Reviewer #2 (Public Review):

The evolution and control of the three-part life history of holometabolous insects have been controversial issues for over a century. While the functioning of broad as a master gene controlling the pupal stage and of E93 as a master gene for the adult stage has been known for about a decade or more, chinmo has only recently been proposed as being the master gene responsible for maintaining the larval stage (Truman & Riddiford, 2022). While the former paper focused on the embryonic and early larval function of Chinmo, this paper explores its metamorphic effects and defines the roles of Broad and E93 in the phenotypes produced by manipulations of Chinmo expression.

Overall, the paper is well presented but in places, readers would be helped if the authors were more explicit about the logic and details of their manipulations. There are a couple of conceptual issues that the authors should address.

The role of Broad in larval tissues:<br /> One intriguing issue relates to the relationship of Chinmo to Broad and E93 in larval versus imaginal tissues prior to metamorphosis. The knock-down of chinmo in imaginal discs results in severe suppression of growth and the lack of metamorphic patterning genes such as cut and wingless. Normal growth and patterning are reestablished though, if broad is also knocked-down, supporting the notion that the effects of the lack of Chinmo are mediated through the premature expression of Broad.<br /> In the salivary glands, by contrast, chinmo knock-down suppresses growth, and this growth suppression is not reversed by simultaneous broad knockdown. They properly conclude that the role of Chinmo in supporting the growth of larval tissues does not involve Broad, but their data on the expression of salivary gland proteins suggest that Broad still plays some role in Chinmo function in salivary glands. Fig. 5E shows the levels of various salivary glue proteins in the glands of Chinmo knock-down larvae. The levels are reduced, as expected by the lack of salivary gland growth, but a significant finding is that they are there at all! The Costantino et al. (2008) paper shows that these genes are only induced in the mid-L3. Ecdysone, acting through Broad isoforms, is necessary for their appearance and these SGS genes can be induced in the L1 and L2 stages by ectopic expression of some Broad isoforms. Their low levels in Fig 5, would be due to the small size of the gland, but the gland's premature expression of Broad likely causes their induction. In larval cells, then, Chinmo may feed into two parallel pathways, one that does not involve broad and regulates growth and the other, utilizing Broad, regulating premetamorphic changes.<br /> It would be useful to look at early larval salivary gland proteins such as ng-1 to -3 that are expressed in salivary glands before the critical weight. Also, it would be interesting if the appearance of the SGS proteins after chinmo knock-down (Fig 5E) is abolished by simultaneous knock-down of broad.

Role of Chinmo and Broad in Hemimetabolous insects:<br /> In the conclusion of their comparative studies on the cockroach (line 342), the authors state that Broad exerts no role in the development of hemimetabolous insects. However, this conclusion is not consistent with the literature. The first study of broad knockdown in a hemimetabolous insect was in the milkweed bug Oncopeltus fasciatus by Erezyilmaz et al. (2006). Surprisingly to Erezyilmaz et al., broad knock-down in early-stage nymphs did not cause premature metamorphosis. However, Broad expression was essential for tissues of the wing pads and dorsal thorax to undergo morphogenetic growth (rather than simple isomorphic growth), and for stage-specific changes in coloration through the nymphal series (but not for the nymph to adult color change). A similar function for Broad on wing growth during the later nymphal stages was later shown in Blattella (Fernandez-Nicolas et al., 2022; Huang et al., 2013). The wing- and genital pads represent "imaginal" tissues in the nymph and the need for Broad in these tissues are the same as seen in imaginal discs as the latter shift from isomorphic growth to morphogenesis at the critical weight checkpoint in the L3.<br /> This would suggest that important roles for Broad and E93 are already established in the hemimetabolous insects with E93 controlling the shift from immature (nymphal) to adult phenotypes and Broad controlling the premetamorphic growth of imaginal tissues in early-stage nymphs. Chinmo might then be needed to keep both in check.

Reviewer #2 (Public Review):

Like humans, Bengalese finches rely on auditory feedback to maintain the acoustic stability of their learned vocalizations, and deafening causes acoustic degradation of their songs. How disruptions to sensory input alter gene expression in brain regions important for singing and song learning remains relatively unexplored. The authors develop an innovative serial laser capture RNA-sequencing method, which allows them to conduct large-scale analyses of gene expression in spatially defined singing-related regions, as well as in surrounding non-singing-related regions. These methods are used to demonstrate that deafening preferentially alters gene expression in song-related regions relative to surrounding song-related areas, and that deafening reduces correlations in gene expression between connected song-related regions. The authors then compare their findings to a previous single-cell RNA sequencing dataset to determine the cell types whose gene expression is likely to be most strongly affected by deafening and song degradation. Finally, the authors repeat their measurements of gene expression changes in RA following unilateral lesion of LMAN and find that LMAN lesions have the largest effect on groups of genes whose expression was also strongly affected by deafening. The study is elegant and rigorous, and its conclusions are well-supported. This work reveals candidate genes that may play a role in stable vocal performance and whose changes in expression may contribute to the acoustic degradation of vocal performance following deafening.

Reviewer #2 (Public Review):

Neurons of the inferior olive exhibit strong subthreshold oscillations, and drive complex spiking through climbing fiber synapses onto Purkinje cells in the cerebellar cortex. This activity plays an essential role in coordinating motor control and the induction of cerebellar plasticity. In this study, the authors make use of optogenetic and electrophysiological approaches to examine the interplay between intrinsic oscillations and two important excitatory and inhibitory input populations to the inferior olive. The authors show that excitation is enhanced when it occurs in the rebound phase of the preceding inhibition. Using a computer model, the authors also show that enhanced excitation can effectively recruit larger populations of neurons, presumably through gap junctional coupling. The strengths of the study include the authors' ability to independently control both excitatory and inhibitory pathways, as well as the rigorous and systematic examination of input timing and amplitude and their effects on spike output. There were some weaknesses; high variability in cell resting potentials raised questions about how cell health impacted the findings, and there needed to be better documentation of recording conditions and parameters. There also needed to be a more extensive discussion about the nature of input timing and frequency under behaviorally relevant conditions. Given these relatively minor issues, the study provides new insight and depth into synaptic integration in the inferior olive and adds to our understanding of how input timing is translated into climbing fiber signals.

Reviewer #2 (Public Review):

The authors report a study comparing self-reported stable and unstable knees with total knee arthroplasty. Advanced imaging methods (dynamic fluoroscopy with model-image registration) and analysis of muscle activities were used to characterize the study subjects during three ambulatory activities (level gait, downhill walking, and stair descent).

The subject cohort all received one design of TKA in a similar time period. The unstable subgroup was >60% female, while the stable knee cohort was 70% male, which is a notable limitation. The measurement methods are state-of-the-art and expertly applied.

The results suggest there may be measurable differences in knee kinematics in subjects with unstable knees, but this was not strongly supported across groups. Rather it was highlighted in 3 individuals who self-reported instability during the test session. The muscle activity analysis supports there being differences between the stable and unstable knee groups.

Despite the limitations of a small subject cohort with only a single TKA design, the study highlights important methods that appear suitable to further study the factors contributing to clinical dissatisfaction with TKA as it relates to joint stability and function during ambulatory tasks.

Reviewer #2 (Public Review):

In this manuscript, Villalobos-Cantor et. al. described a new technique for cell-type specific in vivo labeling of nascent peptides, which they call POPPi. POPPi is based on sequence-independent incorporation of the puromycin analog OPP into an elongating peptide, which also simultaneously terminates the growing peptide. To achieve cell-type-specific labeling, the authors used an OPP derivative, PhAc-OPP, as the labeling substrate. PhAc-OPP contains a blocking group that prevents it from incorporating into the growing peptide, and the blocking group can be cleaved off by the enzyme PGA, which is expressed in the cell type of interest.

The authors validated POPPi in different cell types in the Drosophila brain and showed that this method could be used to image general translation or to biochemically enrich nascent peptides in a cell-type-specific manner. They also showed that with an optimized labeling protocol, it is possible to achieve efficient labeling with minimum effect on animal viability and health. The authors further used POPPi to provide independent support for a previously known phenomenon: age-dependent decline in general translation in the neurons. The results of this work are solid, and the main conclusions are well supported by the data presented. The manuscript is very well written with a clear logic flow and is very easy to read.

What is less clear is how generally useful POPPi will be to the community. The authors pointed out two major cell-type specific applications of POPPi, 1) imaging general translation and 2) biochemically purifying nascent peptides. For application #1, although POPPi might be a more desirable method in some cases, a combination of non-cell-type specific labeling using OPP, and marking the cell type of interest by a fluorescent protein might be simpler. Because labeling with OPP eliminates the enzymatic step that converts non-reactive PhAc-OPP to reactive OPP, the labeling kinetics can be improved, and the toxicity associated with PGA expression can be avoided. For application #2, a currently widely used strategy for a similar purpose is various types of ribosome profiling techniques. Ribosome profiling may be easier to perform than POPPi, and because proteins cannot be amplified, a very large quantity of starting materials will be needed if one wants to use POPPi to characterize cell-type specific nascent proteome. In fact, in this manuscript, the authors used western blots to detect candidate proteins and did not use mass spec to characterize the nascent proteome.

Reviewer #2 (Public Review):

The work from Nakajima-Takagi et al describes the phenotypes and study of a PCGF1 mutant mouse model. PCGF1 is a core component of the non-canonical PRC1.1 complex and specific functions of this complex in hematopoiesis. Using somatic inactivation models, the authors demonstrate that the acute deletion of PCGF1 from adult hematopoiesis leads to a progressive myeloid bias in the bone marrow and peripheral blood. This occurs at the expense of the HSPC compartment, with a reduction in all populations and of the lymphoid committed populations. The myeloid bias is cell intrinsic, as competitive transplant of the PGCF1 deficient bone marrow recapitulates the phenotype. The effect is not due to exhaustion or loss of self-renewal of the HSCs.<br /> To understand the basis for the myeloid bias, the authors first assessed transcriptome signatures and see a shift in gene expression programs related to myeloid development and targets of the key myeloid transcription factor C/EBPa. Further analysis demonstrated an increased expression of Cebp1 in the PCGF1-deficient LSK cells. Reducing the expression of Cebpa could modify the myeloid skewing of Pcgf1 deficient cells in culture. This de-repression of Cebpa correlates with changed local H2AK119ub1 levels in the HPSC populations.

Additional studies assessed how the loss of Pcgf1 changed the response to hemoablation, in this instance with a single dose of 5-FU. This study coupled with scRNA-seq suggested that PRC1.1 was important in regulating the GMP populations, potentially through a self-renewal program. This led to a focussed analysis of the GMPs, with evidence for altered Hoxa9 and b-catenin levels contributing to the altered GMP behaviours. Both have been implicated and demonstrated to have functional roles in these programs in other studies.

Finally, ageing of Pcgf1 deficient mice demonstrated that these mice were predisposed to developing T-ALL and MPN. The authors provide a characterisation of these moribund states and their phenotypes are consistent with the diagnosis.

Overall the work demonstrates a specific requirement for Pcgf1, and therefore PRC1.1, in the regulation of hematopoiesis. I think the authors largely achieved the aims and the results are supportive of the conclusions. The work shows myeloid bias, experimental evidence that this is due to a derepression of a myeloid lineage program in the HPSC and associated chromatin changes, and functions for Pcgf1 in both hematopoietic regeneration and malignancy. This suggests a unique role for non-canonical PRC1.1 compared to canonical PRC 1.

Strengths:<br /> - in vivo experiments and evidence;<br /> - multiple lines of evidence supporting the conclusion;<br /> - mechanistic studies provide direct evidence of the proposed mechanism.

Weaknesses:<br /> - can the authors demonstrate normal maturation of the myeloid lineages as this would be important to differentiate between myeloid bias and a block in myeloid differentiation? This is important to distinguish between.<br /> - include analysis of mature myeloid cells and FACS plots to allow assessment of maturation.

Reviewer #2 (Public Review):

In this manuscript, Niethamer et al. investigate the role of the transcription factor ATF3 in lung regeneration after H1N1 influenza. They focus on endothelial ATF3 which is present in a subset of lung capillaries in the adult mouse lung. Interestingly, they found that influenza infection upregulates endothelial ATF3 and that endothelial deletion of Atf3 results in impaired regeneration, leading to enlarged airspaces after viral infection. They further show that this effect may be due to an increase in apoptosis and a decrease in proliferation, suggesting that endothelial ATF3 is necessary for pulmonary vascular regeneration, as well as recovery of the alveolar architecture.<br /> Given the recent publications in the field describing lung endothelial heterogeneity, as well as its possible role in injury repair, this work is relevant to the community. It also supports the idea that epithelial-endothelial crosstalk is important for lung regeneration and proposes a potential candidate for this process.

Strengths:<br /> The authors identified and tested the role of endothelial Atf3 in lung regeneration using well-established techniques. They identified this transcription factor as a candidate using state-of-the-art scRNA-seq. They also carefully lineage traced ATF3 expressing cells using an inducible reporter before and after infection and then used a pan-endothelial driver Cdh5 to delete Atf3 specifically in the endothelium. Thus, the authors successfully show significant changes in the alveolar structure after infection in their mutant model.

Weaknesses:<br /> Although there is evidence that the author's claims have biological relevance, this paper would benefit from strengthening and/or clarifying some things:

• The scRNA-seq analysis is performed in two separate objects ("control lung" and "H1N1 infected lung 14dpi"). For these two sets of data to be comparable, the authors should have integrated the objects and analyzed them together. This is not only important for deciding the clusters' identities and making sure that the same clusters are compared between control and infected, but also necessary to compare gene expression.<br /> • ATF3 is not only present in Cap1_B, in the infected lung there seems like Cap1_A express less ATF3. The authors should comment on this difference.<br /> • It is unclear how the clusters Cap1_A and Cap1_B were decided. The manuscript would benefit from clarification.<br /> • It would be beneficial to see via immunofluorescence the morphological and spatial differences between ATF3-expressing and non-expressing endothelial cells since this transcription factor is expressed in multiple endothelial cell types.<br /> • The authors mention ATF3 is not endothelial-specific. Expression of ATF3 in other cell types should be evaluated via immunofluorescence.<br /> • The authors should have shown evidence of the deletion in their Atf3EC-KO mouse and addressed whether they had residual ATF3. If there is no antibody available, RNAscope could be used, or Western Blot or RT-PCR on sorted endothelial cells.<br /> • The authors only show the epithelium as evidence that the alveolar region is altered in their mutant after infection. The endothelium should have also been investigated, especially since their mutant is an endothelial-specific deletion. Within this, the different endothelial cells should have been assessed by a method other than RNAscope such as immunofluorescence, given that this method is unable to show morphology and there are antibodies available.<br /> • Bulk RNA-seq from endothelial cells is used in the manuscript. However, because ATF3 is not specific to Cap1_B cells or even capillaries alone, the downstream gene expression analysis of bulk RNA should be placed into the context of lung endothelial heterogeneity.<br /> • Although the authors mentioned that the infection with H1N1 influenza can have regional differences, they do not show how they picked regions for their analysis and quantification, and whether ATF3 upregulation was found in more severely affected regions. Furthermore, since they quantified via FACS, this heterogeneity in the infection itself could have affected their observations.

Reviewer #2 (Public Review):

1) A detailed step-by-step approach to validation of some previously known outcomes.<br /> 2) Useful for more focus to be placed on data from the second half of the paper.<br /> 3) Some reflection on the media used to study paracrine effects is needed - more experiments here would be beneficial.<br /> 4) Path clamp experiments - how does bath solution alter the effect of any limited paracrine effect - we are removing cells from the treatment media and putting them in physiological solutions - an opportunity to recover?

Reviewer #2 (Public Review):

This study assessed the inflammatory and metabolic profiles of a healthy sub-Saharan Africa (Tanzania) population versus a healthy population outside Africa (Dutch). Using plasma samples from these cohorts, an O-Link proteomics inflammatory panel and targeted metabolomics platforms were utilised. The study shows that 'healthy' Tanzanians display an enhanced pro-inflammatory phenotype versus Dutch volunteers. Specific pathways and metabolites identified included - increase activation of the Wnt/Beta catenin pathway, and the metabolites 4E-BP1 and FGF21. The study highlights some interesting findings regarding the impact of diet on inflammatory pathway activation.

Major Strengths & Weaknesses - This is an interesting study and approach that aims to address some challenging questions in underrepresented populations. The findings demonstrate the importance of diet and dietary interventions on metabolic health, as well as key inflammatory proteins. It does raise the question whether anti-inflammatory therapies need to be targeted to specific at-risk populations, more so than other populations.

Impact - The study demonstrates the importance of considering differences between populations and the inclusion of underrepresented populations in such studies. The data suggests that lifestyle changes in sub-Saharan Africa are potentially contributing to altered inflammatory and metabolic profiles. Thus, health initiatives advocating traditional diets may alleviate the NCD epidemic in sub-Saharan Africa.

Reviewer #2 (Public Review):

The authors combine NMR experiments, cell experiments, and molecular simulations to address the question of how lipid interactions of the prolactin receptor contribute to signalling. They assess the interactions of the disordered cytoplasmic tail of the receptor with phosphoinositides among others by chemical shift perturbations from NMR for different PIP2-containing membranes, by coarse-grained simulations, as well as site-directed mutagenesis and subsequent cell signalling experiments to monitor the activation of the mutants. A major result is that PIP2 interactions are functionally important, which so far has not been known for this receptor. Their results are likely relevant for other non-receptor tyrosine kinases.

