimuli and animal behaviors. To find these relationships, the authors fit neural data with artificial neural networks, which are chosen to have an architecture that is tractable and interpretable. To interpret the results, they examine the first- and second-order approximations of the fitted artificial neural network models. They apply their method profitably to two datasets.

The strength of this paper is in the problem it is attempting to solve: it is important for the field to develop more useful ways to analyze and understand the massive neural datasets collected with modern imaging techniques.

The weaknesses of this paper lie in its claims (1) to be model free and (2) to distinguish the method from prior methods for systems identification, including spike triggered averaging and covariance (or rather their continuous response equivalents). On the first claim, the systems identification methods are arguably substantially more model free approach. On the second claim, this reviewer would require more evidence that the presented approach is substantially different from or an improvement on systems identification methods in common use applied directly to the data.

Reviewer #1 (Public Review):

Li et al investigated the behavioral response and fMRI activations associated with deep brain stimulation (DBS) of the lateral habenula (LHb) in 2 distinct rodent models of depression. They found that a) LHb DBS reduces depressive and anxiety behaviors using multiple behavioral tests: sucrose preference, forced swim, and open field. These results held across multiple models of depression and multiple tests, and generally restored results of these behavioral tests to parity with controls. Furthermore, fMRI activations of brain regions with known connectivity to LHb strongly correlated with behavioral responses to LHb DBS, particularly in limbic regions. These behavioral responses clearly depended on electrode location, with more medial placements within the LHb producing a more robust behavioral effect.

The conclusions of this paper are generally well supported by the data, with the primary weaknesses of the study being 1) limited novelty due to LHb already being a well-established target for DBS in depression, and 2) the questionable validity of rodent models of depression in general. The authors deal with the first point (novelty) by extending their study to electrode localization and fMRI correlates with the behavioral response, leading to insight into surgical targeting as well as mechanism of effect, respectively. They also partially mitigate fundamental problems with rodent models of depression by using 2 different models and showing consistent responses to LHb DBS across both. The methods used in this study were sound, with high-quality techniques used for electrode implantation, confirmation of electrode placement, fMRI acquisition, anesthesia and physiological monitoring, as well as an appropriate statistical analytic approach.

Reviewer #1 (Public Review):

Chromosomal aneuploidy in humans causes diseases such as Down syndrome associated with changes in cognitive and metabolic activities, but how extra copies of chromosomes cause the changes remains largely unknown. In this important paper, the authors characterized the metabolisms and physiology of the transgenic mouse with most of human chromosome 21 thoroughly and nicely showed the overexpression of sarcolipin which uncouples Ca2+ import with ATP hydrolysis of sarcoplasmic reticulum Ca2+ ATPase (SERCA), which results in heat production and hyperactive mitochondria activity.

Reviewer #1 (Public Review):

This is a very interesting and timely paper and one of very few that crosses species. Linear multielectrode array recordings are rapidly becoming state-of-the-art. This means that there is a greater need for finding motifs and/or reliable markers that characterize activity in different cortical layers.

Reviewer #1 (Public Review):

This important study by Di et al., focuses on the mechanism by which potassium channels are activated prior to NLRP3 inflammasome activation. Using confocal- and electron-microscopy studies the authors demonstrate that the potassium channel, TWIK2, located in the endosomal compartment during basal conditions, is translocated onto the plasmalemma upon ATP stimulation. The authors suggest that this translocation triggers potassium efflux and subsequent NLRP3 inflammasome activation. Using Rab11a-deficient cells, the authors also show an essential role for Rab11a in this process.

This is a well written mechanistic study that has novel findings that are of interest to the inflammasome field. It addresses a long-standing question in the field, the exact mechanism by which potassium channel is activated upon treatment with NLRP3 stimuli. However, to make the conclusions more convincing, the authors should include additional stimuli such as pore-forming toxins, LPS transfection, and/or infections with bacterial pathogens to show that the Rab11a-dependent TWIK2 translocation is a universal requirement for initiation of potassium efflux by multiple stimuli and not specific to ATP. Similarly, the authors should include important controls in their inhibitor/siRNA experiments to show that the cells are still functional and the defects they observe are specific to NLRP3 inflammasome.

Reviewer #1 (Public Review):

In this paper, Liu et al. analyze a dataset of primate retinal ganglion cell responses to visual stimuli in order to find maximally informative dimensions in the inputs. They use models based on these analyses to examine features of early visual processing that influence predictive coding of visual motion in the early retina. This is an important set of questions because it remains unclear what principles drive sensory encoding and how those principles relate to circuit mechanisms found in sensory systems.

The strength in this paper lies in its rigorous analysis of the maximally informative dimensions (MIDs) of primate retinal ganglion cell signals, and the connections it makes between those dimensions and circuit models for retinal function.

The weakness of this paper lies in drawing strong connections between those analyses and predictive coding by these cells. These analyses of predictive coding are interesting but not tightly related to the MID analysis. This paper also does little to address how the structure of the stimuli affect the conclusions they draw about what circuit features contribute to predictive coding of motion.

Reviewer #1 (Public Review):

In this study, Hara and Kuraku identified the genes lost multiple times across the mammalian phylogenetic tree and termed them "elusive genes." They then investigated the features of these elusive genes in the species where they are well preserved. The authors identified several genomic features that drive gene fates toward loss, in addition to the long-presumed functional dispensability. This analysis explains why some genes are more likely to lose during evolution than others.

This study extends the selection-mutation balance theory from nucleotide substitutions to gene losses. In the context of gene losses, functional dispensability determines the selective coefficient, and the genomic features determine the rate of gene loss mutations. While the selective force has been long presumed to be important, the heterogenous genomic features that led to the mutability of gene losses were not carefully investigated in previous studies. This study fills this gap and shows that some genes are intrinsically prone to be lost (and why).

Strengths:<br /> Identification of gene losses across the phylogenetic tree is not trivial, especially when considering the incompleteness of genomes. The authors conducted their bioinformatic analyses carefully and required two independent gene loss events, each supported by multiple species in a monophyletic group. The accuracy in the identification of elusive genes provides a solid basis for the following analyses.

The authors identified genomic features associated with the gene losses in the species where the gene is preserved. This is an important strategy to avoid identifying genomic features that are formed during the gene losses but to identify the genomic features that likely formed before the gene loss. Using this strategy, the authors were able to recognize the intrinsic properties of elusive genes.

Weaknesses:

Gene expression level as a confounding factor was not well controlled throughout the study. Higher gene expression often makes genes less dispensable after gene duplication. Gene expression level is also a major determining factor of evolutionary rates (reviewed in http://www.ncbi.nlm.nih.gov/pubmed/26055156). Some proposed theories explain why gene expression level can serve as a proxy for gene importance (http://www.ncbi.nlm.nih.gov/pubmed/20884723, http://www.ncbi.nlm.nih.gov/pubmed/20485561). In that sense, many genomic/epigenomic features (such as replication timing and repressed transcriptional regulation) that were assumed "neutral" or intrinsic by the authors (or more accurately, independent of gene dispensability) cannot be easily distinguishable from the effect of gene dispersibility.

Ks was used by the authors to indicate mutation rates. However, synonymous mutations substantially affect gene expression levels (https://pubmed.ncbi.nlm.nih.gov/25768907/, https://pubmed.ncbi.nlm.nih.gov/35676473/). Thus, synonymous mutations cannot be simply assumed as neutral ones and may not be suitable for estimating local mutation rates. If introns can be aligned, they are better sequences for estimating the mutability of a genomic region.

The term "elusive gene" is not necessarily intuitive to readers.

Reviewer #1 (Public Review):

The first synapses of the pain pathway are concentrated in the superficial spinal cord dorsal horn. Here peripheral inputs are processed by local interneuron circuitry before ascending to the brain. The spinal dorsal horn is organized into lamina with the resident interneurons differentiated by their anatomy, physiological and molecular properties. Over the past decade, the restricted expression of select genes has been used to assign potential function to dorsal horn neuron "cell types". This type of work has relied on the genesis of Cre-reporter mouse strains and intersectional tools to generate mice where select sets of neurons can be activated, inhibited, or ablated. The picture that has emerged from these types of experiments is murky but favors the model where there exist genetically defined cell-types play distinct roles in the detection of painful, itchy, thermal, and mechanical stimuli under normal and pathological situations. The current work by Boyle and colleagues concerns itself with the dorsal horn neurons expressing the neuropeptide NPY. This study is directly related to previously published work that demonstrated that ablating spinal cord neurons that express Npy, including those who express this gene transiently during development, resulted in mice that had heightened touch-evoked itch that seemed different from the canonical chemical itch pathways previously identified. A major conclusion from this previous work was that other modalities were unaffected. Subsequent work built on these findings to identify the potential touch inputs and spinal neuron expressing the Npy receptor as part of a mechanical itch circuit.