The hypothesis that the protein complex is regulated by IDR-membrane interactions is very novel. A major strength is the close connection of and feedback between state-of-the-art experiments and simulations.

This is where I see weaknesses:<br /> 1. The motivation of focusing on LID1 is limited.<br /> 2. The data and analysis for the JAK2-PRLR complex appear somewhat superficial, and a connection between conformational states to their functional relevance is lacking. In fact, the majority of the simulation part of the paper is about suggesting different states of the PRLR-JAK2 complex but the states and their hypothesized functional relevance are not further taken up, e.g. by experiments, and yet presented as major results, e.g. in the abstract.<br /> 3. The connection between simulations and mutational study is not very direct.<br /> An open question is if the mutants can distinguish between the effects of PRLR-PIP2 interaction or PRLR-JAK2 interaction, even though this conclusion is still drawn from the data.<br /> 4. The conclusions drawn from the mutagenesis study (lines 547-555) are not directly supported by data. Only a partial correlation between PRLR membrane localisation and STAT5 activation is no reason to attribute the unexplained part of the STAT5 activation to PRLR-JAK2 interactions without further studies.<br /> 5. PIP2 is identified as an important regulator, with very solid support from the presented data. PIP3 is part of the model but not discussed before or as part of the results. The analysis could be similarly applied or the data directly relevant to the understanding of PIP3 plays a similar role, as interactions are likely primarily electrostatically driven.

Reviewer #2 (Public Review):

In A. Ruppel, et al, the authors study the mechanics of one cell, two cells, and cell monolayers upon a transient local activation of contractility. First, the authors characterize the tractions and stress maps (measured via Traction Force Microscopy and Monolayer Stress Microscopy, resp.) for one and two cells in the absence of contractility activation, and found a correlation between the principal stress direction and actin fiber orientation. Next, the authors use the theory of foams to infer, combining traction force data and cell geometry data, the mechanical parameters of cells like the line tension or the force of adherent fibers. Next, the authors activate contractility by means of optogenetic tools on one half of the system and quantify the response on both halves, concluding that the receiver half response is driven by active processes, increasing contractility for two cells, while fluidizing for one cell. Next, the authors estimate the level of active response in cell doublets by comparing the stress maps to numerical simulations of a thin elastic medium with anisotropic contractility. By varying aspect ratios of the H pattern, the authors find a correlation between the principal stress direction and the orientation of stress fibers and find that the previous active response is in general enhanced when the principal stress direction is perpendicular to the orientation of the fibers. Finally, these features are also found in a cell monolayer for a fixed confinement aspect ratio.

Overall, the manuscript contains a broad characterization of the steady state mechanics and the dynamical response to the activation of contractility for one cell, two cells, and cell monolayers.

Reviewer #2 (Public Review):

The authors hypothesized that PTH1R and ZFP467 could constitute a feedback loop that facilitates PTH-induced osteogenesis and that conditional deletion of Zfp467 in osteogenic precursors would lead to high bone mass. Using a number of methods, they have established a regulatory feedback mechanism of this transcription factor and the PTH receptor in osteoblastic precursors as well as showing that PrrxCre deletion of Zfp467 causes an increase in trabecular bone mass, while AdipoCre does not. Nevertheless, they have not established the actual mechanism of action of the transcription factor nor which gene it acts on in the osteoblast. They have mostly achieved their aims and the results partially support their conclusions. However, the work is descriptive and does not address the central issue of how ZFP467 acts. At present, its impact on the field is limited.

Reviewer #2 (Public Review):

This manuscript introduces a novel assay in a 'phenomics' approach to address an important aspect of S. aureus pathogenesis. The authors set out to identify mutations that arise during clinical S. aureus infections that cause a decrease in intracellular host-cell toxicity and increase intracellular persistence. To do this, they use a 'phenomics' approach. For phenotype, they quantify HeLa cell toxicity for each strain in a panel of 387 clinical S. aureus isolates. This is done by measuring HeLa cell death induced by intracellular S. aureus via propidium-iodide uptake. The whole genomes of each of these 387 isolates had previously been sequences. They use the genomic data and phenotype data to carry out a genome-wide association study (GWAS) looking for genetic signatures that correlate with reduced HeLa cell cytotoxicity. As expected, mutations in agr were the strongest locus-level signal, but the study did identify one agr-independent mutation in ausA, which was able to be independently validated, showing that the assay is robust enough to find causal mutations. The analysis is thoughtful, the assay appears robust, and I think the discussion of conclusions and limitations is mostly valid. Thus, my concerns are focused on further understanding the practical utility of the approach and whether or not the HeLa cell model recapitulates what happens in professional phagocytes. For example, it is not clear to me that this system has the statistical power to find novel, biologically relevant rare mutations without first being very mindful in selecting strains that are extremely genetically similar. It is also not clear to me that the toxicity assay captures the important features of the intracellular persistence that occurs in vivo within professional phagocytic cells. Thus, given these practical limitations and a somewhat artificial model system, the impact on the field is likely to be moderate in nature. However, the analysis and approach taken could be re-purposed to any robust quantitative phenotype, and this will certainly be of great interest to others that study bacterial evolution in clinical contexts.

Reviewer #2 (Public Review):

Krishnan, et al describe a unique and powerful approach to assessing the role of genetic variation on mitochondrial and cardiac function and health. Utilizing a panel of inbred mouse strains, on which they performed proteomics on heart samples, they measured 840 mitochondrial proteins and correlated these data to heart function using two heart stress models. This resulted in a number of correlative observations, three of which were explored in more detail to connect three specific genes to cardiac hypertrophy. This is an interesting dataset and there is clearly value in what is presented. The data were largely correlative, however, and there are only a couple of causation-oriented experiments. It's hard to adjudicate between these strengths and weaknesses in determining the overall impact of the manuscript.

Reviewer #2 (Public Review):

This manuscript reports on mermithid nematode fossils from amber which dates from the Cretaceous period. The specimens described in the manuscript consist of insects and associated nematodes which have been trapped in amber and fossilised. The nematodes have been identified as belonging to the Mermithidae family, a family of nematode worm that infect insects.

The findings of this manuscript provide an insight into the evolution history of nematodes and parasitism. Despite the ubiquity of both nematodes and parasites in extant ecosystems, fossil records of both are very rare. This is because nematodes and many parasites are soft bodied, and many are located inside their hosts' bodies, thus they rarely become fossilised. Thus, most of what is known about the evolutionary history of nematodes, and evolution of parasitism are based on what could be inferred from extant examples.

The specimens described in this manuscript provides a valuable contribution to our understanding of parasitism in the geological past. These amber specimens are a snapshot of parasite-host interactions - interactions which are commonly found in nature but are rarely captured in fossils. The identification of the specimens as mermithid nematodes are based on sound scientific reasoning. The worms' morphology and position in relation to the insects are consistent with what have been observed with extant mermithid nematodes.

Additionally, one of the values of such parasite fossils is that they provide us with insight into parasite-host combinations or interactions which may have existed throughout the geological past, but no longer exist today or cannot be inferred from extant taxa. It helps fill in major gaps in our understanding of parasitism. This was the case with the amber fossil that contained a bristletail with its nematode parasite.

Reviewer #2 (Public Review):

This very interesting study uses a combination of high channel count neural recordings and machine learning to characterize neural representations of complex natural and synthetic sounds in the inferior colliculus. The authors use deep neural networks to model sound evoked activity in a large number of IC multiunits with high accuracy in gerbils with normal hearing and hearing loss. They then use the DNNs to simulate activity evoked by a wide range of stimuli and demonstrate systematic differences in latent population representations between normal hearing and hearing-impaired animals. Models for hearing impaired animals show activity consistent with impaired representations of speech in noise. These results lay the groundwork for a potentially valuable approach to improving signal processing in hearing aids and prosthetics.

The large speech dataset and clean hearing loss effects are particularly impressive. While the approach and associated data are novel and likely to be of broad interest, there are some substantial concerns about the study. First, the authors fail to acknowledge substantial previous work on super-threshold activity in cortex of animals with hearing loss, making it appear that they overstate the novelty of the current results. There are also many cases where they fail to clearly report the details of statistics used to support their claims. Finally, while the accuracy of the DNN models is compelling for the speech stimuli in the data set, it is not clear that the comparisons of simulated activity reflect actual neural activity in the stimulus conditions tested.

Reviewer #2 (Public Review):

In the study by Li et al., the authors hypothesize that RELMa, a macrophage-derived protein, plays a sex-dimorphic role as a protective factor in obesity in females vs males. The authors perform largely in vivo studies utilizing male and female WT and RELMa KO mice on a high-fat diet and perform an in-depth analysis of immune cell composition, gene expression, and single-cell RNA Sequencing. The authors find that WT females are protected from obesity and inflammation vs males, and this protection is lost in female RELMa KO mice. Further analysis by the authors including flow cytometry of the visceral fat SVF in female WT mice showed reduced macrophage infiltration, higher levels of eosinophils, and Th2 cytokine expression compared to WT male mice and female KO mice. The authors show that protection from obesity and inflammation in female RELMa KO mice can be rescued with an injection of eosinophils and recombinant RELMa. Lastly, the authors use single-cell RNA-Sequencing to further analyze SVF cells in WT and KO male and female mice on a high-fat diet.

Overall, we find that the study represents an important finding in the immunometabolism field showing that RELMa is a key myeloid-derived factor that helps influence the macrophage-eosinophil function in female mice and protects from diet-induced obesity and inflammation in a sexually dimorphic manner. Overall, the study provides strong and convincing data supporting the authors' hypothesis and conclusion.

Reviewer #2 (Public Review):

Yamaguchi et al. studied the roles of two proteins, Calaxin and Armc4, in the assembly of the outer arm dynein (OAD) docking complex (DC). By combination of the improved cryo-ET analysis and gene knockout zebrafish lacking each of these proteins, they found that Armc4 plays a critical role in the docking of OAD and that Calaxin stabilizes the molecular interaction in the docking.They further showed an evidence that Calaxin changes the conformation of another compartment of DC comprising CCDC151/114. This new information provides an important basis for understanding how the DC is assembled and regulates docking of OAD. The authors' conclusion is well supported by the data but some data presentation and discussion need to be completed.

Gui et al. (2021) already reported on a cryo-EM observation in bovine tracheal cilia, with the conclusion similar to this paper in the structure of OAD/DC on DMT. Using knockout zebrafish strain, the authors present detailed interaction of calaxin with other DC components. They show that the binding of calaxin induces the changes of conformation in N-terminal region of CCDC151/114. The conformation further changes in the presence of Ca2+; specific conformation of N-terminal region of CCDC151/114 becomes undetectable, instead additional structure appears in the vicinity of calaxin.

1) The authors conclude that the Ca2+-dependent conformational change of DC is subtle and not dynamic. This result is eventually valuable information but may be somewhat unexpected from the point of view that calaxin plays an important role in the regulation of flagellar motility in Ciona sperm. The authors found that calaxin changes the conformation of N-terminal CCDC151/114 region but the core dynein structure shows no dynamic change. What about the changes in the interaction between calaxin, core dynein, and DMT? Is this beyond the resolution of cryo-ET analysis?

2) It would be very helpful if the authors could add the cryo-ET images of calaxin-/- axoneme in the presence of 1 mM EGTA in Figure 7. Although these images are thought to be similar or identical to Figure 4F, it would help to confirm that the conformational changes in CCDC151/114 and additional part of DC are induced in a Ca2+-dependent manner.

3) To clarify the molecular interaction of calaxin with other components, it would also be helpful if the authors add the images rotated 80 degree to Figure 4F and G, in similar way in Figure 7,

4) Despite the molecular phylogenetic difference, there are several similarities between calaxin and Chlamydomonas DC3, not only in the in situ structure and configuration but in the phenotype of mutants; Chlamydomonas mutant lacking DC3 shows OAD loss in the distal part of a flagellum (Casey et al, MBC, 2003). It may be a good reference if the authors add the position of DC3 in Figure 4. A', B', and C.

5) There is a significant difference in sperm motility between WT and calaxin-/- or WT and armc4-/- (Figure 2E). However, it is not clear whether immotile sperm were included in the data for VAP (Figure 2F) or BCF (Figure 2G). For example, WT and calaxin-/- show similar VAP, although both are significantly different in the percent of motile sperm.

6) In calaxin-/- mouse, OAD was clearly detected from the base to two-thirds of a flagellum with unclear border (Figure 2A). Typical distribution of OAD+class and OAD-class are shown in Figure 5 in the ~3 micrometer tomograms. Were these taken from around this unclear border? Are proximal most region of a flagellum occupied with OAD+class only? The authors should clearly indicate the region of a flagellum where the tomograms in Figure 5C and D were selected.

7) Line 229~: It is not clear what the authors meant by "probably reflecting the different distance from the sperm head". In relation to this and the comment 6, does the "proximal" in the sentence "OAD loss occurred even in the proximal part of the flagella" (line 232) indicate the region near the base of a flagellum?

8) In conjugation with comment 7, it would be appreciated to show an authors' idea on why distal region of flagella tends to lack calaxin, if they do not discuss anywhere in the text,

9) Immunofluorescence in twister-/- epithelial cilia showed that the localization of calaxin is independent of OAD (line 271-274). Based on the authors' finding, the localization of calaxin requires Armc4, which is preassembled with calaxin in the cytoplasm. If this is true and the localization of calaxin is NOT resulting from diffusion, Armc4 must be localized with calaxin along the entire length of cilia in twister-/- epithelial cilia (Figure 6D). Although Armc4 is shown localized in cryo-ET images (e.g. Figure 1, Figure 7), authors may provide the immunofluorescence of Armc4 along the entire length of sperm flagella and epithelial cilia.

Reviewer #2 (Public Review):

In this study, Rmus and colleagues contribute to the important open question of whether reinforcement learning deficits observed in older adults are due to impairments in basic learning processes, or can be attributed to a decline in working memory function. The authors present cross-sectional behavioral data from a task designed to assess the role of working memory in reinforcement learning. And they use computational modeling in conjunction with MR spectroscopy to demonstrate a relationship between prefrontal glutamate and age-related impairments in learning specific to working memory decay. I found the overall story compelling, the data novel, and the analysis carefully executed. Below I outline some areas in which the claims of the manuscript could be strengthened.

1. I may have missed this, but does glutamate correlate with other model parameters? Or did the authors only focus on the WM parameters because of the age difference? In support of the specificity argument, it would be important to show that glutamate only predicts WM related parameters regardless of whether there was an age difference or not.<br /> 2. As it is somewhat common with these tasks, it seems like the model does not fully capture the performance deficit in OA (Fig. 2B), even when all the individual difference parameters in WM are allowed to vary. Can the authors say more about the discrepancy? This is an interesting datapoint which may give clues to mechanism.<br /> 3. Relatedly, it may not be possible with these data alone, but can authors discuss what the WM decay parameter captures? In particular for OA, the distinction between generating and maintaining a "task set" has been extensively written about. Older adults tend to have difficulty internally generating and flexibly deploying task sets, but somewhat paradoxically can perform better than YA in certain decision situations (e.g. when reward is dependent on previous choices, see Worthy et. Al. 2011). The task in this study necessarily pushes OA in a regime in which relying on familiar decision strategies is sub-optimal, and task sets must be continuously generated. Is there a type of intervention do authors expect would reverse the observed deficit in WM?<br /> 4. There is a wealth of evidence suggesting striatal DA loss in older adults, which served as the basis for many of the original investigations and hypotheses regarding a simple RL deficit in OA (e.g. work by Shu-Chen Li and others). While the authors do not directly measure DA in this study, it would be helpful to place the results in the context of that literature.<br /> 5. Finally, the main argument of the paper as I read it is that PFC glutamate mediates the performance deficits observed in RL because it reflects a compromised WM system. Sample size permitting, it would be helpful to see a formal test of this mediation relationship.

Reviewer #2 (Public Review):

The manuscript by Aderounmu presents an interesting attempt to reconstruct evolution of the function of the helicase domain in ancestral Dicers, RNase III enzymes producing siRNAs from long double-stranded RNA and microRNAs from small hairpin precursors. The helicase has a role in long dsRNA recognition and processing and this function could have an antiviral role. Authors show on reconstructed ancestral Dicer variants that the helicase was losing dsRNA binding affinity and ATPase activity during evolution of the lineage leading to vertebrates while an early divergent Dicer-2 variant in Arthropods retained high activity and seemed better adapted for blunt ended long dsRNA, which would be consistent with antiviral function.

The work is consistent with apparent adaptation of vertebrate Dicers for miRNA biogenesis and two known modes of substrate loading: "bottom up" dsRNA threading through the helicase domain where the helicase domain recognizes the end of dsRNA and feeds it into the enzyme and "top-down" where the substrate is first anchored in the PAZ domain before it locks into the enzyme. Some extant Dicer variants are known to be adapted for just one of these two modes while Dicer in C. elegans exemplifies an "ambidextrous" variant. The reconstruction of the helicase domain complex enabled authors to test how well would be ancestral helicases supporting the "bottom up" feeding of long dsRNA and whether the helicase would be distinguishing blunt-end dsRNA and 3' 2 nucleotide overhang. Although the reconstruction of an ancestral protein from highly divergent extant sequences yields just a hypothetical ancestor, which cannot be validated, the work provides remarkable data for interpreting evolutionary history of the helicase domain and RNA silencing in more general. While it is not surprising that the ancestral helicase was a functional ATPase stimulated by dsRNA, particularly new and interesting are data that the decline of the helicase function started already at the level of the common deuterostome ancestor and the helicase was essentially dead in the vertebrate ancestor. It has been reported two decades ago that human Dicer carries a helicase, which has highly conserved critical residues in the ATPase domain but it is non-functional (10.1093/emboj/cdf582). Recently published mouse mutants showed that these highly conserved residues are not important in vivo (10.1016/j.molcel.2022.10.010). Aderounmu et al. now suggest that Dicer carried this dead ATPase with conserved residues for over 500 million years of vertebrate evolution.

I do not have any major comments to the biochemical analyses and while I think that the ancestral protein reconstruction could yield hypothetical sequences, which did not exist, I think they represent reasonable reconstructions, which yielded data worth of interpretations. My major criticism of the work concerns clarity for the readership and interpretations of some results where I wish authors would clarify/revise the text. The following three examples are particularly significant:

1) It should be explained to which common ancestor during metazoan evolution belongs the ancestral helicase AncD1D2 or at least what that sequence might represent in terms of common ancestry during metazoan evolution.

2) This is linked to the first point - authors work with phylogenetic trees reconstructed from a single protein sequence, which are not well aligned with predicted early metazoan divergence (https://doi.org/10.1098/rstb.2015.0036). While their sequence-based trees show early branching of Dicer-2 as if the two Dicers existed in the common ancestor of almost all animals (except of Placozoa), I do not think there is sufficient support for such a statement, especially since antiviral RNAi-dedicated Dicers evolve faster and Dicer-2 is restricted to a few distant taxonomic group, which might be better explained by independent duplications of ambidextrous ancestral Dicers. I would appreciate if authors would discuss this issue in more detail and make readers more aware of the complexity of the problem.

3) Authors should take more into the account existing literature and data when hypothesizing about sequences of events. Some decline of the helicase activity is apparent in AncD1DEUT suggesting that it initiated between AncD1D2 and AncD1DEUT. This implies that a) antiviral role of Dicer was becoming redundant with other cellular protein sensors by then and b) Dicer was already becoming adapted for miRNA biogenesis, which further progressed in the lineage leading to vertebrates to the unique top-down loading with the distinct pre-dicing state where the helicase forms a rigid arm. Authors even cite Qiao et al. (https://doi.org/10.1016/j.dci.2021.103997) who report primitive interferon-like system in molluscs - this places the ancestry of the interferon response upstream of AncD1DEUT and suggests that this ancestral protein-based system was taking over antiviral role of Dicer much earlier. In fact, a bit weaker performance of AncD1LOPH/DEUT combined with the aforementioned interferon-like system and massive miRNA expansion in extant molluscs (10.1126/sciadv.add9938) suggests that molluscs possibly followed a convergent path like mammals. While I am missing this kind of discussion in the manuscript, I think that the model where "interferon appears ..." in AncD1VERT (Fig. 6) is incorrect and misleading.

Reviewer #2 (Public Review):

In this work, Cunha et al provide an insightful and exhaustive analysis of the role of hypoxia and HIF-1a for T cell activation and function. The work contributes to the field by showing that transient hypoxia occurring simultaneously with T cell stimulation (antigen recognition) induces an effector program in T cells that results in increased cytotoxicity in vivo and in mouse models. Importantly, the induction of this effector phenotype is not necessarily linked with an increase in proliferation in vitro, and in vitro differences are mostly observed upon antigen re-challenging.

The major strengths of the work are the use of different complementary methods to modulate HIF-1a (low oxygen conditions, inhibition of PDH by FG-4592, and deletion of VHL) and the combination of mouse and human models, especially addressing how to implement the findings to the production of CAR-T cells. Besides, the authors not only evaluate T cell function but also dive into the pathways driving the responses observed, which provides mechanistic insight.

While activation of HIF-1a through the different means mentioned before results in similar signatures in terms of T cell effector phenotype and animal response, there are some aspects that differ between the models. This is probably indicating that low levels of oxygen have other effects beyond the regulation of HIF, and that pharmacological modulation of HIF-1a might not be exactly equivalent to HIF-1a stabilization by real hypoxia.

The work is useful to better understand the discrepancies in the field, where it has been previously shown that hypoxia can have both a pro-inflammatory effect and an immunosuppressive effect on T cells. The answer proposed by the authors is that it´s a matter of timing, and not so much the magnitude of the HIF-1a response. Despite this being relatively easy to control ex vivo, the challenge occurs when considering the role of hypoxia in vivo, which probably lasts longer than the transient hypoxia needed for beneficial effects on T cells, causing T cell exhaustion.

From the translational perspective, the study suggests strategies to improve CAR-T cell therapy but also has some limitations. Despite an improvement of cytotoxicity and survival observed in mouse models upon adoptive cell transfer or injection of CAR-T cells with previously increased HIF1a levels, these approaches do not result in curation and survival is still quite low in all groups. Interestingly, improved survival with HER2 CARs exposed ex vivo to low oxygen conditions for 1 day is clear and more promising.

Reviewer #2 (Public Review):

This work formulates a detailed theoretical polymer physics model intended to explain the observed morphology of chromatin in the Drosophila cell nucleus. The model is examined in detail by both analytical calculation and computer simulation. The central premise of the suggested theory is that it is based on equilibrium statistical mechanics. Within this paradigm, authors explore the model that views chromatin fiber as a block copolymer and, most importantly, describes the role of RNA polymerase as it interacts with one of the copolymer blocks and regulates its effective solvent quality. Blocks are assumed to be fixed on the time scale of interest by, e.g., different levels of acetylation or methylation. RNA polymerase is supposed to interact only with one of the chromatin blocks, called active, and assumed interaction is quite peculiar. Namely, RNA polymerase complex may absorb on chromatin fiber and, the model assumes, the fiber decorated with absorbed RNA polymerase molecules is less sticky to itself, or more repulsive than the fiber itself. This peculiar assumption allows authors to make interesting predictions about how proteins can regulate the genome folding architecture.

STRENGTH

The work includes a rather detailed theoretical description of the model and its equilibrium statistical mechanics. As both analytical theory and accompanying simulation indicate, the assumptions put forward in formulating the model do indeed produce the desired morphology, with isolated regions ("micells") of core inactive chromatin surrounded by the less dense shell region in which RNA polymerization may potentially take place. Having such a detailed theory is potentially beneficial for the field and opens up avenues for further exploration.

WEAKNESS

The underlying assumption about the interaction of RNA polymerase complex with the fiber, although important and organic for the model, does not seem easy to justify from a molecular standpoint, especially thinking of the charges and electrostatic interactions.

Reviewer #2 (Public Review):

The paper from Marchal-Duval et al reports for the first time the important role played by the transcription factor PRRX1, expressed specifically in the mesenchyme of the lung, in the context of fibrosis. The authors used a combination of human (Donor and IPF) and mouse lungs (saline and bleomycin treated) as well as associated fibroblasts and PCLS to test the functional role of PRRX1 in the context of proliferation and differentiation induced by TGFb1. The work is supported by an impressive amount of data (7 main figures and 14 supplementary figures).

A main weakness in this work is the counterintuitive result that PRRX1 is downregulated in human lung fibroblasts (from both IPF and Donor) treated with TGFb1. Another smaller weakness is the inactivation of Prrx1 in vivo using ASO starting at d7 post bleomycin treatment.

The strengths of this work are the multiple approaches used by the authors to test the role of PRRX1 in lung fibrosis. The results are statistically solid and informative. The results presented are extremely convincing to support their conclusion that PRRX1, downstream of TGFb1 signaling is important for fibrosis.

Reviewer #2 (Public Review):

The work presented by Jordan and Keller aims at understanding the role of noradrenergic neuromodulation in the cortex of mice exploring a visual virtual environment. The authors hypothesized that norepinephrine released by Locus Coeruleus (LC) neurons in cortical circuits gates the plasticity of internal models following visuomotor prediction errors. To test this hypothesis, they devised clever experiments that allowed them to manipulate visual flow with respect to locomotion to create prediction errors in visuomotor coupling and measure the related signals in LC axons innervating the cortex using two-photon calcium imaging. They observed calcium responses proportional to absolute prediction errors that were non-specifically broadcast across the dorsal cortex. To understand how these signals contribute to computations performed by V1 neurons in layers 2/3, the authors activated LC noradrenergic inputs using optogenetic stimulations while imaging calcium responses in cortical neurons. Although LC activation had little impact on evoked activity related to visuomotor prediction errors, the authors observed changes in the effect of locomotion on visually evoked activity after repeated LC axons activation that were absent in control mice. Using a clever paradigm where the locomotion modulation index was measured in the same neurons before and after optogenetic manipulations, they confirmed that this plasticity depended on the density of LC axons activated, the visual flow associated with running, and the concurrent visuomotor coupling during LC activation. Based on similar locomotion modulation index dependency on speed observed in mice that develop only with visuomotor experience in the virtual environment, the authors concluded that changes in locomotion modulation index are the result of experience-dependent plasticity occurring at a much faster rate during LC axons optogenetic stimulations.

The study provides very compelling data on a timely and fascinating topic in neuroscience. The authors carefully designed experiments and corresponding controls to exclude any confounding factors in the interpretation of neuronal activity in LC axons and cortical neurons. The quality of the data and the rigor of the analysis are important strengths of the study. I believe this study will have an important contribution to the field of system neuroscience by shedding new light on the role of a key neuromodulator. The results provide strong support for the claims of the study.

Reviewer #2 (Public Review):

This work presents an exhaustive study of inferred functional networks from in vitro neuronal cultures across several modalities: primary rat cultures (with varying densities and longitudinal points), and human iPSC monolayers from different cell types and organoids. The authors first estimated the functional connectivity of these networks from their spontaneous activity (recorded with high-density MEAs) and then tried to find which wiring principle could better explain the observations. By deploying generative network models (with 13 different wiring principles) they observed that models with homophilic wiring principles were systematically outperforming the other ones. This proposes a universal rule for how neurons connect, which is that they tend to connect with neurons that have many common neighbors.

One of the major strengths of this study is its scope. They analyzed sparse and dense primary rat cultures at 4 different time points during development (from 7 to 28 DIV in total) as well as with the pharmacological application of GABAA blockers; 3 different cell lines: spinal-cord motor neurons, dopaminergic neurons, and glutamatergic neurons, and organoid slices. However, the big scope of this study is also one of its weaknesses, the techniques presented here to analyze the data are used inconsistently; for some preparations, there's much more detail than in others, and the constant jump between preparations and methodologies makes the findings hard to follow.

Similarly, the number of samples used in some preparations (ranging from 6 to 12) appears to be insufficient, since the study relies on multiple comparisons across the results from 13 different generative models. In many cases, it is not possible to identify which results are significant and which aren't. Most of the methodology used in this study has been used before in the context of the human connectome project (Betzel et al, Neuroimage 2016); in there they used data from 380 total participants, which made the comparisons across all the different models much more robust.

Most previous research with generative models for neural connectivity has focused on structural connectivity. In there, the link between wiring principles, energetic costs, and network topology can be made. This study, however, focuses on functional connectivity (measured by the spike time tiling coefficient), where the link between these quantities is unclear. Although the authors highlight this point in the manuscript, the constant comparisons to structural connectivity concepts and studies often lead to confusion. A clear example of this is the section where the authors explore the effect of chronic GABAA receptor blockade. It is unclear whether the authors are trying to claim that this protocol alters the development of the structural network or only the dynamics. The former could have needed additional controls.

The authors have been diligent and thorough with their statistical testing and their claims are commensurate with that. However, given the large number of different types of results and tests being presented, it is often difficult to find the corresponding explanation in the methods.

This is valuable work for experimental and computational neuroscientists studying the development of neuronal networks and the link between structural and functional connectivity. It would greatly benefit from homogenizing the results, methods, and statistics across the different experimental preparations. The conceptual similarities and differences between structural and functional wiring principles also need to be emphasized.

Reviewer #2 (Public Review):

In the present manuscript, Perrodin et al. investigated which properties of ultrasonic vocalizations determine their attractiveness for female mice. They collected a set of male courtship vocalizations and compared their attractiveness for female mice against a number of conditions, including silence, and a number of modified sequences.

The study has a clear design and used insightful modifications on the vocalization sequences, which allow the present results to be linked to previous results. The most interesting outcome of the study is that female mice prefer regularly timed sequences of vocalizations over less regularly timed sequences. This result is novel and adds to our understanding of the determinants of social communication between mice. Overall the study is likely underpowered, which was, however, hard to assess as animal numbers were largely not reported for the individual tests, and statistical analysis was carried out on the level of sessions only.

The study has a very good discussion embedding the current results with the previous literature, although the discussion steps beyond the results in a few respects, in particular when trying to determine the underlying reasons for the preference for regularly spaced sequences.

Methodologically the study is carried out at the appropriate level, although some improvements could be made to the experimental apparatus to avoid reflections.

The study will likely have a substantial impact on the field of mouse communication because the regularity of spacing has not been a focus of previous research. In addition, the confirmation that a lot of other modifications are less determining for the attractiveness of the vocalizations provides solid data on which to base future work.

Reviewer #2 (Public Review):

The eleven paralogs of SLC26 proteins in humans exhibit a remarkable range of functional diversity, spanning from slow anion exchangers and fast anion transporters with channel-like properties, to motor proteins found in the cochlear outer hair cells. In this study, the authors investigate human SLC26A6, which functions as a bicarbonate (HCO3-)/chloride (Cl-) and oxalate (C2O42-)/Cl- exchanger, combining cryo-electron microscopy, electrophysiology, and in vitro transport assays. The authors provide compelling evidence to support the idea that SLC26A6's exchange anions at equimolar stoichiometry, leading to the electroneutral and electrogenic transport of HCO3-/ Cl- and C2O42-/Cl-, respectively. Furthermore, the structure of SLC26A6 reveals a close resemblance to the fast, uncoupled Cl- transporter SLC26A9, with the major structural differences observed within the anion binding site. By characterizing an amino acid substitution within the SLC26A6 anion binding site (R404V), the authors also show that the size and charge variance of the binding pocket between the two paralogs could, in part, contribute to the differences in their transport mechanisms.

The strength of this work lies in the reductionist, in vitro approach that the authors took to characterize the transport process of SLC26A6. The authors used and developed an array of functional experiments, including two electrogenic transport assays - a fast kinetic (electrophysiology) and a slow-kinetic (fluorescent-based ACMA) - and two electroneutral transport assays, probing for Cl- (lucigenin) and HCO3- (europium), which are well executed and characterized. The structural data is also of high quality and is the first structure of an SLC26 coupled anion exchanger, providing essential information for clarifying our understanding of the functional diversity between the SLC26 family of proteins.

To my knowledge, the outward-facing conformational state has not been determined for any mammalian SLC26 paralog, which limits the mechanistic interpretation of transport and is a weaker point of this manuscript. However, this is a very minor point.

Reviewer #2 (Public Review):

In this work, the authors investigate the role of CRB3 in the formation of the primary cilium both in a mouse model and in human cells. They confirm in a conditional knock-out (KO) mouse model that Crb3 is necessary for the formation of the primary cilium in mammary and renal epithelial tissues and the new-born mice exhibit classical traits of ciliopathies. In the mouse mammary gland, the absence of Crb3 induces hyperplasia and tumorigenesis and in the human mammary tumor cells MCF10A the knock-down (KD) of CBR3 impairs ciliogenesis and the formation of a lumen in 3D-cultures with less apoptosis and spindle orientation defects during cell division.

To determine the subcellular localization of CRB3 the authors have expressed exogenously a GFP-CRB3 in MCF10A and found that this tagged protein localizes in cell-cell junctions and around pericentrin, a centrosome marker, while endogenous CRB3 localizes at the basal body. To dissect the molecular role of CRB3 the authors have performed proteomic analyses after a pull-down assay with the exogenous tagged-CRB3 and found that CRB3 interacts with Rab11 and is present in the endosomal recycling pathway. CRB3 KD also decreases the interactions between components of the γTuRC complex. In addition, the authors showed that CRB3 interacts with a tagged-Rab11 by its extracellular domain and that CRB3 promotes the interaction between Rab11 and CEP290 while CRB3 KD decreased the co-localization of GCP6 with Rab11 and γTub. Finally, the authors showed that CRB3 depletion cannot activate the Hh pathway as opposed to the Wnt pathway.

Reviewer #2 (Public Review):

The manuscript by Minkowicz et al., investigates the presence of neuronal ensembles in the striatum that may encode grooming (as a model of a naturalistic behavior). They implemented a semi-automated detection of grooming, and by recording populations of striatal cells they show that individual neurons in the striatum contain activity modulations around the start, end, or during grooming. Then using this activity they identify ensembles of cells in individual sessions/animals at the start, end or during grooming.