This current work by Boyle and colleagues challenge challenges this view by providing evidence that in adult mice, the dorsal horn neurons expressing Npy function to broadly inhibit pain and itch. The authors use direct injection of viral vectors, chemogenetics and synaptic silencing to probe the behavioral effects of stimulating or silencing Npy-expressing dorsal horn neurons in a variety of assays under normal and pathological conditions known to produce allodynia and hyperalgesia. Overall, this is a rather carefully conducted study with the appropriate controls. The data are clear, the effect sizes robust and the presentation easy to follow. These findings challenge the conclusion that these neurons are involved selectively in mechanical itch and instead reveal a potentially clinical important group of neurons for pain.

Reviewer #1 (Public Review):

Huang C-K. and colleagues in this work address the understudied role of environmental conditions and external forces in cell extrusion as a fundamental part of epithelial homeostasis. They suggest that hydrostatic stress plays a significant role in counteracting cell extrusion forces through the indirect regulation of the focal adhesion kinase (FAK) - protein kinase B (AKT) survival pathway. The team nicely exploits their expertise in fabricating cell culture substrates to control hydrostatic stress on a common epithelial cell model from the kidney (i.e., MDCK). This was done by creating waving surfaces with different lengths from 50µm to 200 µm, thus creating a heterogenous distribution of monolayer forces towards the substrate. Finally, using a specific inhibitor for FAK, they suggest that the survivor pathway FAK-AKT is involved in the observed phenomenon.

In conclusion, the presented data underline the importance of considering external forces and tissue geometry in regulating epithelial homeostasis and the selective transport of water and solutes. These results may have a significant impact on understanding the basic mechanisms of epithelial physiology and pathology, such as in the kidney, intestine, or retina.

Reviewer #1 (Public Review): 

How morphogens spread within tissues remains an important question in developmental biology. Here the authors revisit the role of glypicans in the formation of the Dpp gradient in wing imaginal discs of Drosophila. They first use sophisticated genome engineering to demonstrate that the two glypicans of Drosophila are not equivalent despite being redundant for viability. They show that Dally is the relevant glypican for Dpp gradient formation. They then provide genetic evidence that, surprisingly, the core domain of Dally suffices to trap Dpp at the cell surface (suggesting a minor role for GAGs). They conclude with a model that Dally modulates the range of Dpp signaling by interfering with Dpp's degradation by Tkv. These are important conclusions, but more independent (biochemical/cell biological) evidence is needed.

As indicated above, the genetic evidence for the predominant role of Dally in Dpp protein/signalling gradient formation is strong. In passing, the authors could discuss why overexpressed Dlp has a negative effect on signaling, especially in the anterior compartment. The authors then move on to determine the role of GAG (=HS) chains of Dally. They find that in an overexpression assay, Dally lacking GAGs traps Dpp at the cell surface and, counterintuitively, suppresses signaling (fig 4 C, F). Both findings are unexpected and therefore require further validation and clarification, as outlined in a and b below. 

a) In loss of function experiments (dallyDeltaHS replacing endogenous dally), Dpp protein is markedly reduced (fig 4R), as much as in the KO (panel Q), suggesting that GAG chains do contribute to trapping Dpp at the cell surface. This is all the more significant that, according to the overexpression essays, DallyDeltaHS seems more stable than WT Dally (by the way, this difference should also be assessed in the knock-ins, which is possible since they are YFP-tagged). The authors acknowledge that HS chains of Dally are critical for Dpp distribution (and signaling) under physiological conditions. If this is true, one can wonder why overexpressed dally core 'binds' Dpp and whether this is a physiologically relevant activity. 

b) Although the authors' inference that dallycore (at least if overexpressed) can bind Dpp. This assertion needs independent validation by a biochemical assay, ideally with surface plasmon resonance or similar so that an affinity can be estimated. I understand that this will require a method that is outside the authors' core expertise but there is no reason why they could not approach a collaborator for such a common technique. In vitro binding data is, in my view, essential. 

In a subsequent set of experiments, the authors assess the activity of a form of Dpp that is expected not to bind GAGs (DppDeltaN). Overexpression assays show that this protein is trapped by DallyWT but not dallyDeltaHS. This is a good first step validation of the deltaN mutation, although, as before, an invitro binding assay would be preferable. Nevertheless, the authors show that DppDeltaN is surprisingly active in a knock-in strain. At face value (assuming that DeltaN fully abrogates binding to GAGs), this suggests that interaction of Dpp with the GAG chains of Dally is not required for signaling activity. This leads to authors to suggest (as shown in their final model) that GAG chains could be involved in mediating the interactions of Dally with Tkv (and not with Dpp. This is an interesting idea, which would need to be reconciled with the observation that the distribution of Dpp is affected in dallyDeltaHS knock-ins (item a above). It would also be strengthened by biochemical data (although more technically challenging than the experiments suggested above). 

In an attempt to determine the role of Dally (GAGs in particular) in the signaling gradient, the paper next addresses its relation to Tkv. They first show that reducing Tkv leads to Dpp accumulation at the cell surface, a clear indication that Tkv normally contributes to the degradation of Dpp. From this they suggest that Tkv could be required for Dpp internalisation although this is not shown directly. The authors then show that a Dpp gradient still forms upon double knockdown (Dally and Tkv). This intriguing observation shows that Dally is not strictly required for the spread of Dpp, an important conclusion that is compatible with early work by Lander suggesting that Dpp spreads by free diffusion. These result show that Dally is required for gradient formation only when Tkv is present. They suggest therefore that Dally prevents Tkv-mediated internalisation of Dpp. Although this is a reasonable inference, internalisation assays (e.g. with anti-Ollas or anti-HA Ab) would strengthen the authors' conclusions especially because they contradict a recent paper from the Gonzalez-Gaitan lab. 

The paper ends with a model suggesting that HS chains have a dual function of suppressing Tkv internalisation and stimulating signaling. This constitutes a novel view of a glypican's mode of action and possibly an important contribution of this paper. As indicated above, further experiments could considerably strengthen the conclusion. Speculation on how the authors imagine that GAG chains have these activities would also be warranted.

Reviewer #1 (Public Review):

This study by Cao et al. demonstrates role of Neutrophil in clearing apoptotic hepatocytes by directly burrowing into the apoptotic hepatocytes and ingesting the effete cells from inside without causing inflammation. The authors applied intravital microscopy, Immunostaining and electron microscopy to visualize perforocytosis of neutrophil in hepatocytes. They also found that neutrophil depletion impairs the clearance of apoptotic hepatocytes causing impaired liver function and generation of autoantibodies, implying a role of defective neutrophil- mediated clearance of apoptotic cells in Autoimmune Liver disease. The experiments were well designed and conducted, the results were reasonably interpreted, and the manuscript was clearly written with logical inputs.

One weak point is that the signals/mechanisms that determine why neutrophil specifically target apoptotic hepatocytes in liver and no other organs or cells is not clearly understood.

Reviewer #1 (Public Review):

In this manuscript, Mastrototaro et al. perform a series of experiments in transgenic murine models assessing the function of Palladin (PALLD) in the heart. Global PALLD KOs are embryonic lethal, precluding the assessment of the roles of this protein in adulthood. To circumvent this limitation, the authors generated a floxed Palld allele and ablated it with two cardiomyocyte-specific Cres: the constitutively active Myh6-Cre and the tamoxifen-inducible aMHC-MerCreMer. Interestingly, ablation with the constitutive Cre (cKO) did not produce any overt phenotype, but ablation in adulthood (cKOi) resulted in compromised cardiac function. These observations suggest a compensation mechanism that takes place when cardiomyocytes develop in the complete absence of this protein but not when cardiomyocytes develop in a wild-type background and are deprived of this protein after achieving full maturation. These experiments were complemented with yeast two-hybrid techniques to identify novel partners that bind to a region of PALLD for each no interactants had been previously identified. Experiments in human samples revealed an upregulation of PALLD transcripts in the hearts of patients.