The behavioral tracking and recordings are remarkable, the manuscript is clearly written and the finding mostly sound with the proposed conclusions, providing original findings in the field. Nonetheless some points are raised that need further clarification

1. When claiming that the findings show encoding of transitions into or out of grooming (and duration of grooming) one could expect to see specific regressions between the neuronal activity (of individual cells or ensembles) and the parameters mentioned besides the analysis shown in figure 3 and 5.<br /> 2. Was the detection of ensembles presented in figure 4 sensible to use less than 5 seconds before/after grooming. I am thinking that 5 seconds are times that could contain behaviors that may have their own ensembles. Why 5 seconds?<br /> 3. According to Figure 2-figure supplement 1. The recordings were performed covering the lateral and in some cases the central part of the striatum. Shall it be specified along the text where the specific recordings come from?

Reviewer #2 (Public Review):

The authors embarked on a study to identify SNPs in clinical isolates of S. aureus that influence sensitivity to serum killing. Through a phenotypic screen of 300 previously sequenced S. aureus bacteremia (SAB) isolates, they identified ~40 SNPs causing altered serum survival. The remainder of the study focuses of tcaA, a gene with unknown function. They show that when tcaA is disrupted, it results in increased resistance to glycopeptides and antimicrobial components of human serum.

They perform an elegant series of experiments demonstrating how a tcaA knockout is more resistant to killing by whole serum. arachadonic acid, LL-37 and HNP-1. They provide compelling evidence that in the absence of tcaA resistance to arachidonic acid is mediated through release of wall teichoic acids from the cell wall, which acts as a decoy and sequesters the fatty acid.

Similarly, they suggest that resistance to cationic antimicrobial peptides is through alteration of the net charge of the cell wall due to loss of negatively charged WTAs based on reduced cytochrome C binding.

They continue to show that tcaA is induced in the presence of human serum, which causes increased resistance to the glycopeptide teichplanin.

They propose that tcaA disruption causes altered cell wall structure based on morphologic changes on TEM and increased sensitivity to lysostaphin and increased autolysis via triton x-100 assay.

5, Finally, they propose that tcaA influences mortality in SAB based on raw differences in 30-day morality. Interestingly they do decreased fitness during murine bacteremia model compared to wild-type.

Strengths:

1. The manuscript is well-written and easy to follow<br /> 2. The identification of SNPs leading to altered serum killing is convincing and valuable data<br /> 3. The mechanism for tcaA-mediated resistance to arachadonic acid and AMPs is compelling and novel<br /> 4. The murine infection data demonstrating that tcaA mutants exhibit reduced virulence is important data

Weaknesses:

1. Some of the conclusions are not supported by the data shown (either missing or incomplete)<br /> 2. The authors conclude that tcaA mutants show reduced peptidoglycan crosslinking. This conclusion is based on qualitative TEM images and increased sensitivity to lysostaphyin/autolysis. While these data are suggestive. it is difficult to draw such a conclusion without analysis of the cell wall by LC-MS (such as http://doi.org/10.1371/journal.ppat.1009468).<br /> 3. The authors conclude "TcaA contributes to increased disease severity in mice and humans". While it seems biologically plausible that a polymorphism known to increase glycopeptide MIC affects mortality, the human data presented is based on raw 30-day mortality numbers. It is misleading to make the association with mortality without adjusting for confounding variables known to influence mortality in SAB (e.g. age, comorbidities, presence of sepsis, endocarditis, duration of bacteremia). Also, with just 12 patients in the SNP group, this is likely underpowered to detect any difference.

Overall, I think this is a good submission and the majority of their conclusions are supported by the data. The mechanism behind the clinically relevant tcaA mutation is important, given its known role in glycopeptide resistance and therefore likely clinical outcomes. This manuscript would benefit with the inclusion of some additional experiments to help support their finding.

Reviewer #2 (Public Review):

In this work, the authors reported cryo-EM structures of four types of zinc-binding site mutants of a bacterial Zn2+/H+ antiporter YiiP, and proposed distinct structural/functional roles of each of the binding sites in the intramolecular Zn2+ relay and the integrity of the homodimeric structure of YiiP. MST analysis using the mutants with a single Zn2+-binding site at different pH further clarified the pH dependence of Zn2+ binding affinity of each site. Moreover, the inverse Multibind approach refined the CpHMD pKa values of the key Zn2+-binding residues so that they agreed with the MST data. Consequently, energetic coupling of Zn2+ export to the proton-motive force has been suggested. These findings definitely provide new mechanistic insight into this Zn2+/H+ antiporter.

Regrettably, the resolutions of the cryo-EM structures presented in this work are, overall, not high enough to describe detailed structure of some specific regions including the Zn2+-binding sites, and the density is missing for some important regions including the kinked segment of TM5. Further attempts toward higher resolution cryo-EM maps would be beneficial to corroborate their conclusions. Additionally, it may, in a sense, appear that the MD simulations have been carried out forcibly so that the outcomes are compatible with or nicely explain the experimental data. Although this is not unusual or unacceptable, I am concerned that the determined pKa values of some residues, especially of Asp residues at Site A, are unusually high. These outcomes seem to need careful interpretation and discussion.

Reviewer #2 (Public Review):

Zacharopoulos et al. investigated the relationship between MR spectroscopy-detected neurotransmitter concentrations (GABA/glutamate) in the intra-parietal sulcus (IPS) and middle frontal gyrus (MFG), behaviourally measured indices of sensory and cognitive processing, and fMRI measured functional connectivity within the frontoparietal network. They find that increased IPS glutamate concentration is related to poorer visuomotor processing in younger participants and better performance in older participants, while IPS GABA predicts the opposite pattern. They further show that these relationships are mediated by frontoparietal functional connectivity. Finally, they show that IPS GABA and glutamate concentration are related to fluid intelligence and that this relationship is mediated by visuomotor processing and moderated by the developmental stage. These data add to our understanding of the dynamic role of excitatory and inhibitory neurotransmitter systems in cognitive processes throughout development.

Strengths:

The study employs an impressively large cross-sectional, multimodal, dataset, with almost 300 participants ranging from 6 to 18+ years old.

The main finding (i.e., the interaction between GABA/Glu, visuomotor processing, and age) is found across three behavioural tasks and replicated in a second dataset collected 1.5 years after the first.

The authors extensively report the results of the numerous analyses performed in the supplementary material.

Weaknesses:

Pre-registration of experimental and analytical plans should be the norm, e.g., to reduce so-called 'p-hacking'. I am by no means asserting that this behaviour has occurred in the current study; however, it is disappointing that there is no reported pre-registration for such a large-scale study, where the selection and order of analyses (and the subsequent corrections applied) can meaningfully influence the pattern of results.

Many tests were performed in the study using frequentist statistics, and the way the results are reported makes it difficult to discern how distinguishable those that were reported as meaningful are from those that were disregarded.

Insufficient analyses were conducted to describe the relationships between a) GABA and glutamate, b) repeated behavioural measures, and c) test-retest reliability. This reduces the strength of the claims, some of which could be accounted for by simpler, potentially less interesting, explanations.

Reviewer #2 (Public Review):

The mechanisms of action potential firing were studied by whole-cell patch-clamp recordings in acute brain slices of the zebra finch. The study builds on the initial finding by Zemel et al. (2021) that the action potentials of robustus arcopallialis projection neurons (RAPNs) have an exceptional small half-duration of about 0.2 ms at 40C. The authors, therefore, set out to investigate the mechanisms of action potential repolarization. They use an impressive set of complementary techniques including voltage clamp and current clamp recordings, pharmacological interventions with classical and novel subunit-specific blockers, in situ hybridization, and comparative genomics of the KCNC/Kv3 potassium channel genes. The data convincingly demonstrate that the Kv3.1 but not Kv3.2-Kv3.4 nor Kv1.1/1.2/1.6, Kv7, or BK channels mediate the rapid repolarization. The manuscript is clearly written and the data and the presentation of the data are of the highest scientific quality. The study is of interest to a broad readership because the zebra finch is a fascinating and novel model to investigate the mechanisms of rapid motor control. The similarities of these neurons of the zebra finch with the specialized Betz cells in the motor cortex of humans and other primates demonstrates the exciting advantages of this animal model in comparison with well-established rodent models to investigate the mechanisms of complex sensory-motor control in vertebrates.

Reviewer #2 (Public Review):

This paper is an interesting and novel addition to our understanding of the link between ER stress and lipid homeostasis. Utilizing a genetic screen to determine modulators of the UPRER, Garcia, G., et al., determine C. elegans cannot activate the UPRER as strongly with knockdown of the putative hydroxysteroid dehydrogenase let-767. Additionally, let-767 knockdown results in smaller lipid droplets and changes to ER morphology. Both lipid droplet size and ER morphology size can be restored with supplementation of lipids, while the defect to UPRER activation persists. The authors elegantly show that one impact of let-767 knockdown on UPR is downstream of XBP1 splicing. The authors then go on to show that in mammalian cells, the lipid precursor 3-oxoacyl-CoA can cause a similar reduction to UPRER activation to that seen in C. elegans with let-767 knockdown. Some limitations of this study are that let-767 exact role in lipid metabolism is not well understood and it is unclear what the impact of let-767 knockdown in C. elegans has on lipid composition. It is also unclear mechanistically how let-767 is able to effect UPRER, as the authors show one potential mechanism is by blocking activation of the UPR downstream of XBP1 splicing. While the authors demonstrate that high levels of 3-oxoacyl-CoA can cause a reduction in the UPR response in mammalian cells, this finding is not recapitulated in C. elegans, nor does the study determine whether this compound accumulates in a let-767 knockdown.

Reviewer #2 (Public Review):

Aimon et al. used fast whole-brain imaging to investigate the relationship between walking and neural activity in adult fruit flies. They find that increases in brain-wide activity are tightly correlated with walking behavior, and not with grooming or flailing, and are independent of visual input. They reveal that excitatory, inhibitory, and neuromodulatory neurons all contribute to brain-wide increases in neural activity during walk. Aimon et al. extend their observations of brain-wide activity to reveal that activity in some inferior brain regions is more correlated with walk than in other brain regions. The authors further analyzed their imaging dataset to identify candidate brain regions and cell types that may be important for walking behavior, which will be useful in hypothesis generation in future studies. Finally, the authors show that brain-wide activity is similar between spontaneous and forced walk and that severing the connection between the ventral nerve cord and central brain abolishes walk-related increases in brain activity. These results suggest that increases in brain-wide activity during walking may be largely attributed to sensory and proprioceptive feedback ascending to the central brain from the ventral nerve cord rather than to top-down executive and motor control programs. The observations presented in this study suggest hypotheses that may be tested in future studies.

Strengths: This paper presents a rich imaging dataset that is well-analyzed and cataloged, which will be valuable for researchers who use this paper for future hypothesis generation. The comparison of many different reagents, imaging speeds, and behavioral conditions suggests that the observed increases in brain-wide activity during walking are quite robust to imaging methods in adult fruit flies.

Weaknesses: This study is largely observational, and the few experimental manipulations presented are insufficient to support the author's broad claims about the generation of brain-wide neural activity.

Notably, the authors suggest that their image analysis can reveal individual cell types that are important for walking by matching their morphologies to registered components from whole-brain imaging experiments. While these predictions are a useful starting point for future experiments, they have not convincingly shown that their method can identify individual cell types in genetic reagents with more restricted expression patterns. Adding further validation to show that genetically subtracting the candidate neurons from the overall expression pattern of the calcium indicator abolishes that component from the response would strengthen this claim. Furthermore, imaging the matched candidate neuronal cell type to show that it recapitulates the activity dynamics of the proposed component would add additional evidence.

In addition, increases in neural activity prior to walk onset in specific brain regions are intriguing but insufficient to demonstrate the neurons in these regions trigger walking. This claim should await further studies that employ targeted and acute manipulation of neural activity, as noted by the authors. Furthermore, that activity in these brain regions is significantly increased prior to walk onset awaits more rigorous statistical testing, as do the authors' claims that spontaneous versus forced walking alters these dynamics. The suggestion that walking increases brain-wide activity via feedback from the ventral nerve cord is an interesting possibility and would also benefit from additional experimental validation. Activating and silencing neurons that provide proprioceptive feedback from the legs and determining the effect of this manipulation on brain-wide neural activity would be a good starting point.

Reviewer #2 (Public Review):

In this study, Tomasi et al identify a series of tRNA modifying enzymes from Mtb, show their function in the relevant tRNA modifications and by using at least one deleted strain for MnmA, they show the relevance of tRNA modification in intra-host survival and postulate their potential role in pathogenesis.

Conceptually it is a wonderful study, given that tRNA modifications are so fundamental to all life forms, showing their role in Mtb growth in the host is significant. However, the authors have not thoroughly analyzed the phenotype. The growth defect aspect or impact on pathogenesis needs to be adequately addressed.

- The authors show that ΔmnmA grows equally well in the in vitro cultures as the WT. However, they show attenuated growth in the macrophages. Is it because Glu1_TTC and Gln1-TTG tRNAs are not the preferred tRNAs for incorporation of Glu and Gln, respectively? And for some reason, they get preferred over the alternate tRNAs during infection? What dictates this selectivity?

- As such the growth defect shown in macrophages would be more convincing if the authors also show the phenotype of complementation with WT mnmA.

An important consideration here is the universal nature of these modifications across the life forms. Any strategy to utilize these enzymes as the potential therapeutic candidate would have to factor in this important aspect.

Reviewer #2 (Public Review):

In this publication, the authors provide a comprehensive trajectory of transcriptional changes in Müller glia cells (MG) in the regenerating retina of zebrafish. These resident glia cells of the retina can differentiate into all neural cell classes following injury, providing full regenerative capabilities of the zebrafish retina. The authors achieved this by using single-cell RNA sequencing of Müller glia, progenitors, and regenerated progeny, comparing uninjured and light-lesioned retinae.

The isolation strategy involves using two transgenic strains, one labelling dividing cells and their immediate progeny, and the other Müller glia cells. This allowed them to separate injury-induced proliferating and non-reactive Müller glia cells. Subsequent single-cell transcriptomics showed that MG could be non-reactive under both uninjured and lesioned conditions and reactive MG give rise to a cell population that both replenishes the pool of MG and replenish neurogenic retinal precursor cells. These precursor cells produce regenerated neurons in a developmental time series with ganglion cells being born first and bipolar cells being born last. Interestingly hybrid populations have been detected that co-share characteristics of photoreceptor precursors and reactive glia.

This is the first study of its kind following the dynamic changes of transcriptional changes during retinal regeneration, providing a rich data source of genes involved in regeneration. Their finding of transcriptionally separable MG populations is intriguing.

This study focuses on the light-lesioned retina and leaves open the question if the observed transcriptional trajectories of regenerating neurons are generalizable to other lesion models (e.g. chemical or mutational lesions) or are specific to the light-damaged retina.

Reviewer #2 (Public Review):

The authors sought to characterize normal placental aging to better understand how the molecular and cellular events that trigger the labor process. An understanding of these mechanisms would not only provide insight into term labor, but also potential triggers of preterm labor, a common pregnancy complication with no effective intervention. Using bulk transcriptomic analysis of mouse and human placenta at different gestational timepoints, the authors determined that stabilization of HIF-1 signaling accompanied by mitochondrial dysfunction and cellular senescence are molecular signatures of term placenta. They also used in vitro trophoblast (choriocarcinoma) and a uterine myocyte culture system to further validate their findings. Lastly, using chemically induced HIF-1 induction in vivo in mice, the authors showed that stabilization of HIF-1 protein in the placenta reduced the gestational length significantly.

The major strength of this study is the use of multiple model systems to address the question at hand. The consistency of findings between mouse and human placenta, and the validation of mechanisms in vitro and in vivo modeling are strong support for their conclusions. The rationale for studying the term placentas to understand the abnormal process of preterm birth is clearly explained. Although the idea that hypoxic stress and placental senescence are triggers for labor is not novel, the comprehensiveness of the approach and idea to study the normal aging process are appreciated.

There are some areas of the manuscript that lack clarity and weaknesses in the methodology worth noting. The rationale for focusing on senescence and HIF-1 is not clearly given that other pathways were more significantly altered in the WGCNA analysis. The placental gene expression data were from bulk transcriptomic analyses, yet the authors do not explicitly discuss the limitations of this approach. Although the reader can assume that the authors attribute the mRNA signature of aging to trophoblasts - of which, there are different types - clarification regarding their interpretation of the data and the relevant cell types would strengthen the paper. Additionally, while the inclusion of human placenta data is a major strength, the differences between mouse and human placental structure and cell types make highlighting the specific cells of interest even more important; although there are correlations between mouse and human placenta, there are also many differences, and the comparison is further limited when considering the whole placenta rather than specific cell populations.

Additional details regarding methods and the reasons for choosing certain readouts are needed. Trophoblasts are sensitive to oxygen tension which varies according to gestational age, and it is unclear if this variable was taken into consideration in this study. Many of the cellular processes examined are well characterized in the literature yet the rationale for choosing certain markers is unclear (e.g., Glb1 for senescence; the transcripts selected as representative of the senescence-associated secretory phenotype; mtDNA lesion rate).

Overall, the findings presented are a valuable contribution to the field. The authors provide a thoughtful discussion that places their findings in the context of current literature and poses interesting questions for future pursuit. Their efforts to be comprehensive in the characterization of placental aging is a major strength; few placental studies attempt to integrate mouse and human data to this extent, and the validation and presentation of a potential mechanism by which fetal trophoblasts signal to maternal uterine myocytes are exciting. Nevertheless, a clear discussion of the methodologic limitations of the study would strengthen the manuscript.