This manuscript adds important information to our understanding of sarcomeric proteins. Data are generally of good quality and well presented in figures. The numbers of animals in echocardiographic studies are also adequate for proper conclusions. Authors achieve most of their goals, including the identification of novel partners of PALLD and the identification of a requirement for PALLD in cardiomyocytes for normal heart function. However, given that all experiments performed in this study were focused on the loss-of-function of PALLD, it is not clear what is the relevance of the PALLD upregulation observed in human patients. Authors should clearly state this limitation in their results.

Considering that authors have observed evidence for nuclear PALLD, which could hint at potential major gene expression changes when this protein is ablated, it would be interesting to perform an unbiased assessment of transcriptional alterations (RNA-seq) in cardiomyocytes isolated from control and cKOi hearts. In addition, to test if the compensation observed in the embryonic cKO involves mechanisms of transcriptional adaptation, it would be interesting to compare RNA-seq results from cKOi and cKO (genes encoding proteins similar to PALLD that are upregulated in cKO but not cKOi cardiomyocytes would be very strong candidates). However, these transcriptomic data are not essential to support current findings and can be performed in follow-up studies.

Reviewer #1 (Public Review):

The authors set out to analyse the pattern of movement of T cells in different tissues- lymph nodes, villi, and inflamed/infected lungs. The authors are comparing data sets from multiple sites in different studies but acquired using similar instruments, preparations, and imaging conditions.

The more confined movement pattern in the lung that has a turning angle distribution with more incidence of angles near 180 degrees is striking.

T cells in the infected inflamed lung search a smaller volume over time but will explore it more extensively.

The measurements of T cell movement are context-free such that obstacles and tissue boundaries that could account for some of the confined behaviours in the lung parenchyma are not discussed.

Nonetheless, the work will motivate further study of the biological significance of the different T cell movement patterns in the lung, which may also be considered in the context of recent data on changes in B cell motility- a potential interacting cell.

Reviewer #1 (Public Review):

This study presents a resource aiming to unify language and rules used in the literature to describe, curate and assess biology experiments, published or not. Focusing on host-pathogen interactions, the work presents a new ontology and controlled vocabulary, as well as rules to describe 'metagenotypes', a term coined for the joint description of interacting host-pathogen genotypes. 'PHI-Canto' extends a previous resource by also enabling using UniProtKB IDs to curate proteins. Among other important by-products, PHI-Canto could contribute to damping proliferating names and acronyms for genes, processes, and interactions; a chronic annoyance in the biosciences.

The tool does give the impression that, with sufficient time and usage, it could become a rich and robust resource. Just addressing the Uniprot IDs issue is a nice move.

Reviewer #1 (Public Review):

Motivated by the premise that Alzheimer's disease (ADD) and major depressive disorder (MDD) have shared underlying environmental and genetic risk factors, Petrican and Fornito combine non-imaging risk factors and executive task-based functional network change indices into latent variables of resilience to AD and MDD. The authors find two latent variables (LVs): LV1 represents change in network membership over time of distributed nodes during task, which is associated with greater genetic MDD risk, less psychopathology, and more advanced puberty, all while adjusting for age and indices of environmental stressors. LV2 represents occipital lobe nodal flexibility across task and time, decreased AD genetic risk, increased MDD genetic risk and less psychopathology, again adjusted for age and environmental stressors. The authors validate the latent network variables by assessing their overlap with genes for which SNPs have been associated with both depression risk and change in gene expression. Finally, the authors create simple path models in order to break down the relationships between genetic risk, latent variables, and what the authors term "resilience", finding distinct path for MDD and (non-APOE) AD genetic risk. All of these analyses are then re-run using a different brain parcellation. LV2 replicates, while a new LV1 emerges with similar non-imaging variables now being correlated with a different set of distributed network nodes.

The authors conclude from this work that they have identified imaging indices of resilience manifest during adolescent brain development, and that they have found further evidence linking MDD to AD. However, the analyses do not fully support the conclusions. The premise of this work - to examine links between MDD and AD and to try to define indices of resilience during development - is fascinating and will hopefully motivate future work in this direction. However, the impact of this work as currently presented may be limited.

*STUDY STRENGTHS*

There are two premises motivating this study that deserve praise for their innovation and creativity. First, in the introduction the authors present several fairly new papers showing shared environmental and risk factors between AD and MDD. This is a very interesting line of study that certainly deserves more attention. Second, the authors are interested in finding aspects of adolescent brain development that may be helpful to understanding resilience to genetic or environmental risk later in life. The AD resilience community is very interested in contributions of early life experiences and development, but there is still very little research in this domain. I hope the authors continue to conduct research in the direction of these pursuits.

The authors demonstrate great methodological and statistical rigor in some aspects of data preprocessing and analysis. This is particularly salient in null modeling and permutation, graph-based analysis, treatment of motion for functional imaging, using eQTLs to inform disease-relevant genes, statistical considerations in PLS and path modeling, processing of Allen Brain Atlas gene expression data, and validating certain study variables. The methodology of these steps displays great attention to detail and a mastery of certain data types.

The authors reproduce all analyses using a second parcellation and carefully report the results. This type of painstaking analysis is nonetheless important in the context of network-based graph analysis that is reliant on nodal information.

*STUDY LIMITATIONS*

1) The overarching limitation of this study is that the study variables, both independent and dependent, are abstracted to the point where interpretations are challenging. The authors' own interpretations are not sufficiently justified and are often taken at face value rather than supported by analysis. These are further combined into latent variables with weak conceptual foundation, which are then abstracted even further to other analyses with cortical molecular data maps. It is not clear that the conclusions drawn are convincingly supported by this highly abstracted analysis.

2) The other major limitation of this study is that several PLS models are run but, while appropriate null modeling is used to identify "significant" LVs, none of the LVs are cross-validated. Null modeling can help to protect against overfitting to noise in data, but it does not necessarily provide a good index of generalizability nor reliability. Without cross-validation, I question the reliability of the LVs irrespective of how they are interpreted. This is once again partially driven by the fact that changing the atlas resulted in a different imaging LV.

3) The study notes that participants were selected based on "having contributed high-quality data on all measures of interest". This is of course meritorious from a methodological perspective, but the authors should be aware that this may create an important selection bias (10.1007/s11682-022-00665-2, 10.1016/j.ynirp.2022.100085, 10.1016/j.neuroimage.2022.119296)

4) The premise of this paper was interesting, as described in the Strengths section above. However, what was missing was a clear theory or hypothesis as to how resilience to AD and MDD are related, and how the analyses in this study were conducted in order to support that hypothesis. The relevance of the results to AD was not clear; a clear biological model would help put the pieces together.

5) The selection of relevant features involved in LVs was inconsistent. At several points, the authors use an arbitrary threshold of bootstrap ratio (BSR) > 4, which they equated to a p-value. A p-value doesn't make sense in this context, since bootstrap samples are not independent samples. Instead, features should be selected based on 95% CIs that don't cross 0, which the authors do in some places but not in others.

Reviewer #1 (Public Review):

The present study combines quantitative histomorphometry, live cell imaging and tracking, functional analyses, and computational modeling to define potentially pathologic interactions between lung CD8 T cells and fibrocytes in human COPD. The authors use multiple technical approaches to establish the close proximity of CD8 T cells with fibrocytes in peri-bronchial tissue in COPD subjects that notably correlate with functional disease parameters (FEV1/FEV). Their follow-on studies identify specific chemokine pathways and inflammatory consequences of these interactions. Collectively, these seminal data acquired in a unified experimental context, provide support for pathogenic interactions between lung CD8 T cells and fibrocytes and now offer the consideration of mediators and pathways that may be amenable to therapeutic targeting. The strength of the study is the integration of the multi-modality approach, the quality of the quantitative data, and the creation of a tenable model for the interaction role in COPD of CD8 T cells and fibrocytes. While both have been previously implicated in COPD, this new study is more definitive by using this integrated approach.

Reviewer #1 (Public Review):

While the mechanism about arm-races between plant and specialist herbivores has been studied, such as detoxification of specific secondary metabolites, the mechanism of the wider diet breadth, so-called generalist herbivores have been less studied. Since the heterogeneity of host plant species, the experimental validation of phylogenetic generalism of herbivores seemed as hard to be conducted. The authors declared the two major hypotheses about the large diet breadth ("metabolic generalism" and "multi-host metabolic specialism"), and carefully designed the experiment using Drosophila suzukii as a model herbivore species.

By an untargeted metabolomics approach using UHPLC-MS, authors attempted to falsify the hypotheses both in qualitative- and quantitative metabolomic profiles. Intersections of four fruit (puree) samples and each diet-based fly individual samples from the qualitative data revealed that there were few ions that occur as the specific metabolite in each diet-based fly group, which could reject the "multi-host metabolic specialism" hypothesis. Quantitative data also showed results that could support the "metabolic generalism" hypothesis. Therefore, the wide diet breadth of D. suzukii seemed to be derived from the general metabolism rather than the adaptive traits of the diverse host plant species. On the other hand, the reduction of the metabolites (ions) set using GLM seemed logical and 2-D clustering from the reduced ions set showed that quantitative aspects of diet-associated ions could classify "what the flies ate". These interesting results could enhance the understanding of the diet breadth (niche) of herbivorous insects.

The authors' approach seemed clear to falsify the hypotheses based on the appropriate data processing. The intersection of shared ions from the qualitative dataset could distinguish the diet-specific metabolites in flies and commonly occurring metabolites among flies and/or fruits. Also, filtering on the diet-specific ions seemed to be a logical and appropriate way. Meanwhile, the discussion about the results seemed to be focused on different points regarding the research hypotheses which were raised in the introduction part. Discussion about the results mainly focused on the metabolism of D. suzukii itself, rather than the research hypotheses and questions that were raised from the evolution of the wide diet breadth of generalist herbivores. In particular, the conclusion seems to be far from the main context of the authors' research; e.g. frugivory. It makes the implication of the study weaker.

Reviewer #1 (Public Review):

Much experimental work on understanding how the visual system processes optic flow during navigation has involved the use of artificial visual stimuli that do not recapitulate the complexity of optic flow patterns generated by actual walking through a natural environment. The paper by Muller and colleagues aims to carefully document "retinal" optic flow patterns generated by human participants walking a straight path in real terrains that differ in "smoothness". By doing so, they gain unique insights into an aspect of natural behavior that should move the field forward and allow for the development of new, more principled, computational models that may better explain the visual processing taking place during walking in humans.

Strengths:<br /> Appropriate, state-of-the-art technology was used to obtain a simultaneous assessment of eye movements, head movements, and gait, together with an analysis of the scene, so as to estimate retinal motion maps across the central 90 deg of the visual field. This allowed the team to show that walkers stabilize gaze, causing low velocities to be concentrated around the fovea and faster velocities at the visual periphery (albeit more the periphery of the camera used than the actual visual field). The study concluded that the pattern of optic flow observed around the visual field was most likely related to the translation of the eye and body in space, and the rotations and counter-rotations this entailed to maintain stability. The authors were able to specify what aspects of the retinal motion flow pattern were impacted by terrain roughness, and why (concentration of gaze closer to the body, to control foot placement), and to differentiate this from the impact of lateral eye movements. They were also able to identify generalizable aspects of the pattern of retinal flow across terrains by subsampling identical behaviors in different conditions.

Weaknesses:<br /> While the study has much to commend, it could benefit from additional methodological information about the computations performed to generate the data shown. In addition, an estimation of inter-individual variability, and the role of sex, age, and optical correction would increase our understanding of factors that could impact these results, thus providing a clearer estimate of how generalizable they are outside the confines of the present experiments.

Reviewer #1 (Public Review):

Habituation to noxious insults is a conserved mechanism that may act through varying pain-sensitivity thresholds based on previous sensory experience. Impaired regulation of nociceptive habituation may lead to a chronic pain condition. In the current manuscript, the authors identified additional structural elements of the CaM kinase-1 that regulate the protein shuttling between the cytosol and nucleus during nociceptive habituation. Based on the presented findings, we get a more complex regulatory model and a better understanding of the CMK-1 protein redistribution during stimulation-dependent nociceptive plasticity.

The data is carefully planned and results conclusively support the claims of the authors. The performed experiments are easy to follow and the results obtained are robust and statistically well-powered. The complex regulatory model presented in the manuscript is well supported by the reported data. Finally, the presented data presents a complex and dynamic mechanism of nuclear import and export rates of the CMK-1 protein to control nociceptive plasticity.

Reviewer #1 (Public Review):

In this work, the authors propose a phenomenological grounded theoretical framework to explain why microbial taxonomic richness can show positive, unimodal, as well as negative diversity-temperature gradients. They thus propose to introduce a temperature dependence in the form of the Boltzmann-Arrhenius equation in both species' competitive interaction and growth rates. By means of a mean-field-like approximation, they estimate the probability of having N feasible coexisting species as a function of the normalized growth rate, and average competition strength, which in turn depends on temperature. They find that the shape of the microbial community temperature-richness relationship depends on how rapidly the strength of competition between species pairs increases with temperature relative to an increase in the variance of their growth rates. Furthermore, the mean-field result predicts that the position of richness peak depends on the sign of the covariance between the two main parameters of the Boltzmann-Arrhenius law. Finally, they show that the real-world community-level temperature-richness responses observed are qualitatively reproduced by their model.

I found the work interesting and stimulating, surely tackling a relevant research question such as the effect of thermal physiology on biodiversity patterns through a simple, but quantitative model. Overall, I like the proposed approach.

At the same time, the central mathematical results are not clear in my view, some strong approximations are not discussed, but they hold only in very specific conditions. A lot of important details are missing or scattered here and there, the notation is a little sloppy, and in general, it has been difficult for me to reproduce their finding.

The overall structure and flow of the manuscript can be remarkably improved.

Reviewer #1 (Public Review):

The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is a G4C2 repeat expansion within the first intron of the C9ORF72 gene. However, how this repeat contributes to disease pathology is still an active area of research. This study takes a targeted approach to analyzing specifically how the C9ORF72 antisense transcript (C4G2) may be contributing to FTD/ALS.

Using an artificial (C4G2)75 antisense cassette, the authors show in both HEK293T cells and cultured neurons that the C4G2 antisense transcript leads to elevated levels of activated PKR and increased phosphorylated eIF2alpha. This then leads to a decreased level of translation, the formation of stress granules, and decreased survival, phenotypes that can be suppressed through the knockdown of PKR. The authors nicely demonstrate that PKR activation upon transfection with their antisense cassette is independent of toxic dipeptide repeat proteins by using reporter constructs that do not create these dipeptides but are still able to activate PKR. Furthermore, using a construct that expresses both sense and antisense transcripts, the authors show that knockdown of the antisense, but not the sense transcript, abrogates the PKR response (demonstrating the specificity of this stress pathway for the antisense RNA). The authors additionally show the relevance of PKR activation in FTD/ALS through the presence of activated PKR and elevated eIF2alpha in ALS postmortem brain tissue.

This paper shows that, at least in model systems, the C4G2 transcript can have cytotoxic effects through the stimulation of PKR. The experiments are well-controlled and fairly comprehensive. The claim that PKR activation occurs via the antisense RNA, and not the sense, is well supported by the data. However, some limitations exist, some of which the authors explicitly recognize. They are as follows:<br /> 1. It is not clear how the results from these reporter constructs inform on the repeat expansion RNAs produced in disease, which can be significantly longer, and might be expressed at different levels. Perhaps if the C4G2 repeat used in this work were expressed at levels comparable to what the antisense transcript is expressed in an actual disease, or in a similar RNA context, PKR would not be activated. This is important to keep in mind.<br /> 2. It is still unclear how PKR is being activated in the presence of C4G2 (it could be direct or indirect). The authors list a variety of explanations in the discussion. A prior study has shown that a similar repeat expansion leads to the accumulation of cytoplasmic dsRNA inclusions marked by TDP-43 (Rodriguez et al., 2021). It would be interesting to see if these inclusions are present upon expression of the antisense construct.<br /> 3. In the context of C9ORF72 FTD/ALS disease, it is still difficult to say how much of the disease pathology is on account of antisense triggered stress responses as opposed to dipeptide repeat, RBP titration, etc. This study nevertheless provides a new perspective to consider for how the C9ORF72 repeat expansion contributes to the diseased state.