Reviewer #2 (Public Review):

This preprint presents a compelling study examining the relationship between genotypic changes and phenotypic traits in bacteria over an extended period using the Long-Term Evolution Experiment (LTEE) as a model. The primary advances in methodology include employing high-resolution mass spectrometry for comprehensive metabolic profiling and combining it with previous gene expression and DNA sequencing datasets. This approach provides insight into how specific genetic mutations can alter metabolic pathways over 50,000 generations, enabling a deeper understanding of how genetic changes lead to observed differences in evolved bacterial strains. The findings reveal that evolved bacteria possess more diverse metabolic profiles compared to their ancestors, suggesting that these populations have uniquely adapted to their environment. The work also attempts to uncover the molecular basis for this adaptive evolution, demonstrating how specific genetic changes have influenced the bacteria's metabolic pathways.

Overall, this is a significant and well-executed research study. It offers new insights into the complex relationship between genetic changes and observable traits in evolving populations and utilizes metabolomics in the LTEE, a novel approach in combination with RNA-seq and mutation datasets.

However, the paper's overall clarity is lacking. It is spread too thin and covers many topics without a clear focus. I strongly recommend a substantial rewrite of the manuscript, emphasizing structure and readability. The science is well executed, but the current writing does not do it justice.

Reviewer #2 (Public Review):

Hersperger et al. investigated the importance of Drosophila immune cells, called hemocytes, in the response to oxidative stress in adult flies. They found that hemocytes are essential in this response, and using state-of-the-art single-cell transcriptomics, they identified expression changes at the level of individual hemocytes. This allowed them to cluster hemocytes into subgroups with different responses, which certainly represents very valuable work. One of the clusters appears to respond directly to oxidative stress and shows a very specific expression response that could be related to the observed systemic metabolic changes and energy mobilization. However, the association of these transcriptional changes in hemocytes with metabolic changes is not well established in this work. Using hemocyte-specific genetic manipulation, the authors convincingly show that the DNA damage response in hemocytes regulates JNK activity and subsequent expression of the JAK/STAT ligand Upd3. Silencing of the DNA damage response or excessive activation of JNK and Upd3 leads to increased susceptibility to oxidative stress. This nicely demonstrates the importance of tight control of JNK-Upd3 signaling in hemocytes during oxidative stress. However, it would have been nice to show here a link to systemic metabolic changes, as the authors conclude that it is tissue wasting caused by excessive Upd3 activation that leads to increased susceptibility, but metabolic changes were not analyzed in the manipulated flies. The overall conclusion of this work, as presented by the authors, is that Upd3 expression in hemocytes under oxidative stress leads to tissue wasting, whereas in fact it has been shown that excessive hemocyte-specific Upd3 activation leads to increased susceptibility to oxidative stress (whether due to increased tissue wasting remains a question). The DNA damage response ensures tight control of JNK-Upd3, which is important. However, what role naturally occurring Upd3 expression plays in a single hemocyte cluster during oxidative stress has not been tested. What if the energy mobilization induced by this naturally occurring Upd3 expression during oxidative stress is actually beneficial, as the authors themselves state in the abstract - for potential tissue repair? It would have been useful to clarify in the manuscript that the observed pathological effects are due to overactivation of Upd3 (an important finding), but this does not necessarily mean that the observed expression of Upd3 in one cluster of hemocytes causes the pathology.

Reviewer #2 (Public Review):

In whole exome sequencing of two patients suffering from MMAF syndrome, mutations of CCDC146 gene that result in premature stop codons were identified. The position of mutations could result in a truncated form of protein, thus whether these patients do indeed lack CCDC146 protein or if present, whether the truncated protein is functional, is unanswered by showing the CCDC146 protein localization only in the sperm from healthy donors. The main claim that CCDC146 protein is microtubule associated protein in the axoneme is well supported imaging expanded sperm flagellum to increase spatial resolution. However, the author's claim that the signal in the mid-piece is not specific is less supported by experimental evidence. The detection of CCDC146 in the sperm head is not further explored while TEM images show spermatogenesis defects in the manchette and acrosome formation. Increased detection of the CCDC146 protein in mouse sperm with sarkosyl supports its association with microtubules but does not exclude its potential role in the formation of sperm head. Overall, this study provides valuable information on CCDC146 function in male germ cells during spermatogenesis.

Reviewer #2 (Public Review):

This study visualizes a specific localized form of cell-to-cell communication and conveys very well with what dynamics and sensitivity this biological phenomenon occurs.

Using a FRET-based PKA biosensor, the authors observed that radial localized kinase activity changes spontaneously occur in adjacent cells of certain cell density. This phenomenon of radial propagation of PKA activity changes in groups of cells was further mechanistically elucidated and characterized. Interestingly, the authors found that individual cells in the cell groups form spontaneous Ca2+ transients, which at a certain strength can trigger the biosynthesis and release of prostaglandin E2 (PGE2). PGE2 then acts on the neighboring cells and triggers the increase of cAMP levels and the associated activation of the PKA via G-protein-coupled receptors (EP2 and EP4). In systematic, well-structured experiments, it was then found that the frequency of occurrence of such radial activations depends not only on the cell density but also on the activation state of the ERK MAP kinase pathway. The authors skillfully used various modern genetically encoded biosensors and other tools such as optogenetic tools to visualize and characterize an interesting biological phenomenon of cell-to-cell communication. The insights gained with these investigations produce a better understanding of the dynamics, sensitivity, and spatial extent with which such communications can occur in a cell network. It is also worth noting that the authors have not limited the studies to 2D cell culture in vitro, but were also able to confirm the findings in an animal model.

Reviewer #2 (Public Review):

In this study, Huang et al. investigated Bacillus velezensis, a species that colonizes plant roots as part of the rhizosphere. They showed that clone of B. velezensis SQR9 retains a division of labor of motile, planktonic subpopulation that do not produce extracellular matrix (ECM) and biofilm-forming sessile subpopulation that do produce ECM. Specifically, the sessile subpopulation secret toxins named bacillunoic acids (BAs) to kill some, but not all, of the planktonic subpopulation. The killing mechanism is mediated by a global regulator Spo0A, which co-activates BAs production and immunity, as well as ECM production. A strain that has a disrupted policing system revealed reduced biofilm formation, lower resistance to environmental stresses and alleviated ability to colonize plant roots. Overall, the toxin-mediated policing system is important for B. velezensis to mediate division of labor for enhancing population stability and ecological fitness when required (e.g., cell transition from a planktonic style to a multicellular style).

Reviewer #2 (Public Review):

Raykov et al. reported that TrafE, a member of the E3 ubiquitin ligase family similar to the TRAF proteins in mammalian cells, is essential for Dictyostelium discoideum to effectively respond to endolysosomal damage and defend itself against Mycobacterium marinum infection. First, the authors demonstrate that TrafE is recruited to the site of Mycobacterium-Containing Vacuole (MCV) damage along with ubiquitin molecules. This recruitment is necessary for the effective suppression of M. marinum growth in the cells. They also found that this response was not limited to the damage caused by M. marinum, but was also triggered by sterile damage caused by chemical compounds. Furthermore, the authors revealed that TrafE plays a role in the recruitment of Vps4 to sites of membrane damage and regulates the disassembly of ESCRT subunits. While TRAF6 has been previously implicated in ubiquitination in response to invaded bacteria in mammalian cells, this study provides solid data that furthers our understanding of the mechanism behind xenophagy. The authors conducted a thorough analysis to contribute to this field of research.

Reviewer #2 (Public Review):

The manuscript "Rapid and precise genome engineering in a naturally short-lived vertebrate" describes the development of a CRISPR- based knock-in technology in Nothobranchius furzeri, or the African turquoise killifish, an innovative model species for studying aging and age-related disorders. While Tol2 systems had been demonstrated to be successful in generating reporter killifish lines, endogenous reporters via knock-in had not been reported so far. The major strength of the paper is that the authors show that they have been successful in developing 5 different knock-in fish lines with large inserts (up to 1.8kb) with high efficiency. They have inserted single or dual fluorescent reporters and demonstrated expression in line with the expected pattern. This is a breakthrough in the field and this method can be instrumental for many researchers working with unusual model species, and in particular, will expand the killifish community toolbox.

While this is very promising, the paper would benefit from a more rigorous validation of the KI lines that were generated. The authors did not show a co-localisation of the target gene expression with the reporter to prove bona fide reporting. In addition, it was not clear whether the KI affects the endogenous expression level of the target genes. The targeting efficiency of the method is high, but the quantifications are based on rather limited numbers of animals, which might not yet be very robust. A larger number of animals would have strengthened the efficiency conclusion.

The figures of the manuscript are well designed and support the conclusions, but several contain information that is not discussed in the main text, such as (un)expected bands on gels, reporter staining in WT animals, and unusual staining patterns. The body text seems to ignore these and only discusses findings that are in line with the story. A key point to the efficiency of the method seems to be a chemical modification of the repair template, which was not disclosed in the method section which at the moment hampers replication.

Finally, the discussion is brief and does not benchmark the method to other CRISPR-based KI methods in Xenopus or more typical model species such as mouse.

In conclusion, this paper describes a breakthrough method for a rising animal model that would benefit from a more thorough validation. Full disclosure of the methodology will boost the generation of genetically edited killifish lines and aid in the establishment of this promising animal model.

Reviewer #2 (Public Review):

The goal of this study was to determine whether heartbeat-evoked responses measured at the scalp level with EEG, which followed regularity violations, could potencial help inform the diagnosis of patients with altered states of consciousness.

The authors use high density EEG and an oddball paradigm that probes violations of both local and global regularities. Four groups were considered including unresponsive wakefulness syndrome patents, minimally consciousness patients, emerging minimally consciousness patients and healthy controls. A difference was found between unresponsive and minimally conscious patients in the amplitude of the heartbeat evoked responses measure with EEG following a sound that violated a global regularity. Similarly, differences were found between the variance of these responses between the two above mentioned groups (N=58 and N=59), but no differences were found in relation to the healthy control group, which appear to be "in between" the two other groups (at least for global effect of HER). I thought this was a little counterintuitive and raises some questions about what this neural signature can tell us about the state of consciousness. Having said that, the healthy control sample was very small, more than 5 times smaller (only N=11).

In general, I thought the Discussion section was a little light on the implications of the findings, what they tell us about the brain mechanisms of consciousness and their different levels/states. A question is raised about whether it is necessary to lock EEG to heartbeats to find differences between patients. The data appeared to say that this is not the case but the discussion does not appear to reflect that very clearly.

Reviewer #2 (Public Review):

A distinguishing feature of live cells is that intracellular organelles move powered by molecular motors. However, the arsenal of molecular motors is limited relative to the vast variety of cargoes and processes involving long-distance movement. Cells cope with this mismatch by using adaptors that "bridge" a given molecular motor with a specific cargo, whose identity is dictated by peripheral membrane proteins, such RABs, or identity-determining lipids, such as PtdIns3P. Cytoplasmic dynein walks towards the minus end of the microtubules. A score of cellular processes is dependent on dynein, such that deficient regulation of the motor has deep consequences in cellular homeostasis, and the identification of new adapters is of broad interest, both basic and, potentially, clinical.

Dynein adaptors usually stabilize the binding of dynactin to dynein using coiled-coil regions to longitudinally embrace dynactin, holding it to the elongated dynein cap of the super-complex. Not only do they adapt cargo but additionally increase the processivity and speed of the motor. In this manuscript, Julie and collaborators present evidence that a protein denoted kazrin, which is involved in a variety of processes, is actually an adaptor connecting endosome domains specialized in recycling cargo back to the surface of the cell by way of the RAB11 perinuclear recycling endosome. The topic is important, experiments have been carefully conducted and well controlled and display items faithfully guide readers through the main findings. However, I feel that the evidence that kazrin is a dynein adaptor is somewhat thin and that it could be improved with relatively little additional work. The manuscript would also benefit from better integration of the conclusions in the current state of the art in the dynein field.

Reviewer #2 (Public Review):

In the current study, Mondoloni and colleagues investigate the neural correlates contributing to nicotine aversion and its alteration following chronic nicotine exposure. The question asked is important to the field of individual vulnerability to drug addiction and has translational significance. First, the authors identify individual nicotine consumption profiles across isogenic mice. Further, they employed in vivo and ex vivo physiological approaches to defining how antiparticle nuclei (IPn) neuronal response to nicotine is associated with nicotine avoidance. Additionally, the authors determine that chronic nicotine exposure impairs IPn neuronal normal response to nicotine, thus contributing to higher amounts of nicotine consumption. Finally, they used transgenic and viral-mediated gene expression approaches to establish a causal link between b4 nicotine receptor function and nicotine avoidance processes.

The manuscript and experimental strategy are well designed and executed; the current dataset requires supplemental analyses and details to exclude possible alternatives. Overall, the results are exciting and provide helpful information to the field of drug addiction research, individual vulnerability to drug addiction, and neuronal physiology. Below are some comments aiming to help the authors improve this interesting study.

1. The authors used a two-bottle choice behavioral paradigm to investigate the neurophysiological substrate contributing to nicotine avoidance behaviors. While the data set supporting the author's interpretation is compelling and the experiments are well-conducted, a few supplemental control analyses will strengthen the current manuscript.<br /> a. The bitter taste of nicotine might generate confounds in the data interpretation: are the mice avoiding the bitterness or the nicotine-induced physiological effect? To address this question, the authors mixed nicotine with saccharine, thus covering the bitterness of nicotine. Additionally, the authors show that all the mice exposed to quinine avoid it, and in comparison, the N-Av don't avoid the bitterness of the nicotine-saccharine solution. Yet it is unclear if Av and N-Av have different taste discrimination capacities and if such taste discrimination capacities drive the N-Av to consume less nicotine. Would Av and N-Av mice avoid quinine differently after the 20-day nicotine paradigm? Would the authors observe individual nicotine drinking behaviors if nicotine/quinine vs. quinine were offered to the mice?<br /> b. Metabolic variabilities amongst isogenic mice have been observed. Thus, while the mice consume different amounts of nicotine, changes in metabolic processes, thus blood nicotine concentrations, could explain differences in nicotine consumption and neurophysiology across individuals. The authors should control if the blood concentration of nicotine metabolites between N-Av and Av are similar when consuming identical amounts of nicotine (50ug/ml), different amounts (200ug/ml), and in response to an acute injection of a fixed nicotine quantity.

2. Av mice exposed to nicotine_200ug/ml display minimal nicotine_50ug/ml consumption, yet would Av mice restore a percent nicotine consumption >20 when exposed to a more extended session at 50ug/kg? Such a data set will help identify and isolate learned avoidance processes from dose-dependent avoidance behaviors.

3. The author should further investigate the basal properties of IPn neuron in vivo firing rate activity recorded and establish if their spontaneous activity determines their nicotine responses in vivo, such as firing rate, ISI, tonic, or phasic patterns. These analyses will provide helpful information to the neurophysiologist investigating the function of IPn neurons and will also inform how chronic nicotine exposure shapes the IPn neurophysiological properties.

Reviewer #2 (Public Review):

The authors successfully show that how EPAC and PKCε work together to recruit presynaptic proteins for neurotransmitter release instead of synaptic vesicle formation since the absence of EPAC and PKCε does not affect the number of synaptic vesicles. In addition, the data clearly demonstrate that EPAC and PKCε function specifically at the presynaptic terminals and thus is required for induction of presynaptic LTP. Their suggested EPAC- PKCε module is also essential for proper cerebellar motor performance and motor learning.

Furthermore, the order of data analysis perfectly matches the logical explanation of the entire story. The authors first prove that EPAC and PKCε, together with RIM1a, are necessary for neurotransmitter release at the presynaptic terminal. Then, by using specific knockdown mice of presynaptic granule cells, both proteins contribute to the release of synaptic vesicles in that only the frequencies of EPSC have changed. In addition, presynaptic LTP is only induced with the presence of EPAC and PKCε, highlighting the important role of the EPAC- PKCε module. Ultimately, the impact of EPAC and PKCε is shown by conducting the behavior tasks including OKR, VOR, and VVOR.

The authors suggest the missing link between EPAC and RIM1 is PKCε. Phosphorylation of RIM1 by PKCε is a novel signaling cascade found in this paper. The authors' data from the heterologous expression system and cerebellar granule cell-specific PKCε KO mice indicate that PKCε can regulate RIM1Threonine phosphorylation.<br /> The EPAC-PKCε unit is essential to both presynaptic neurotransmitter release and presynaptic LTP in parallel fiber-Purkinje cell synapse. Future work is necessary to dissect which is responsible for cerebellar motor performance and motor learning.

The study provides the necessity of exploring the new part of the motor learning circuit since the significant focus of cerebellar motor learning has been only confined to postsynaptic plasticity. Generally, postsynaptic plasticity is affected by the presynaptic properties, such as presynaptic vesicle release and recycling of neurotransmitters at the synapse. Also, the presynaptic terminal, which can be referred to as an inducing force of the postsynaptic plasticity, does not merely release the neurotransmitters at a constant rate; they also change as a result of incoming stimuli. Such change is called presynaptic plasticity. Therefore, it should be further scrutinized how presynaptic plasticity is conducted and determined.

operator norm is actually a type of norm. Linear operators as homomorphism between the vector spaces are indeed a vector space itself. See here.

The properties of the operator norm basically interits the properties from the normed vector space. This is direct from the definition. However, these results are only for the bounded linear operators.

Reviewer #2 (Public Review):

The authors sought to define the molecular structure of autoinhibited Kinesin-1, which is the major kinesin providing plus-end directed transport on microtubules. The paper reports a structural model of full-length kinesin-1 which builds on the known folded conformation of kinesin-1 and describes its autoinhibitory mechanism using cryo-EM, alphafold structural predictions, cross-linking and mass spectrometry. The authors study the conformation of dimeric Kinesin Heavy Chain (KHC) and tetrameric KHC bound to the Kinesin Light Chains (KLCs), where KLC stabilize the autoinhibited conformation. The combination of these various approaches leads to an integrated molecular model of autoinhibited Kinesin-1. Until now, there was some debate over the role of the small coiled coil 3 (a and b) and where the hinge region of Kinesin-1. The authors resolve this question and present data indicating the hinge is between cc3a and cc3b.

In some places the absence of crosslinks is reported as a lack of interaction, however it could also be that there are no residues that can be crosslinked in this region. The distance is also not reported in the figures so we do not know how valid these model are. For example for TRAP binding to KHC, there are not many crosslinks but it is not clear if there was an issue with the complex assembly or crosslinking reaction-as there is no EM data of this complex. There is also a structural model of KHC and KLC (Fig 4) where the domains are too far apart for the crosslinks to be allowed, raising a question about whether that model is correct or not. The structural data are supported by single molecule motility assays with various mutants of Kinesin-1, which greatly help characterising the domains functionally.

Overall there are some interesting novel data on the autoinhibitory mechanism of Kinesin-1, with well performed and analyzed data with KLC and TRAP. The topic and paper will be of interest to many.

Reviewer #2 (Public Review):

To date, only a handful of studies have addressed the importance of AGS3, a paralog of the relatively well-characterized spindle orientation factor LGN. The authors now show that AGS3 acts as a negative regulator of LGN and propose that this activity could work through competition for binding partner(s). Remarkably, regulation is temporally restricted in such a way that the conserved role played by LGN in metaphase spindle orientation is unaffected. Instead, AGS3 regulates a post-metaphase function for LGN, namely Telophase Correction.

The article is well-written, the experiments are performed at a high level, and the claims are generally supported by the data. Two main points of confusion are raised in the current version. 1) The authors show that AGS3 regulates cortical localization of LGN, but would need to clarify how LGN is being affected. 2) The authors propose in the discussion that AGS3 might exert its regulatory effect through competition for NuMA, an important binding partner for LGN, but would need to clarify how and why NuMA would be involved in Telophase Correction.

Reviewer #2 (Public Review):

This manuscript by Walker et al describes an elegant study that synergizes our knowledge of virulence gene regulation of Vibrio cholerae. The work brings a new element of regulation for CRP, notably that CRP and the high density regulator HapR co-occupy the same site on the DNA but modeling predicts they occupy different faces of the DNA. The DNA binding and structural modeling work is nicely conducted and data of co-occupation are convincing. The work could benefit from doing a better job in the manuscript preparation to integrate the findings into our current state of knowledge of HapR and CRP regulated genes and to elevate the impact of the work to address how bacteria are responding to the nutritional environment. Importantly, the focus of the work is heavily based on the impact of use of GlcNAc as a carbon source when bacteria bind to chitin in the environment, but absent the impact during infection when CRP and HapR have known roles. Further, the impact on biological events controlled by HapR integration with the utilization of carbon sources (including biofilm formation) is not explored. The rigor and reproducibility of the work needs to be better conveyed.

Specific comments to address:

1) Abstract. A comment on the impact of this work should be included in the last sentence. Specifically, how the integration of CRP with QS for gene expression under specific environments impacts the lifestyle of Vc is needed. The discussion includes comments regarding the impact of CRP regulation as a sensor of carbon source and nutrition and these could be quickly summarized as part of the abstract.<br /> 2) Line 74. This paper examines the overlap of HapR with CRP, but ignores entirely AphA. HapR is repressed by Qrrs (downstream of LuxO-P) while AphA is activated by Qrrs. WithLuxO activating AphA, it has a significant sized "regulon" of genes turned on at low density. It seems reasonable that there is a possibility of overlap also between CRP and AphA. While doing an AphA CHIP-seq is likely outside the scope of this work, some bioinformatic or simply a visual analysis of the promoters known AphA regulated genes would be interest to comment on with speculation in the discussion and/or supplement.<br /> 3) Line 100. Accordingly with the above statement, the focus here on HapR indicates that the focus is on gene expression via LuxO and HapR, at high density. Thus the sentence should read "we sought to map the binding of LuxO and HapR of V. cholerae genome at high density".<br /> 4) Line 109. The identification of minor LuxO binding site in the intergenic region between VC1142 and VC1143 raises whether there may be a previously unrecognized sRNA here. As another panel in figure S1, can you provide a map of the intergenic region showing the start codons and putative -10 to -35 sites. Is there room here for an sRNA? Is there one known from the many sRNA predictions / identifications previously done? Some additional analysis would be helpful.<br /> 5) Line 117. This sentence states that the CHIP seq analysis in this study includes previously identified HapR regulated genes, but does not reveal that many known HapR regulated genes are absent from Table 1 and thus were missed in this study. Of 24 HapR regulated investigated by Tsou et al, only 1 is found in Table 1 of this study. A few are commented in the discussion and Figure S7. It might be useful to add a Venn Diagram to Figure 1 (and list table in supplement) for results of Tsou et al, Waters et al, Lin et al, and Nielson et al and any others). A major question is whether the trend found here for genes identified by CHIP-seq in this study hold up across the entire HapR regulon. There should also be comments in the discussion on perhaps how different methods (including growth state and carbon sources of media) may have impacted the complexity of the regulon identified by the different authors and different methods.<br /> 6) The transcription data are generally well performed. In all figures, add comments to the figure legends that the experiments are representative gels from n=# (the number of replicate experiments for the gel based assays). Statements to the rigor of the work are currently missing.<br /> 7) Line 357-360. The demonstration of lack of growth on MurNAc is a nice for the impact of the work. However, more detailed comments are needed for M9 plus glucose for the uninformed reader to be reminded that growth in glucose is also impaired due to lack of cAMP in glucose replete conditions and thus minimal CRP is active. But why is this now dependent of hapR? A reminder also that in LB oligopeptides from tryptone are the main carbon source and thus CRP would be active.<br /> 8) A great final experiment to demonstrate the model would have been to show co-localization of the promoter by CRP and HapR from bacteria grown in LB media but not in LB+glucose or in M9+glycerol and M9+MurNAc but not M9+glucose. This would enhance the model by linking more directly to the carbon sources (currently only indirect via growth curves)<br /> 9) Discussion. Comments and model focus heavily on GlcNAc-6P but HapR has a regulator role also during late infection (high density). How does CRP co-operativity impact during the in vivo conditions? Does the Biphasic role of CRP play a role here (PMID: 20862321)?

Reviewer #2 (Public Review):

DNA gyrase is an essential enzyme in bacteria that regulates DNA topology and has the unique property to introduce negative supercoils into DNA. This enzyme contains 2 subunits GyrA and GyrB, which forms an A2B2 heterotetramer that associates with DNA and hydrolyzes ATP. The molecular structure of the A2B2 assembly is composed of 3 dimeric interfaces, called gates, which allow the cleavage and transport of DNA double stranded molecules through the gates, in order to perform DNA topology simplification.<br /> The article by Germe et al. questions the existence and possible mechanism for subunit exchange in the bacterial DNA gyrase complex.

The complexes are purified as a dimer of GyrA and a fusion of GyrB and GyrA (GyrBA), encoded by different plasmids, to allow the introduction of targeted mutations on one side only of the complex. The conclusion drawn by the authors is that subunit exchange does happen, favored by DNA binding and wrapping. They propose that the accumulation of gyrase in higher-order oligomers can favor rapid subunit exchange between two active gyrase complexes brought into proximity.<br /> The authors are also debating the conclusions of a previous article by Gubaev, Weidlich et al 2016 (https://doi.org/10.1093/nar/gkw740). Gubaev et al. originally used this strategy of complex reconstitution to propose a nicking-closing mechanism for the introduction of negative supercoils by DNA gyrase, an alternative mechanism that precludes DNA strand passage, previously established in the field. Germe et al. incriminate in this earlier study the potential subunit swapping of the recombinant protein with the endogenous enzyme, that would be responsible for the detected negative supercoiling activity.

Accordingly, the authors also conclude that they cannot completely exclude the presence of endogenous subunits in their samples as well.

Strengths

The mix of gyrase subunits is plausible, this mechanism has been suggested by Ideka et al, 2004 and also for the human Top2 isoforms with the formation of Top2a/Top2b hybrids being identified in HeLa cells (doi: 10.1073/pnas.93.16.8288).<br /> Germe et al have used extensive and solid biochemical experiments, together with thorough experimental controls, involving :<br /> - the purification of gyrase subunits including mutants with domain deletion, subunit fusion or point mutations.<br /> - DNA relaxation, cleavage and supercoiling assays<br /> - biophysical characterization in solution (size exclusion chromatography, mass photometry, mass spectrometry)

Together the combination of experimental approaches provides solid evidence for subunit swapping in gyrase in vitro, despite the technical limitations of standard biochemistry applied to such a complex macromolecule.

Weaknesses

The conclusions of this study could be strengthened by in vivo data to identify subunit swapping in the bacteria, as proposed by Ideka et al, 2004. Indeed, if shown in vivo, together with this biochemical evidence, this mechanism could have a substantial impact on our understanding of bacterial physiology and resistance to drugs.

Reviewer #2 (Public Review):

I believe the authors succeeded in finding neural evidence of reactivation during REM sleep. This is their main claim, and I applaud them for that. I also applaud their efforts to explore their data beyond this claim, and I think they included appropriate controls in their experimental design. However, I found other aspects of the paper to be unclear or lacking in support. I include major and medium-level comments:

Major comments, grouped by theme with specifics below:<br /> Theta.<br /> Overall assessment: the theta effects are either over-emphasized or unclear. Please either remove the high/low theta effects or provide a better justification for why they are insightful.

Lines ~ 115-121: Please include the statistics for low-theta power trials. Also, without a significant difference between high- and low-theta power trials, it is unclear why this analysis is being featured. Does theta actually matter for classification accuracy?

Lines 123-128: What ARE the important bands for classification? I understand the point about it overlapping in time with the classification window without being discriminative between the conditions, but it still is not clear why theta is being featured given the non-significant differences between high/low theta and the lack of its involvement in classification. REM sleep is high in theta, but other than that, I do not understand the focus given this lack of empirical support for its relevance.

Line 232-233: "8). In our data, trials with higher theta power show greater evidence of memory reactivation." Please do not use this language without a difference between high and low theta trials. You can say there was significance using high theta power and not with low theta power, but without the contrast, you cannot say this.

Physiology / Figure 2.<br /> Overall assessment: It would be helpful to include more physiological data.

It would be nice, either in Figure 2 or in the supplement, to see the raw EEG traces in these conditions. These would be especially instructive because, with NREM TMR, the ERPs seem to take a stereotypical pattern that begins with a clear influence of slow oscillations (e.g., in Cairney et al., 2018), and it would be helpful to show the contrast here in REM. Also, please expand the classification window beyond 1 s for wake and 1.4 s for sleep. It seems the wake axis stops at 1 s and it would be instructive to know how long that lasts beyond 1 s. The sleep signal should also go longer. I suggest plotting it for at least 5 seconds, considering prior investigations (Cairney et al., 2018; Schreiner et al., 2018; Wang et al., 2019) found evidence of reactivation lasting beyond 1.4 s.

Temporal compression/dilation.<br /> Overall assessment: This could be cut from the paper. If the authors disagree, I am curious how they think it adds novel insight.

Line 179 section: In my opinion, this does not show evidence for compression or dilation. If anything, it argues that reactivation unfolds on a similar scale, as the numbers are clustered around 1. I suggest the authors scrap this analysis, as I do not believe it supports any main point of their paper. If they do decide to keep it, they should expand the window of dilation beyond 1.4 in Figure 3B (why cut off the graph at a data point that is still significant?). And they should later emphasize that the main conclusion, if any, is that the scales are similar.

Line 207 section on the temporal structure of reactivation, 1st paragraph: Once again, in my opinion, this whole concept is not worth mentioning here, as there is not really any relevant data in the paper that speaks to this concept.

Behavioral effects.<br /> Overall assessment: Please provide additional analyses and discussion.

Lines 171-178: Nice correlation! Was there any correlation between reactivation evidence and pre-sleep performance? If so, could the authors show those data, and also test whether this relationship holds while covarying our pre-sleep performance? The logic is that intact reactivation may rely on intact pre-sleep performance; conversely, there could be an inverse relationship if sleep reactivation is greater for initially weaker traces, as some have argued (e.g., Schapiro et al., 2018). This analysis will either strengthen their conclusion or change it -- either outcome is good.

Unlike Schönauer et al. (2017), they found a strong correspondence between REM reactivation and memory improvement across sleep; however, there was no benefit of TMR cues overall. These two results in tandem are puzzling. Could the authors discuss this more? What does it mean to have the correlation without the overall effect? Or else, is there anything else that may drive the individual differences they allude to in the Discussion?

Medium-level comments<br /> Lines 63-65: "We used two sequences and replayed only one of them in sleep. For control, we also included an adaptation night in which participants slept in the lab, and the same tones that would later be played during the experimental night were played."

I believe the authors could make a stronger point here: their design allowed them to show that they are not simply decoding SOUNDS but actual memories. The null finding on the adaptation night is definitely helpful in ruling this possibility out.

Lines 129-141: Does reactivation evidence go down (like in their prior study, Belal et al., 2018)? All they report is theta activity rather than classification evidence. Also, I am unclear why the Wilcoxon comparison was performed rather than a simple correlation in theta activity across TMR cues (though again, it makes more sense to me to investigate reactivation evidence across TMR cues instead).

Line 201: It seems unclear whether they should call this "wake-like activity" when the classifier involved training on sleep first and then showing it could decode wake rather than vice versa. I agree with the author's logic that wake signals that are specific to wake will be unhelpful during sleep, but I am not sure "wake-like" fits here. I'm not going to belabor this point, but I do encourage the authors to think deeply about whether this is truly the term that fits.

Reviewer #2 (Public Review):

The authors reexamine the effects of depth on crowding, using a clever display that presents at three depths at once, and find that placing the target or flanker at far depth greatly increases crowding, contrary to what might have been expected by prior work with small depth differences. These stimuli avoid creating conflicting cues to depth and are thus the most relevant to viewing in daily life, indicating more crowding than was expected.

Reviewer #2 (Public Review):

This study was designed to study the cortical response to violations in auditory temporal sequences during wakefulness and sleep. To this end, the study had three levels of temporal sequence, a regular temporal sequence, an auditory tone that was yoked to the cardiac signal, and an irregular tone. The authors show significant EEG differences to an omitted tone when the auditory tone was predictable both during wakefulness and sleep.

The authors analyze the ERP to the omitted tone as well as when aligned to the R-peak of the HEP. The analysis was comprehensive and the effects reported align with the interpretation given. Of particular interest was the fact that a deceleration of the heart rate was present for omissions when the auditory tone was yoked to the R-peak (synch) in all stages of wakefulness and sleep.

However, one weakness was the rationale for the current study and how the results link to current theoretical frameworks for the role of interoception in perception and cognition. This was in contrast to the clear background and explanation to study the response to omissions for a predictable auditory sequence in wakefulness and sleep. It was unclear why the authors selected the cardiac signal to yoke their auditory stimuli. What is the specific motivation for the cardiac signal rather than the respiratory signal? This was not clear.

Reviewer #2 (Public Review):

The authors tackled a longstanding question for brain evolution: if the brain regions change based on functional constraints or developmental constraints.

The strength of this study is that the authors introduced an automated method for brain segmentation based on the zebrafish tool, which is a highly advanced technology. They also performed the volume and landmark-based shape analyses in a surface, cave and their F1 and F2 hybrid, highlighting genetic regulations, and revealed 3 genetically correlating clusters of brain regions, which are brand new as far as I know. This study needs intense effort, fine skills to conduct, and intellectual efforts to summarize the vast dataset. I simply admire how the authors achieve their study at this level.

The weakness of this study is that the method/approach used in this study is difficult to test the functional constraint hypothesis although the authors nicely tested the developmental constraint hypothesis, which was highlighted in their correlation studies (volumetric and shape: Fig 4 and 5). I am also a little concerned with the accuracy of the automated segmentation algorithm shown in Figure 1-figure supplement 2. The original zebrafish paper (CobraZ) showed a similar accuracy (cross-correlation as 80%). If this level of accuracy is accepted in the field, I am OK with it.<br /> Their data support the conclusion 'brain-wide evolution occurs in distinct developmental modules' because of their correlation study. However, I am not so positive at the point that one of two central hypotheses were directly tested in this study: the functional constraint hypothesis - to test it, for example, the authors need to address the functional connectivities (calcium imaging, etc) and then test if the correlation between calcium-transients and the size/shape of each pair of brain regions.