Reviewer #1 (Public Review):

In the manuscript "Staphylococcus aureus FtsZ and PBP4 bind to the conformationally dynamic N-terminal domain of GpsB", Sacco et. al. solved the crystal structure of S. aureus GpsB, an essential cell growth and division protein. The authors also identified its interactions with the master regulator of cell division FtsZ and a penicillin-binding protein PBP4 that is implicated in B-lactam insensitivity. Although GpsB is essential for growth in S. aureus the reason for its essentiality is poorly understood. The authors used biochemical, biophysical, and crystallographic methods to determine the structure of GpsB and characterized its binding with FtsZ and PBP4. The authors also solved the co-crystal structure of GpsB with the C-terminal peptide of PBP4. These results are significant because it details the interactions of an essential growth protein in S. aureus with known cell division proteins. However, the impact of the work could be further enhanced if the authors had more functional studies to demonstrate the importance of the new hinge motif, the binding with FtsZ C-terminal tail, and PBP4.

Reviewer #1 (Public Review):

The authors present a study to test the relationships between a measured dopamine marker in the brain - so-called, dopamine synthesis capacity - and various other measures purported to index dopamine function. These measures include questionnaire answers about behaviour, and measured behaviour. Various studies have used these other measures as indices or proxies of dopamine function with some evidence to support this. However, some of the evidence is in small groups or indirect.

The major strength of this study is the size of the sample (n=66-94) compared to other studies and the three different analytical strategies employed - frequentist, Bayesian, and predictive modelling.

Areas, where the study is more limited, are the use of only one marker of dopamine neurochemistry ([18F]FDOPA) and this does not discount relationships with other markers such as pre-synaptic receptors, post-synaptic receptors, and dynamic release. The authors acknowledge that this study does not speak to the general principle of dopamine relationships with other measures. While the numbers are impressive for this type of study the use of correlation means their power is for correlations of 0.32-0.37 and higher (G*power). It is possible genuine relationships between markers do exist but all studies to date, including this one, are underpowered. The Bayesian analysis conducted speaks to this and is a welcome addition. It is also possible that the conclusions are restricted by the participants recruited as they are limited to the ages of 18-43 and it is not clear how representative they are of the general community from the information provided.

The dopamine system is not one entity in terms of system components (pre-synaptic, post-synaptic, etc), but also in terms of subcortical area with a gradient of input from the brainstem and a distinct connectionist anatomy between the striatum and the cortex (via other structures). Here the authors use a segmentation of the striatum to test the relationships. While this is embedded in the methods and results the introduction's treatment of the subcortical dopamine system is as a single entity. This could be improved.

The results of this work have an important impact in that they strongly suggest one cannot use proxies to estimate endogenous neurochemistry (at least in the dopamine system). However, this implies that any other proxy for any other system needs to be (re-)assessed using similar methods. This is not to say that the proxies are not sensitive to dopamine manipulations, but that they cannot by themselves be used instead of direct measurement. Given the number of studies which suggest that a measure of baseline state may predict the effects of dopaminergic drugs, one must question what the baseline state is being measured.

Despite these limitations, the authors have provided the largest assessment of the relationships between [18F] FDOPA-assessed dopamine synthesis capacity and various markers previously linked to dopamine function. In this respect, it is an important negative. This does mean that the assessments used cannot be used to assess 'baseline' states in relation to dopaminergic drug effects, but the mechanism through which this baseline dependency operates is not well understood.

Reviewer #1 (Public Review):

The manuscript by Lujan and colleagues describes a series of cellular phenotypes associated with the depletion of TANGO2, a poorly characterized gene product but relevant to neurological and muscular disorders. The authors report that TANGO2 associates with membrane-bound organelles, mainly mitochondria, impacting in lipid metabolism and the accumulation of reactive-oxygen species. Based on these observations the authors speculate that TANGO2 function in Acyl-CoA metabolism.

The observations are generally convincing and most of the conclusions appear logical. While the function of TANGO2 remains unclear, the finding that it interferes with lipid metabolism is novel and important. This observation was not developed to a great extent and based on the data presented, the link between TANGO2 and acyl-CoA, as proposed by the authors, appears rather speculative.

1. The data with overexpressed TANGO2 looks convincing but I wonder if the authors analyzed the localization of endogenous TANGO2 by immunofluorescence using the antibody described in Figure S2. The idea that TANGO2 localizes to membrane contact sites between mitochondria and the ER and LDs would also be strengthened by experiments including multiple organelle markers.

2. The changes in LD size in TANGO2-depleted cells are very interesting and consistent with the role of TANGO2 in lipid metabolism. From the lipidomics analysis, it seems that the relative levels of the main neutral lipids in TANGO2-depleted cells remain unaltered (TAG) or even decrease (CE). Therefore, it would be interesting to explore further the increase in LD size for example analyze/display the absolute levels of neutral lipids in the various conditions.

3. Most of the lipidomics changes in TANGO2-depleted cells are observed in lipid species present in very low amounts while the relative abundance of major phospholipids (PC, PE PI) remains mostly unchanged. It would be good to also display the absolute levels of the various lipids analyzed. This is an important point to clarify as it would be unlikely that these major phospholipids are unaffected by an overall defect in Acyl-CoA metabolism, as proposed by the authors.

Reviewer #1 (Public Review):

The manuscript provides a comprehensive analysis of the consequences of a mutation in WDR62 in human pluripotent stem cell-derived progenitor cells and neurons. The experiments are logical and presented well. The data support the conclusion that WDR62 dysfunction causes impaired cell cycle progression and defective neuronal differentiation. The data corroborate previous findings in mouse and human cells and cell lines and extend knowledge to cells that are relevant to the microcephaly characteristic of individuals with WDR62 mutations. The major shortcoming of the data is that it relies on cells from a single donor and so requires additional validation to support the generalization of the conclusions. In addition, limited mechanistic insight is provided.

Reviewer #1 (Public Review):

The authors attempted to delete a rhodopsin allele with single-nucleotide mutation seen in a Chinese subpopulation of autosomal dominant retinitis pigmentosa patients, (Rho-T17M). This was done in vitro and in vivo, while keeping the Rho wild type allele intact in vitro and in vivo using CRISPR-SaCAS9 guide RNA-specific approach, a previously established technique. In this study, solid in vitro data was presented showing that one of the tested guide RNAs was effective to specifically delete targeted the Rho-T17M sequence of synthetic DNA as well as in iPSCs from RP patients. However, the in vivo part of this study is incomplete. The issues are: 1. confusing choice of disease animal model (Rho-5m mice that carry 4 additional rhodopsin mutations other than the targeted T17M); 2. no proof of gene editing efficiency at the cellular level of the targeted cell type (i.e. what percentage of rod photoreceptors lose the T17M disease mutation?); and 3) lack of evidence of therapeutic potential (i.e. is there any rescue of vision in the mouse disease model or any toxicity due to the vector itself?).

Reviewer #1 (Public Review):

According to current knowledge, zebrafish neurons maintain the capacity of regenerating with the exception of adult cerebellar Purkinje cells (PC), which are thought to have lost this property. Regeneration instead occurs at larval stages but whether newly generated PC form fully functional circuits is still unclear. This elegant and well-performed study takes advantage of a transgenic zebrafish line that enables inducing apoptosis under a tamoxifen-inducible system and at the same time visualizes PCs morphology through a membrane tagged RFP. Using this line (and other lines that tag radial glial and ventricular progenitors) in combination with morphological and functional analysis, the authors show that ventricular progenitors retain the lifelong ability to regenerate PCs. At larval stages, the newly regenerated PCs form fully functional circuits that lead to normal behavior. In adults, PC regeneration is less efficient (and PCs are also less prone to undergo apoptosis) but sufficient to support exploratory behavior. This study resolves the controversial issue of whether adult PC regeneration is possible and demonstrates that newly formed PCs at larval and adult stages can form functional circuits that support normal behavior.

This is a well-performed and carefully executed and quantified study. There is however a point that needs clarification:

The authors state that acute regeneration occurs between 5-10dpt. However, the graphs in Fig 1D, F, and 2F indicate that most PC generation occurs from 20-30 days. What happens in this period? Does proliferation increase? Can the authors perform BrdU incorporation between 6 days and 1 month? Related to this, as the authors indicate in lines 129-131, the regeneration of new PCs overlaps with normal development. Are other neuronal cell types generated in appropriate numbers?

Reviewer #1 (Public Review):

In this manuscript, Gonzalez et al investigated the dynamics of dopamine signals, measured with optophysiological methods in the lateral shell of the nucleus accumbens (LNAc), in response to different types of visual stimuli. Contrary to most current theories of dopamine signaling, the authors found that LNAcc dopamine transients tracked sensory transitions in visual stimulation rather than any immediately apparent motivational variable. This unorthodox finding is of potential interest to the field, as it suggests that dopamine in this particular area of the striatum supports a very different, albeit unclear behavioral function than what has been previously attributed to this neuromodulator. Many of the approaches used by the authors were very elegant, like the careful selection of visual stimuli parameters and the use of Gnat1/2 KO mice to demonstrate that the dopamine responses were directly dependent on the visual stimulation of rods and cones. That said, the authors did not discuss how their findings relate to much previously published work, many of which offer potential alternative explanations for their results. It is also not clear from the manuscript text which mice were used for which experiments, and how testing history might affect the results.

Reviewer #1 (Public Review):

The endothelin ETB receptor is a G-protein coupled receptor activated by vasoactive peptide endothelins, causing vaslorelaxtion in smooth muscle. By determining the Cryo EM structure of human ETB in complex with the vasoconstricting peptide ET-1 and the inhibitory G-protein (Gi), the study represents a convincing insight into agonist-induced receptor activation and transducer-coupling. The complex structure is solid and will appeal to the GPCR and pharmacology communities.

Strengthens: The authors have managed to obtain the first G-protein complex structure of an ETB receptor by working with a receptor that still retains G-protein coupling (i.e. not a thermostabilized mutant) and by developing new methodologies into how the G-protein is remotely tethered to the GPCR. The Cryo EM structural details highlight clear differences into how the G-protein binds that also includes the more downward movement of TM7.

Weaknesses: While it is technically challenging to obtain an endothelin-1-ETB-Gi complex, the fusion approach means that there is equilibrium is already pushed towards a complex that may otherwise require lipids, such as PIP2. Whilst I don't know what may alter how alpha 5 interacts with ETB, this cannot be ruled out either.

Reviewer #1 (Public Review):

This manuscript describes efforts to understand how independence from ribonucleotide reduction might evolve in obligate intracellular bacterial pathogens using E. coli as a model for this process. The authors successfully deleted the three ribonucleotide reductase (RNR) operons present in E. coli and showed that growth of this knockout strain can be achieved with deoxyribonucleotide supplementation. They also performed evolutionary experiments and analysis of cell growth and morphology under conditions of low nucleotide availability. In this work, they established that certain genes are consistently mutated to compensate for the loss of RNR activity and the low availability of deoxynucleotides. Comparison to genomes of intracellular pathogens that lack RNR genes shows that these patterns are largely conserved.

While the experimental results support the conclusions of the study, the authors do report changes in cell morphology upon the growth of the RNR knockout strains with low concentrations of nucleotides. It would be ideal to note this complication earlier in the manuscript. And to clarify how the possibility of cell elongation might affect the OD measurements in Figure 3 describing the experiments to establish that dC is necessary for growth in the knockout strain. It would also be ideal to provide a more detailed explanation for that observation in the discussion.

Reviewer #1 (Public Review):

This work presents a unification model (of sorts) for explaining how the flow of evidence through networks can be controlled during decision-making. The authors combine two general frameworks previously used as neural models of cortical decision-making, dynamic normalization (that implement value encoding via firing activity) and recurrent network models (which capture winner-take-all selection processes) into a unified model called the local disinhibition-based decision model (LDDM). The simple motif of the LDDM allows for the disinhibition of excitatory cells that represent the engagement of individual actions that happens through a recurrent inhibitory loop (i.e., a leaky competing accumulator). The authors show how the LDDM works effectively well at explaining both decision dynamics and the properties of cortical cells during perceptual decision-making tasks.

All in all, I thought this was an interesting study with an ambitious goal. But like any good study, there are some open issues worth noting and correcting.

MAJOR CONCERNS

1. Big picture

This was a comprehensive and extremely well-vetted set of theoretical experiments. However, the scope and complexity also made the take-home message hard to discern. The abstract and most of the introduction focus on the framing of LDDM as a hybrid of dynamic normalization models (DNM) and recurrent network models (RNMs). This is sold as a unification of value normalization and selection into a novel unified framework. Then the focus shifts to the role of disinhibition in decision-making. Then in the Discussion, the goal is stated as to determine whether the LDDM generates persistent activity and does this activity differ from RNMs. As a reader, it seems like the paper jumps between two high-level goals: 1) the unification of DNM and RNM architectures, and 2) the role of disinhibition. This constant changing makes it hard to focus as the reader goes on. So what is the big picture goal specifically?

Also, the framing of value normalization and WTA as a novel computational goal is a bit odd as this is a major focus of the field of reinforcement learning (both abstractly at the computational level and more concretely in models of the circuits that regulate it). I know that the authors do not think they are the first to unify value judgements with selection criteria. The writing just comes across that way and should be clarified.

2. Link to other models

The LDDM is described as a novel unification of value normalization and winner-take-all (WTA) selection, combining value processing and selection. While the authors do an excellent job of referencing a significant chunk of the decision neuroscience literature (160 references!) the motif they end up designing has a highly similar structure to a well-known neural circuit linked to decision-making: the cortico-basal ganglia pathways. Extensive work over the past 20+ years has highlighted how cortical-basal ganglia loops work via disinhibition of cortical decision units in a similar way as the LDDM (see the work by Michael Frank, Wei Wei, Jonathan Rubin, Fred Hamker, Rafal Bogacz, and many others). It was surprising to not see this link brought up in the paper as most of the framing was on the possibility of the LDDM representing cortical motifs, yet as far as I know, there does not exist evidence for such architectures in the cortex, but there is in these cortical-basal ganglia systems.

3. Model evaluations

The authors do a great job of extensively probing the LDDM under different conditions and against some empirical data. However, most of the time there is no "control" model or current state-of-the-art model that the LDDM is being compared against. In a few of the simulation experiments, the LDDM is compared against the DNM and RNM alone, so as to show how the two components of the LDDM motif compare against the holistic model itself. But this component model comparison is inconsistently used across simulation experiments.

Also, it is worth asking whether the DNM and RNM are appropriate comparison models to vet the LDDM against for two reasons. First, these are the components of the full LDDM. So these tests show us how the two underlying architectural systems that go into LDDM perform independently, but not necessarily how the LDDM compares against other architectures without these features. Second, as pointed out in my previous comment, the LDDM is a more complex model, with more parameters, than either the DNM or RNM. The field of decision neuroscience is awash in competing decision models (including probabilistic attractor models, non-recurrent integrators, etc.). If we really want to understand the utility of the LDDM, it would be good to know how it performs against similarly complex models, as opposed to its two underlying component models.

4. Comparison to physiological data

I quite enjoyed the comparisons of the excitatory cell activity to empirical data from the Shadlen lab experiments. However, these were largely qualitative in nature. In conjunction with my prior point on the models that the LDDM is being compared against, it would be ideal to have a direct measure of model fits that can be used to compare the performance of different competing "control" models. These measures would have to account for differences in model complexity (e.g., AIC or BIC), but such an analysis would help the reader understand the utility of the LDDM in connecting with empirical data much better.