Reviewer #2 (Public Review):

Jangir et al test the hypothesis that resistance to the antimicrobial peptide (AMP) colistin can simultaneously increase resistance to other AMPS with related modes of action. Because AMPS comprise part of innate immunity, their central concern is that colistin resistance may compromise host defenses and thereby increase bacterial virulence. Their results show that MCR-1, whether expressed from naturally circulating or synthetic plasmids, can increase the MIC to AMPS from humans, pigs, and chickens, and impart fitness benefits at sub-MIC concentrations. In addition, they find that MCR-1-containing strains have increased survival in human plasma and are more lethal in an insect infection model.

The conclusions of the paper are generally well supported by the results, but some aspects could be clearer and better defended with a few small additional experiments.

Strengths:<br /> Using both synthetic and natural plasmids makes it possible to cleanly separate the effects of MCR-1 from the effects of other plasmid-borne genes or plasmid copy numbers. This helps confirm the causal role of MCR-1 on altered AMP susceptibility.

Testing the survival of transformed isolates in human serum and in insects points to relevance in the more immunologically complex host environment where cells are exposed to a suite of factors that reduce bacterial survival.

Weaknesses/suggestions:<br /> Although increases in MIC are evident for different AMPS, the effects are generally modest. To address this, it might be helpful to use pairwise competition assays, as in Figure 1, to establish that even small changes to MIC are associated with clear selective benefits. This would be especially helpful in assays with human serum and in Galleria where the concentrations of AMPS or other immune components are unknown.

Assays using human serum are interesting but challenging to interpret given the diverse causes of bacterial killing, including complement. Although this was partly addressed in Supplementary Figure 6, I found the predictions of these experiments unclear. First, I think these experiments are too central to be relegated to the supplemental materials; they belong in the main text. Secondly, it is important to explicitly spell out the expectations of using heat-killed serum (which will degrade any heat-labile components) or complement-deficient serum. It should be clearer under which conditions MCR-1-containing strains are predicted to do better or worse than controls.

Galleria is a useful infection model for virulence, but it is unclear what drives differences between strains. First, bacterial numbers aren't measured in this assay, so it isn't known if increased virulence is due to increased bacterial growth or decreased bacterial clearance. As above, I think these assays would be stronger using the competition-based approach in Figure 1. This would indicate bacterial numbers through time and directly show the selective benefit associated with MCR-1. Second, it would be useful to elaborate on why MCR-1 increases virulence, especially any known similarities between Galleria AMPS and those tested in Figures 1 and 2. Overall, it would help if Galleria were less of a black box.

Reviewer #2 (Public Review):

The study by Yanase et al. investigated the details of the 3D architecture of Leishmania haptomonad promastigote's adhesion to the midgut of the insect vector. The authors generated a dataset of images that reveal intricate details of the formed adhesion plaque and expanded the study with in vitro alternatives for the exploration of how Leishmania promastigotes strong adhesion by hemidesmosomes to surfaces can happen and be maintained. They show with unprecedented detail the ultrastructure of the attachment plaque. The in vitro dataset of the paper adds to the specific literature important details on how to explore micro/nanostructures involved in an important attachment step for this eukaryotic parasite. However, the in vitro data should be reconsidered in its discussion and conclusions as it does not support direct comparison with in vivo Leishmania forms as pictured by the authors. In general, the dataset presented in this manuscript adds valuable data and resources for the study of Leishmania promastigotes to surfaces, especially to the thoracic midgut parts of its insect vector.

The dataset of this paper is well-collected and robust, but some aspects of image analysis need to be clarified and extended. Also, the in vitro data from the manuscript will benefit from an extensive adjustment in its discussion. Points to focus on:

1) The haptomonad promastigote is indeed a possible critical form for transmission, but it lacks formal demonstration still in all literature available. This should not be claimed without proper formal demonstration.

2) Literature available and cited in this manuscript regarding in vitro adhesion of culture Leishmania promastigotes does not provide direct evidence for haptomonad differentiation. Haptomonads are still a largely unknown promastigote form with no defined ontogeny. With that, to propose an in vitro haptomonad differentiation protocol, more detailed direct evidence of in vivo haptomonads will be necessary. The in vitro experiments available show how cultured promastigotes attach to surfaces. Detailed studies in vivo will be needed still to attribute the findings in vitro to haptomonads.

3) This manuscript will benefit by having a detailed description of how to analyze and get to the 3D models presented. This has a strong potential for usage beyond the Leishmania/sand fly field. Statistics should be made available with ease across the manuscript and with a dedicated section on methods.

Reviewer #2 (Public Review):

The manuscript by Sampaio and colleagues utilizes an elegant and delicate approach to manipulate fluid dynamics in zebrafish Kupffer's vesicle (KV) to answer a long-standing question in the field - is it fluid movement or something in the fluid that governs the break in symmetry?

The researchers extract fluid from KV at different times during somitogenesis and find this procedure results in left-right organ defects when fluid is removed from the 3 to 5 somite stage, peaking at 5 somites. The effect on left-right patterning by this manipulation is not significant from the 6 somite stage onward. This technique is non-trivial and the researchers have used it with great effect.

Fluid extraction in this sensitive time window (3-5 somites) did not affect cilia number, length, or distribution within KV suggesting the effect on left-right patterning is due to disruption of the fluid. There is a clear effect of the manipulation on dand5 RNA asymmetry as expected. Manipulated embryos that developed left-right defects also showed a decrease in angular velocity of particle movement in the anterior LRO. Increasing the viscosity of the fluid in KV with methylcellulose also results in left-right patterning defects. Taken together, these results are in strong support of fluid movement and detection being important in breaking symmetry in a ciliated left-right organizer. They also argue against the idea that there are signals in the fluid that are being moved asymmetrically to signal to the "left" to break the symmetry. Importantly, they help set a time window when fluid flow is critical for this process.

Reviewer #2 (Public Review):

The authors conducted a wide-ranging series of experiments which lead to the conclusion that NBR1 is involved in the clearance of photodamaged chloroplasts. It is a novel finding because the role of NBR1 in this process was never documented. Notably, the NBR1-mediated clearance is only one of the several possible mechanisms responsible for chloroplast turnover. It is not surprising, considering that the nbr1 mutants are viable. The work is arranged very well. The rationale of the subsequent experiments is logically justified and the outcomes and followed by clear conclusions. In consequence, the authors managed not only to observe the association of NBR1 with the chloroplasts but they threw some light on the corresponding mechanisms. The manuscript contains numerous high-quality images from a confocal microscope and from a transmission electron microscope. All images are accompanied by statistical analysis of the respective microscopic observations, which greatly improves the credibility of the conclusions. Shortly, the authors demonstrated that NBR1 decorates not only the exterior but also the interior of damaged chloroplasts in an ATG7-independent way. Next, they establish that NBR1 and ATG8 are recruited to different populations of damaged chloroplasts, and they document differences in chloroplasts turnover, differences in chlorophyll abundance and chlorophyll photochemical properties, as well as differences in the total proteome of the nbr1 mutant in comparison to the wild type and atg7 mutant in two light regimes (low light and high light). Finally, they exclude the requirement for the known E3 ligases PUB4 and SP1 for NBR1-mediated degradation and show that the NBR1 internalization relies rather on the chloroplastic membrane rupture than on the TIC-TOC-dependent processes. In summary, the authors postulate that NBR1-mediated chloroplast clearance is a novel, not yet described mechanism and summarize it in a clear diagram.

The work is interesting, the figures are convincing and the conclusions are justified by the results. It provides novel data on the function of selective autophagy receptors NBR1 in plant cells, however, it also leaves the reader with some unanswered questions. The most important is the relative contribution of each of the chloroplast's degradation routes to the turnover of these organelles in different stresses, light regimes, plant growth stages, etc. This is a difficult problem because the mutations in relevant genes have pleiotropic effects and it is difficult to separate the functions of the individual turnover routes. For example, the defects in core autophagy genes (like the atg7 mutant used in this study) result in an increased level of NBR1. These issues are not sufficiently addressed in the discussion.

Reviewer #2 (Public Review):

In this work Xia et al have generated CRISPR resources for genome-wide gain-of-function genetic perturbation in the Drosophila genome and have used them to identify novel genes that cause Rapamycin resistance in Drosophila cells. To do so, they have used the SAM system, already established to work well in flies (Jia et al., PNAS 2018). 3 of these candidate genes they discovered in the screen, were further characterized to study how they affect the mTOR pathway leading to Rapamycin resistance. Since genome-wide libraries for GOF studies do not exist for Drosophila, these resources will be very useful for a wider Drosophila community.

Strengths

1. GOF CRISPR library does not exist currently to be used in Drosophila and hence this is going to be useful for the wider Drosophila community<br /> 2. Authors have used already established and currently the most effective SAM system for gene activation for a genome-wide genetic screen.<br /> 3. From this screen they have found candidate genes overexpression which leads to Rapamycin resistance. They have validated 3 of these genes by multiple methods and have also tried to elucidate the mechanism by which these genes might regulate mTOR signaling and confer resistance to Rapamycin. The authors have shown the strength and usefulness of the resource that they have generated and this resource will be complementary to loss-of-function screens of similar nature.

Weaknesses

1. Authors have taken a number of measures to maintain the integrity of the CRISPRa library, including multiple gRNA targets per gene, 1000 cells per gRNA, and deep sequencing. However, do the authors have an idea of what percentage of the gRNA vectors are functional? Looking at the data they show for the 3 candidate genes, at least half of them are not functional, which could be either because of gRNA location or efficiency. Considering this to be an average situation, there might be a large number of genes for which all gRNAs might not function at all. I understand this might be a caveat for all such studies, but an estimate of some kind in discussion might be useful for anyone who might want to use these resources.<br /> 2. As the authors mention that ~32% of genes in Drosophila have transcription start side <1kb apart, off-targeting (neighbouring genes getting activated in addition to the intended gene) will be an issue. To address this, the authors describe one example of genes where although the genes TSS are within one kb of each other, the sgRNA specifically activated only one gene and not the other. However, since following this, authors have generated genome-wide resources keeping 500bp upstream as their benchmark, a large percentage of these 32% genes might have off-targets. It would be useful to know the estimates of off-targeting for such a resource. In addition, have authors looked at the transcripts of genes close to the specific genes they have studied? CG9932 is in close proximity to (although not within the 1 kb range) a few genes including mTOR.

Reviewer #2 (Public Review):

The authors conduct a structure-function analysis of an uncharacterized gene, DltE, which was found by a genetic screen to be involved in the growth promotion of Drosophila larvae by Lactiplantibacillus plantarum, a bacterium that is consistently associated with Drosophila. They find that DltE is a D-Ala carboxylesterase that removes D-Ala from lipoteichoic acids in the cell envelope and that D-alanylated lipoteichoic acids stimulate Drosophila larval growth. The result that D-Ala LTA stimulates larval growth is compelling, although some minor experimental details to do with biological replicates are not shown and the tracking of bacterial abundances should be addressed to make the conclusions more solid. Additionally, I think the use of the terms "direct" and "symbiotic" is inappropriate in the manuscript, but this can be resolved by removing them or performing additional experiments.

The authors make these claims:<br /> - DltE is not a carboxypeptidase modifying Lp peptidoglycan;<br /> - DltE is a D-Ala esterase acting upon D-Ala-LTA;<br /> - only LTAs but not WTAs are D-alanylated in LpNC8 cell envelopes;<br /> - D-Ala-LTAs, in addition to PG, are direct symbiotic cues supporting<br /> (1) intestinal peptidase expression and<br /> (2) juvenile growth in Drosophila.<br /> I find all of the claims to be well supported by data except the suggestion that these are "direct symbiotic" cues. I think the authors provide the support that D-Ala LTAs are nutritional cues, not symbiotic ones.

Overall, I find the work compelling.

Reviewer #2 (Public Review):<br /> <br /> MAPKs are key fundamental enzymes and out of the 14 MAPKs, ERK3 and ERK4 remain less studied. The authors have made some interesting discoveries on ERK3, especially in the context of chemotaxis and tumourigenesis previously (Bogucka et al eLife 2020). Here they investigated the role of ERK3 in the control of cell architecture. Loss of ERK3 led to a reduction in the formation of actin-rich protrusions which led the authors logically to look for the activation of RhoGTPases. Intriguingly, they found that ERK3 functioned as a GEF for Cdc42 but not for Rac1. Further, they identified that Rac-WAVE and Arp2/3 were present at endogenous levels in a heteromeric complex in cells. As ERK3-deficient breast epithelial cells exhibit less F-actin content, this has led the authors to check for Arp2/3-dependent events here. By employing a variety of knockdown and complementation approaches, the authors convincingly demonstrate that the kinase activity of ERK3 is not required for the total F-actin content but for the formation of actin-rich protrusions. Finally, loss of ERK3 reduced random cell motility in vitro and in vivo, which was accomplished by intravital imaging of breast cancer cells in mice. Many protein kinases have catalysis-dependent and -independent functions (catalytic activity versus allosteric activity) and here is another example that deserves further investigation and opens new lines of investigation.

Reviewer #2 (Public Review):

De Filippo et al. investigated the spatiotemporal dynamics of the ripples propagation in the hippocampus of head-fixed mice. By leveraging the LFP and the isolated units of an open dataset of 49 animals with ~6 Neuropixels probes in the longitudinal axis of the hippocampus, they found: first, that stronger ripples (>ninth decile of power) originated in the most septal pole of the hippocampus (medially, anatomically) tend to travel more (M to L) than more lateral ripples (closer to the temporal pole). Second, while strong ripples were mainly local, the authors found that they are most likely to be generated in the temporal pole of the hippocampus, from where they can travel with relatively small attenuation. Finally, they found that strong/septal ripples elicit high spiking activity along the entire mediolateral axis of the hippocampus. Longer/stronger ripples have been proposed to be important in situations with high memory load, and these analyses increase our understanding of their physiology and mechanisms of generation.

The conclusions of this paper are mostly well supported by data, but some aspects of interpretation and data analysis need to be clarified and extended.

1) High amplitude ripples preferentially occur in distal CA1, and ripples can propagate at a higher degree on the proximo-distal than in the septo-temporal axis of the hippocampus (Kumar and Deshmuckh, 2020). Therefore, a proximo-distal bias in the Neuropixel positioning could explain part of the variance the authors report. Authors should consider (or control for) the proximodistal positioning of the electrodes.

2) In my opinion, the dynamics of the ripple-induced spiking activity for the events generated in the medial or lateral section of the hippocampus are very striking, more even considering that only a minority of the detected ripples are strong/long events (less than 5% in a familiar environment, Fernandez-Ruiz et al, 2019), while, according to the authors, majority of the ripples (grouped as 'common' by the authors) travel on the opposite direction (from the lateral section towards the septal pole, figure 2). Moreover, in the 50-120ms window, the most lateral positions (>3500um) seem to be more influenced by the medial ripples than relatively more central electrodes (~3000um). How can the authors explain this? To understand a little bit more how ripple features relate to the spiking dynamics, authors could try to generate heatmaps of the differential spiking between medial and lateral ripples (as they did in Fig. 4D-E) for 'strong' and 'common' ripples, or for local and propagating ripples.

Reviewer #2 (Public Review):

Lloyd et al examine the relationship between pupil size and fMRI signals in six brain nuclei responsible for providing the four major neuromodulators in the brain: norepinephrine from the locus coeruleus (LC), dopamine from ventral tegmental area (VTA) and substantia nigra, serotonin from the dorsal and median raphe nuclei, and acetylcholine from the cholinergic basal forebrain. Importantly, the authors focus on the relationship between these nuclei in the ascending arousal system (AAS) and the pupil at rest, outside of the context of any task, to determine the extent that small changes in pupil size are predictive of AAS activity.

Very few previous studies have examined this relationship at rest, perhaps in part because of the increased sensitivity required in the absence of event-based averaging. These nuclei are small (especially the LC), and thus are difficult to measure with standard fMRI.

The authors use a number of data collection and processing techniques to increase the sensitivity and precision of their recordings targeted to small ROIs. They find robust correlations between multiple AAS nuclei and pupil size with a time course that is not well captured by a standard hemodynamic response function (HRF).

The latter methodological finding is likely to be useful to the field for future studies focused on extracting useful signals from these nuclei, and the observed relationship between multiple AAS nuclei and the pupil support an emerging consensus from animal research that pupil fluctuations are correlated with neuromodulators besides norepinephrine.

Reviewer #2 (Public Review):

Mansur et al highlight interesting aspects of KLHL40-mediated proteostatic mechanisms in secretion and skeletal muscle development in zebrafish. They propose that KLHL40-mediated ubiquitylation of functional modules in the muscle proteome, particularly membrane traffic components, regulates protein abundance to control development. The authors present solid evidence for the role of KLHL40-mediated ubiquitylation and degradation of the cellular proteome but would benefit from further supporting evidence for their direct consequences on protein secretion.