Reviewer #1 (Public Review):<br /> <br /> The pH-dependent conformational change of the envelope protein in flaviviruses is required for the infection process, thus it represents an attractive target for drug development. In this study, the authors conducted extensive atomistic simulations for models for the envelope in six flaviviruses. Using a benzene-mapping approach, they were able to identify several cryptic binding sites that can be targeted for drug development. One of the cryptic binding site was observed in a previous study to be occupied by a detergent molecule, while the other cryptic binding site is located at domain interface. The second binding site involves a cluster of ionizable residues. Using constant pH simulations, the authors suggested that the cluster of ionizable residues contribute to the pH dependent conformational rearrangements. This cluster model helps to explain the inconsistencies reported in the literature regarding the role of several key histidine residues as pH sensors. Overall, the study has provided new mechanistic insights that can be taken advantage of in future drug developments that target flaviviruses. The work also highlights the importance of constant pH simulations to the analysis of pH sensitive biological processes.

Reviewer #1 (Public Review):

This work introduces a new computational model of healthy blood cell formation and chronic myeloid leukemia (CML). By combining data from the literature, animal experiments and patients the authors aim to develop a detailed description of the regulatory mechanisms governing healthy blood cell formation and CML therapy response. The model is used to derive hypotheses explaining why some patients respond to tyrosine kinase inhibitors (TKI) better than others. Based on the model simulations the authors seek predictors of TKI efficacy and for concepts to improve CML therapy.

Strengths:

(1) The authors start from all possible ordinary differential equation models which describe positive and negative regulations of proliferation rates and self-renewal/differentiation probabilities. The models account for hematopoietic stem cells, multipotent progenitors, terminally differentiated myeloid cells, and terminally differentiated lymphoid cells. Using an automated approach referred to as design space analysis (DSA) the authors exclude models with unfeasible qualitative dynamics. Using data from mouse experiments the authors exclude all regulatory configurations except one. This systematic approach combining model analysis and data from various sources is clearly a strength of the work.

(2) The authors consider a large number of parameter sets that are in line with physiological steady-state cell counts and realistic responses to system perturbations. Thus the authors can potentially account for inter-patient differences.

(3) The model predictions are compared to experimental and published data. The proposed predictors of TKI efficacy are tested on retrospective patient data.

Weaknesses:

(1) In my opinion the sub-model of leukemic cells requires a more solid justification. The authors assume that the configuration of regulatory loops and most key parameters are identical for normal and leukemic cells. The only difference the proposed model accounts for is that leukemic cells exhibit a weaker response to the feedback signal acting on stem cell self-renewal. The weaker response of leukemic stem cells is justified by data from the literature supporting differential responses to CCL3. However, the authors propose no justification for the assumption that all other parameters, such as proliferation rates or maximal self-renewal probabilities, are identical or have minor impacts.

(2) The authors come to the conclusion that "a key predictor of refractory response to TKI treatment is an increased probability of self-renewal of normal hematopoietic stem cells" (Abstract). This conclusion is, in my opinion, not fully supported by the model as it is. In the model, it is assumed that normal and leukemic stem cells have the same maximal self-renewal probability. Only the regulation of self-renewal by feedback signals is different. The parameter which is a predictor in the presented analysis (p_{0,max}) is the maximal self-renewal probability of both normal AND leukemic stem cells. Therefore, the conclusion that normal stem cell self-renewal is a predictor of TKI response is, in my opinion, questionable. If I understand the analysis correctly, the authors show the following: Under the assumptions that the maximal self-renewal probability of normal and leukemic stem cells is identical and that the feedback inhibition of self-renewal is weaker in leukemic stem cells compared to normal stem cells, the maximal self-renewal probability of the two stem cell populations is a predictor of TKI response. Notably, if the value of maximal self-renewal probability is increased, the self-renewal probability of leukemic and normal stem cells increases simultaneously at all time points. Therefore, I find it difficult to argue that normal stem cell self-renewal [as opposed to leukemic stem cell self-renewal] is the relevant quantity.

(3) The simulation of differentiation therapy is interesting, however, due to a lack of knowledge in the field, the specific impacts of such therapy on normal versus leukemic cell differentiation have to be rather hypothetical.

(4) The used patient cohort is very small (n = 21).

The proposed model of the regulations governing blood cell formation is a valuable contribution to the fields of computational modeling and experimental hematology. The derived predictors of TKI efficacy are potentially useful.

Reviewer #1 (Public Review):

This is a fascinating effort from the Ryan laboratory, revisiting fundamental issues of calcium-dependent release probability at cultured synapses. The authors point out that our basic understanding of mammalian synapses rests on a foundation of older research that was not acquired at physiological temperature, and represented a statistical interpretation of data acquired electrophysiologically without direct knowledge of release at individual active zones. The authors employ techniques of calcium imaging and glutamate sensing and argue that single synapses can be 'silenced' by a moderate drop in extracellular calcium, a drop that is within the range of calcium channel inhibition following activation of GABAergic signaling. While fascinating, the conclusions are most powerful when the data can be distilled to direct observation of single release sites and this is not uniformly the case.

Reviewer #1 (Public Review):

In this manuscript the authors describe the development and application of hierarchical machine learning model to identify the likely source of S. Enteritidis using whole genome sequence data. The application makes use of a collection of 2,313 genomes from 4 continents, 11 sub-regions and 38 countries. The approach is, to the best of my knowledge, novel and represents a substantial advance over previous approaches. The model is demonstrated to have good performance at the continental level and - where sufficient training data were available - also at the country level.

Strengths of the work include the clear exposition of the methods, application to a large and detailed genomic database of clinical S. Enteritidis isolates, and the use of five independent validation data-sets.

Limitations include lack of validation using post-pandemic data (as the authors state, the model may need retraining in light of changes to the global food network). Also, claimed novelties of the work include greater geographic granularity and faster turnaround time compared to alternative methods, but no explicit comparison to other methods is made.

Overall, the authors achieve their aims in describing a hierarchical machine learning model for source attribution using pathogen whole genome sequences. The approach is likely to be of broad relevance and considerable public health utility.

Reviewer #1 (Public Review):

The author constructed a novel rat model with a clinically relevant PLS3 hemizygous E10-16del mutation (PLS3E10-16del/0), which presents a classic form of early-onset osteoporosis, which recapitulate the osteoporotic phenotypes. Treatment with alendronate and teriparatide significantly improved bone mass and bone microarchitecture. Their results showed alendronate and teriparatide treatment could be a potential treatment for early-onset osteoporosis induced by PLS3 mutation.

This experiment is very interesting and has clinical relevance. The authors used common clinical drugs to treat osteoporosis caused by PLS3 mutation and achieved certain results. This result will give a way to the treatment of osteoporosis induced by PLS3 mutation.

Reviewer #1 (Public Review):

The authors have achieved a demonstration of different stellate ganglion nerve cell functions and transmitter subtypes, of potential cardiac importance. They employ viral tracing techniques. These convincingly make this demonstration. The work will be key to our understanding of sympathetic function at the transmitter and physiological levels.

Reviewer #1 (Public Review):

Pedigo et al, apply statistical modeling to a complete brain nanoscale network - a synaptic connectome of an insect brain: the Drosophila larva. They use a series of approaches to explore the symmetry of the right and left hemispheres. First, they compare network densities and find significant differences between the two hemispheres, with the right hemisphere having a higher density. They further grouped neurons by cell type to determine whether the differences were distributed across the entire brain or to specific connections and find the differences involving neurons in the learning and memory center, the mushroom body. Finally, they explored different definitions of an edge by using different thresholds either based on synaptic counts or proportions of synaptic inputs to a downstream neuron and found that when using the proportion of synaptic inputs, removing fewer edges (compared to when using synaptic count) was necessary to achieve left and right symmetry. The presentation of the methodology and writing is very clear and effective and is accessible to scientists from various backgrounds: both biologists and theoreticians. The methodology and approach used in this paper on the assessment of the degree of bilateral symmetry will serve as a basis for comparing networks and connectomes in general by providing a clear framework for statistical network modeling. This work is particularly timely as an increasing number of synaptic connectomes is being generated giving opportunities for various connectome comparisons. It will be of interest to neuroscientists in order to address various biological questions: to evaluate the degree of inter-individual variability/stereotypy of connectivity in the brain and how it relates to behavioral variability/stereotypy, to characterize changes in network connectivity due to different diseases, etc.

Reviewer #1 (Public Review):

Tornini et al. investigate the function of long non-coding RNAs in vivo. In the manuscript, the authors show that two of these molecules linc-mipep and linc-wrb encode for a micropeptide that regulates zebrafish behavior. In the absence of this peptide, zebrafish larvae show dysregulation of NMDA receptor and glucocorticoid receptor-mediated signaling and immediate early gene induction. Given the homology of linc-mipep and linc-wrb encoded peptides with homology to chromosome binding and chromatin unwinding domain of HMGN1 the authors explore the altered chromatin accessibility in the mutant animals. This analysis revealed a broad dysregulation of 3D chromatin structure with some enrichment at loci regulating the expression of immediate early response genes. Finally, single cell analysis revealed that oligodendrocyte progenitor cells and cerebellar granule cells are more affected in the mutants.

This work represents a technical tour-de-force with extensive genomics data to characterize the molecular phenotype of linc-mipep and linc-wrb loss of function. This data show interesting findings in part consistent with the behavioral phenotype observed.

The manuscript provides compelling evidence that micropeptides encoded by what were previously identified as long non-coding RNAs have a precise biological function.

Reviewer #1 (Public Review):

This study presents a useful study, proposing the modelling of Buruli ulcer occurrence in humans based on detection of M. ulcerans in Australia. The data were collected and analyzed using solid and validated methodology and can be used as a starting point for the elucidation of M. ulcerans transmission in Australia.

Reviewer #1 (Public Review):

Francou et al. examine the dynamics of cell ingression at the primitive streak during mouse gastrulation and correlate this with the localization of elements of the apical Crumbs complex and the actomyosin cytoskeleton. Using time-lapse live imaging, they show that cells at the primitive streak ingress in a stochastic manner, by constricting their apical surface through a ratcheting shrinkage of individual junctions. Meticulous evaluation of immunofluorescent staining for many elements of the actomyosin contractile process as well as junctional and apical domain elements reveals anisotropic localization of Crumbs2, ZO1, and ppMLC. In addition, the localization of two groups of proteins showed a close correlation - actomyosin regulators and apical and junctional components - but there was a lack of correlation of localization of these two groups of proteins to each other. The localization of actomyosin and its activity, was altered and more homogeneous in Crumbs2-/- embryos, and there was a significant decrease in aPKC and Rock1. The authors conclude from these observations that Crumbs2 regulates anisotropic actomyosin contractility to promote apical constriction and cell ingression.

The strengths of this manuscript are the very detailed observations on the process of apical constriction and the meticulous evaluation of the localization of the many proteins likely to be involved in the process. While many of the general observations are not new, Francou et al. provide a much richer understanding of this process, as well as a paradigm with which to evaluate the effects of mutations on the gastrulation process. The figures are beautiful, clear, and informative, and support the conclusions made by the authors. The data provide a very compelling picture of both the dynamics of cell behavior and the anisotropies in protein localization associated with it.

However, much of the Crumbs2 mutant phenotype is not sufficiently explained by the authors' data or conclusions. First, the loss of Crumbs2 does not prevent ingression, as there are mesoderm cells evident between the epiblast and endoderm (Ramkumar et al., 2016, Xiao et al., 2011). There are certainly fewer, and the biggest effect appears to be during the elongation of the axis from E7.75 onward and not during the earlier migratory period (E6.5-E7.75) according to data from both previously published work (Xiao et al., 2011; Ramkumar et al., 2015, 2016) and the data presented here. Nor does the loss of Crumbs2 prevent apical constriction. Ramkumar et al. in their 2016 paper show by live imaging that the major effect of the Crumbs2 mutation is to prevent the cells from detaching from the epithelium, but that the apical domain does undergo constriction, leading to many elongated flask-shaped cells still attached at the apical end. These observations do not fit well with the model proposed by the authors of Crumbs2 regulating anisotropic actomyosin contractility to promote apical constriction and suggest a more complicated story. However, the complications of the Crumbs2 mutant do not detract from the value of the basic observations presented in this manuscript, which are solid and well-documented, and will be a valuable resource for the field.

Reviewer #1 (Public Review):

In this study, Sapiro et al sought to develop technology for a transcriptomic analysis of B. burgdorferi directly from infected ticks. The methodology has exciting implications to better understand pathogen RNA profiles during specific infection timepoints, even beyond the Lyme spirochete. The authors demonstrate successful sequencing of the B. burgdorferi transcriptome from ticks and perform mass spectrometry to identify possible tick proteins that interact with B. burgdorferi. This technology and first dataset will be useful for the field. The study is limited in that no transcripts/proteins are followed-up by additional experiments and no biological interactions/infectious-processes are investigated.

Critiques and Questions:

This study largely develops a method and is a resource article. This should be more directly stated in the abstract/introduction.

Details of the infection experiment are currently unclear and more information in the results section is warranted. State the species of tick and life-stage (larval vs nymphal ticks) used for experiments. For RNA-seq, are mice are infected and ticks are naïve or are ticks infected and transmitting Borrelia to uninfected mice?

What is the limit of detection for this protocol? Experimental data should be provided about the number of B. burgdorferi required to perform this approach.

More information regarding RNA-seq coverage is required. Line 147-148 "read coverage was sufficient"; what defines sufficient? Browser images of RNA-seq data across different genes would be useful to visualize the read coverage per gene. What is the distribution of reads among tRNAs, mRNAs, UTRs, and sRNAs?

My lab group was excited about the data generated from this paper. Therefore, we downloaded the raw RNA-seq data from GEO and ran it through our RNA-seq computational pipeline. Our QC analysis revealed that day 4 samples have a different GC% pattern and that a high percentage of E. coli sequences were detected. This should be further investigated and addressed in the paper: Are other bacteria being enriched by this method? Why would this be unique to day 4 samples? Does this affect data interpretation?

Comprehensive data comparisons of this study and others are warranted. While the authors note examples of known differentially expressed genes (like lines 235-241), how does this global study compare to other global approaches? Are new expression patterns emerging with this RNA-seq approach compared to other methods? What differences emerged from day 1 to day 4 ticks compared to differences observed in unfed to fed ticks or fed ticks to DMC experiments? Directly compare to the following studies (PMID: 11830671; PMID: 25425211; PMID: 36649080).

Details about the categorization of gene functions should be further described. The authors use functional analysis from Drechtrah et al., 2015, but that study also lacks details of how that annotation file was generated. Here, the authors have seemed to supplement the Drechtrah et al., 2015 list with bacteriophage and lipoprotein predictions - which are the same categories they focus their findings. Have they introduced a bias to these functional groups? While it can be noted that many lipoproteins are upregulated (or comment on specific genes classes), there are even more "unknown" proteins upregulated. I argue that not much can be inferred from functional analysis given the current annotation of the B. burgdorferi genome.

Reviewer #1 (Public Review):

In this manuscript, the authors used an unbiased method to identify proteins from porcine oocyte extracts associated with permeabilised boar spermatozoa in vitro. The identification of the proteins is done by mass spectrometry. A previous publication of this lab validated the cell-free extract purification methods as recapitulating early events after sperm entry in the oocyte. This novel method with mammalian gametes has the advantage that it can be done with many spermatozoa at the time and allows the identification of proteins associated with many permeabilised boar spermatozoa at the time. This allowed the authors to establish a list of proteins either enriched or depleted after incubation with the oocytes extract or even only associated with spermatozoa after incubation for 4h or 24h. The total number of proteins identified in their test is around 2 hundred and with very few present in the sample only when spermatozoa were incubated with the extracts.

The list of proteins identified using this approach and these criteria provide a list of proteins likely associated with spermatozoa remnants after their entry and either removed or recruited for the transformation of spermatozoa-derived structures.

Using WB and histochemistry labelling of spermatozoa and early embryos using specific antibodies the authors confirmed the association/dissociation of 6 proteins suspected to be involved in autophagy.

While this unique approach provides a list of potential proteins involved in sperm mitochondria clearance it's (only) a starting point for many future studies and does not provide the demonstration that any of these proteins has indeed a role in the processes leading to sperm mitochondria clearance since the protein identified may also be involved in other processes going-on in the oocyte at this time of early development.

Concerning the localisation of the 6 proteins further analysed, the authors must add how much the presented picture represents the observed patterns. They must include the details on the fraction of spermatozoa and embryos displaying the presented pattern.