Reviewer #2 (Public Review):

The availability of large collections of Mycobacterium tuberculosis (Mtb) isolates has enabled many important studies looking to identify mycobacterial genetic polymorphisms associated with anti-tuberculosis (TB) drug resistance, including both classical "resistance-conferring" mutations and novel "resistance-enabling" mutations. Importantly, these studies have expanded our understanding of mycobacterial genetic adaptations undermining chemotherapy, in many cases allowing for improved diagnostic tests and predictions of treatment failure. In this submission, Gao and colleagues adopt a different approach to the problem: although also applying a GWAS-type analysis, they instead attempt to elucidate polymorphisms implicated in poor outcomes of TB patients undergoing treatment for the drug-susceptible disease. Starting with a large dataset comprising 3496 samples with corresponding clinical (host) metadata, the authors generate Mtb whole-genome sequence data for 91 samples obtained from patients with "poor" outcomes and 3105 patients with "good" outcomes. These are used to identify 14 fixed and >230 unfixed mutations that might be associated with "poor" treatment outcomes, a conclusion which they argue is plausible given transcriptional evidence implicating many of the identified genes in the mycobacterial response in vitro to first-line drug exposure and/or hypoxia, both of which are considered relevant to clinical disease. Notably, they also identify a tendency for a greater proportion of "ROS mutational signatures" in unfixed mutations from "poor" outcome samples. Finally, incorporating these observations in a prediction model, the authors observe that the mycobacterial factors aren't adequate on their own but, when combined with key host factors - including patient age, sex, and duration of diagnostic delay (which have stronger predictive value) - they enhance predictive capacity. In summary, this paper reports a novel approach yielding observations that offer tantalizing insight into the mycobacterial factors which might influence TB treatment outcomes independent of drug resistance, however, the following must be considered:

(i) The manuscript provides little to no detail about how the samples were obtained, other than the fact that they comprise "pre-treatment" samples: are they all sputum samples? Were they induced? Similarly, no information is provided about sample propagation: were the samples cultured to achieve sufficient biomass for whole-genome sequencing? If so, in what growth media, for how long, and how many passages? Were all samples treated identically? And were they plated to single colonies - or are the "isolates" referred to throughout the manuscript actually heterogenous populations of potentially different Mtb clones obtained - and propagated - as a mixed sample? This information is critical given the potential that the identified polymorphisms - both fixed and (perhaps even more so) unfixed - might have arisen as a consequence of in vitro (laboratory) manipulation under standard aerobic conditions.

(ii) A key question that arises from this study (and others like it) is whether causation has been adequately established. Ideally, the Mtb genotypes contained within samples obtained pre-treatment should be compared with samples obtained from the same patients following treatment - that is, when the "poor" outcome was manifest. The expectation is that the polymorphisms identified prior to initiation of therapy - especially the 14 fixed mutations - should be evident (even dominant) at the later stage when therapy failed (or at the subsequent presentation in cases of relapse). Recognizing that this is not easily accomplished, though, it seems fair to suggest that the perceived relevance of the identified mutations would be strengthened if the authors were able to provide any other evidence - perhaps from studies of drug-resistant Mtb isolates - supporting their inferred role in undermining frontline treatment.

(iii) Related to the above, the authors make the valid point that their intention here was different from other studies which have deliberately utilized drug-resistant Mtb isolates to identify resistance-conferring and resistance-enabling mutations (such as in the study they cite by Hicks et al). It would be interesting to know, however, if any of the mutations identified in those other studies were also picked up in this work - and, if not, why that might be the case.

(iv) Finally, the analyses presented in this study are heavily dependent on the use of appropriate statistical methods to identify potentially rare genetic polymorphisms. However, as noted for sample processing (see my earlier comment above), there is very little detail provided about the methodology applied. This omission detracts from the interpretation, especially given that the predominance of lineage 2 (which contributes >75% of the isolates, with sublineage 2.3 constituting >50%) risks a lineage-specific association, rather than a more generalizable pathogenicity phenotype. Similarly, the heavy skew in the numbers of "good" (3105 samples) versus "poor" (91 samples) collections (approximately 34x difference in sample size) raises the possibility that mutations identified in the "poor" category might be artificially over-represented. More clarity in detailing the statistical methods is required to allay any concerns about the identification of candidate polymorphisms.

Reviewer #2 (Public Review):

Acute lung injury (ALI) and ARDS are major causes of morbidity and mortality in critically ill patients and patients infected with Sars-Cov-2. There are no effective therapies for ALI/ARDS, and the 28-day mortality rate is ~40%. One of the main pathological features of ALI/ARDS is a vascular injury characterized by endothelial dysfunction, inflammation, and in situ thrombosis. Using a murine model of ALI/ARDS triggered by diphtheria toxin (DT) mediated endothelial specific ablation, the authors apply sc-RNA-seq analysis to study how lung cell populations respond to injury and identify two main endothelial subpopulations responsible for regenerating lung vasculature over seven days. The study's implications are exciting as they provide evidence of intrinsic repair mechanisms that could be targeted for vascular regeneration and recovery of lung function in the context of ALI/ARDS. In particular, the apelin pathway rises as a prime therapeutic candidate given its role in coordinating the behavior of general and aerocyte capillary cells in lung vascular repair.

While the results of this study are exciting and novel, it must be recognized that several limitations need to be properly addressed to facilitate the translation of the findings toward medical care. For instance, the animal model used in this study (DT mediated EC ablation) does not fully recapitulate all the pathological hallmarks of ALI/ARDS, the most important of which is that repair proceeds at a very slow pace as a result of multiple factors that are not recapitulated in this made. Since the authors use only one model of ALI/ARDS, it is not entirely clear whether the current findings can be generalized to other models. Since no one model truly recapitulates the complexity of human ALI/ARDS, it is important to use at least two or more models that can narrow genetic and molecular mechanisms fundamental to lung injury and recovery. Another important aspect is the lack of validation in human samples and cells, which could strengthen the conclusions raised by the authors in the discussion. Finally, the authors appropriately emphasize how this study could help efforts to understand Sars-Cov2 mediated ALI/ARDS. Still, no studies explore any overlap with currently available Omics data from COVID lungs.

Despite these weaknesses, this study is the first to apply rigorous scRNA-seq analysis to this unique model of ALI/ARDS. It also provides data to support the importance of the two newly discovered endothelial cell subpopulations (gCap and aCap) in lung repair and regeneration, which hold the potential to offer unique mechanistic insights into the genetic and molecular mechanisms responsible for vascular repair and offers the opportunity to consider apelin based therapeutic approaches to treat ALI/ARDS. In conclusion, this study is expected to contribute to our lung biology understanding greatly. It provides the research community with novel resources and tools that greatly aid efforts to understand ALI/ARDS and identify therapeutics to treat this devastating disease.

Reviewer #2 (Public Review):

The authors utilized a label-free LC-MS/MS analysis in formalin-fixed paraffin-embedded (FFPE) tumors from 143 LNM-negative and 78 LNM-positive patients with T1 CRC to identify protein biomarkers to determine LNM in T1 CRC.

The authors used a fair number of clinical samples for the proteomics investigation. The experimental design is reasonable, and the statistical methods used in this manuscript are solid.

The authors largely achieved their aims and the results supported their conclusion. The method used in this proteomic study can also be used for the proteomics analysis of other cancer types to identify diagnostic and prognostic biomarkers. In addition, the 9 marker panel has a potential clinical diagnosis practice in determining LNM in T1 CRC.

Nevertheless, the authors need to justify their standards in selecting the biomarkers. For example, a p-value cut-off of 0.1 is not a usual criterion in similar proteomic studies. In addition, an identification frequency of 30% in patients seems not preferable for biomarker identification. The authors also need to justify the definition of fold change in the three subtypes with Kruskal-Walli's test. The authors need to describe more details on how they identified the 13 proteins from a 55-protein database. In addition, what is the connection between the final 9 proteins and the 19 proteins? What is the criterion to select 5 proteins for IHC validation from the 9 proteins?

Reviewer #2 (Public Review):

The paper by Laurenz Lammer and colleagues used cohort data to investigate the cross-sectional and longitudinal association between loneliness and brain structure and cognitive function. The main finding was that baseline social isolation and change in social isolation were associated with smaller hippocampus volumes, reduced cortical thickness, and poorer cognitive function. Given that more and more people feel lonely nowadays (e.g., due to the pandemic), the study by Lammer and colleagues addresses a highly relevant health concern of our time.

Significant strengths of the study:

- large cohort;<br /> - the cross-sectional and longitudinal analyses confirmed the findings;<br /> - the study was preregistered;<br /> - the study included men and women;<br /> - analyses were sound and controlled for essential confounders.

The major weaknesses of the study:

- it is unclear whether loneliness causally contributes to brain structure and cognitive function;<br /> - the factors that may cause loneliness are unclear.

Reviewer #2 (Public Review):

Overall, this manuscript provides a thorough characterization of the role of microtubules in the movement of GLUT4 in muscle fibers, and demonstrates the need for an intact microtubule network for GLUT4 responsiveness but only after the initial round of response.<br /> The study poses a very interesting question, rooted in studies in the literature studying the effects of Nocodazole (Noco) and C2-ceramide on GLUT4 traffic in cell systems. It is important to validate or refute predictions from those studies and, largely through this group's work, the quest to examine these questions in isolated muscle fibers and intact muscles as feasible is commendable. The authors develop very interesting imaging approaches to this end, and quantify the results in a convincing and elegant fashion. The system to measure 2-DG uptake and glucose uptake by electrochemical sensing in isolated fibers using the microfluidic pump is very ingenious.<br /> The main conclusion that microtubules are important for GLUT4 proper localization is important and adds mechanistic insight beyond that obtained from work in myoblasts and pre/adipocytes. The observation that microtubules are not engaged in GLUT4 traffic in the first round of insulin action but it is thereafter is also very revealing and should lead to more insights into the first and subsequent rounds of GLUT4 translocation.

Reviewer #2 (Public Review):

Schild et al. investigate the regulation of temporal control during neuroblast migration in the roundworm C. elegans. The authors find that expression of the Wnt pathway receptor Mig1 is regulated early through a specific noncoding conserved intronic element and later through two specific upstream conserved DNA elements. The expression levels of Mig1 in QR.pa cells are further regulated through Ced-3 and pig-1. The variability in the timing of later expression of Mig1 in QR.pa cells through bar-1 or a terminally truncated version of Bar1 was modulated but the mean expression did not change.

The single molecule RNA-FISH data is strong, and this method is sensitive enough to detect differences between different single cells and mutants. The mutants are very precise and straightforward to interpret. An additional strength is that many cells and replicas have been measured. The data analysis is simple.

The proposed model is simple with few intuitive parameters. This makes parameter identification straightforward. The qualitative predictions do make sense and are consistent with most experimental observations.

Overall the manuscript addresses the important question of timing regulation in transcription.

Reviewer #2 (Public Review):

The normally T cell restricted Src family tyrosine kinase Lck is ectopically expressed in most B cell Chronic Lymphocytic Leukemias. This, along with the fact that ectopic expression of other SFKs, such are Fyn and Fgr, are not seen, suggests that Lck may have some unique function, distinct from the endogenous Lyn SFK, that promotes malignant transformation. Using inducible expression in a human B cell lymphoma, the study explores this possibility. Studies reveal no qualitative functional differences in Lck and Lyn that are likely to explain its unique ectopic expression of Lck in CLL.

The strengths of this study include the use of Lentiviral transfer of genes encoding SFKs in conjunction with Doxycycline inducible expression. This allows comparative analysis of acute Lyn and Lck overexpression effects, free of cell resetting artifacts consequent to long term expression of the SFK. Strength is also seen in the authors fluorescent tagging of the SFK so analysis could be gated on ectopic expression level. Strength exists in the authors dissection of SFK effects on early events in the BCR signaling pathway, which reveal the ability of both overexpressed SFKs to drive receptor ITAM tyrosine phosphorylation and initiating BCR signaling. These studies reveal little difference in the function of the SFKs, though it appears that Lck may be less sensitive to phosphatase regulation.

It is unclear from the material and methods whether the overexpressed Lyn is LynA or Lyn B. It appears in the text (lines 130-133) that they overexpress LynB specifically. A recent paper from Tania Freedman (Sci Adv 2022 PMID:35452291) suggests that LynA is more activating whereas LynB is more balanced with an inhibitory bias. The point is that it is important to discuss this because they may not be making a relevant comparison.

If Lck promotes pathophysiology by transduction of a qualitatively unique signal, one would expect that transcriptome analysis should reveal this difference. The authors look for this signal using transcriptome analysis of bulk populations expressing similar levels of SFK. Although differences were seen in the transcriptome, finding were not consistent with a qualitatively unique function. However, bulk transcriptomic analysis may miss important differences. Single cell RNAseq, e.g., by 10x, may have been more incisive because gene expression could have been normalized to SFK expression in individual cells.

Finally, while some interesting differences are seen in the biology of Lyn and Lck, weakness exists in the failure to explore the causality of these differences in driving CLL phenotype. A final thought relevant to this comment. It is a truism that "absence of proof is not proof of absence".

Reviewer #2 (Public Review):

In this work, the authors aimed to understand the ion selectivity mechanism of a plant NRAMP-related aluminum transporter by structural and biochemical characterizations.

The authors successfully identified SiNRAT as a promising candidate for structural and biochemical analyses, showed that SiNRAT transport various divalent cations as well as binding to trivalent cations, determined the cryo-EM structure of SiNRAT, and performed structure-based mutational analysis to identify a potential binding site for metal ions. Unfortunately, the authors failed to show direct evidence of Al3- transport, due to technical problems. Furthermore, the structure of SiNRAT in complex with Al3+ was also not shown.

Despite such weakness, the structural comparison with other NRAMP members with different ion selectivity properties together with the extensive biochemical analyses would support the statement by the authors on a mechanism of ion selectivity for Al3+.

In the discussion section, the authors posed an important question. Considering the weak ion selectivity of SiNRAT over divalent cations, it is still unclear how NRAT proteins can function as an Al3+ transporter in a physiological condition where other divalent cations are also abundant. This would be an important question to be addressed in the related research field in the future.

The methods section is well written and the atomic coordinates and EM map file will be available to the community.

Reviewer #2 (Public Review):

The authors use data from 3 cross-sectional age-stratified serosurveys on Enterovirus D68 from England between 2006 and 2017 to examine the transmission dynamics of this pathogen in this setting. A key public health challenge on EV-D68 has been its implication in outbreaks of acute flaccid myelitis over the past decade, and past circulation patterns and population immunity to this pathogen are not yet well-understood. Towards this end, the authors develop and compare a suite of catalytic models as fitted to this dataset and incorporate different assumptions on how the force of infection varies over time and age. They find high overall EV-D68 seroprevalence as measured by neutralizing antibodies, and detect increased transmission during this time period as measured by the annual probability of infection and basic reproduction number. Interestingly, their data indicate very high seroprevalence in the youngest children (1 year-olds), and to accommodate this observation, the authors separate the force of infection in this age class from the other groups. They then reconstruct the historical patterns of EV-D68 circulation using their models and conclude that, while the serologic data suggest that transmissibility has increased between serosurvey rounds, additional factors not accounted for here (e.g., changes in pathogenicity) are likely necessary to explain the recent emergence of AFM outbreaks, particularly given the broader age-profile of reported AFM cases. The Discussion mentions important current unknowns on the biological interpretation of EV-D68 neutralizing antibody titers for protection against infection and disease. The analysis is rigorous and the conclusions are well-supported, but a few aspects of the work need to be clarified and extended, detailed below:

1) Due to the lack of a clear single cut-point for seropositivity on this assay, the authors sensibly present results for two cut-points in the main text (1:16 and 1:64). While some differences that stem from using different cut-points are fully expected (i.e., seroprevalence being higher using the less stringent cut-point), differences that are less expected should be further discussed. For instance, it was not clear in Figure 2 why the annual probability of infection decreased after 2010 using the 1:64 cut-point, while it continued to increase using the 1:16 cut-point. It would also be helpful to explain why overall seroprevalence and R0 continue to increase over this time period using the 1:64 cut-point. Lastly, it would be useful to see the x-axis in Figure 4 extended to the start of the time period that FOI is estimated, with accompanying credible intervals.

2) Additional context of EV-D68 in the study setting of England would be useful. While the Introduction does mention AFM cases "in the UK and elsewhere in Europe" (line 53), a summary of reported data on EV-D68/AFM in England prior to this study would provide important context. The Methods refers to "whether transmission had increased over time (before the first reported big outbreak of EV-D68 in the US in 2014)" (lines 133-134), rather than in this setting. It would be useful to summarize the viral genomic data from the region for additional context - particularly since the emergence of a viral clade is highlighted as a co-occurrence with the increased transmissibility detected in this analysis.

Reviewer #2 (Public Review):

This manuscript explores a novel technique to use dyes co-injected with various pharmaceutical reagents, like chemotherapic agents, to assess cellular effects in a cell culture model.

The major premise is that dye diffusion can be detected through fluorescent microscopy and be used as a measure of co-injected drug concentration. In chemotherapy commonly multiple drugs are given simultaneously, however, understanding how to tailor the concentrations of a multi-drug cocktail to each individual is largely trial and error. The authors surmise that perhaps using a cell culture model whereby cancer cells are cultured and then exposed to dye-tracked molecules an optimal multi-drug combination and concentrations can be determined. In other words, the intermixing of various connected drugs can then be fluorescently monitored to elucidate optimal concentrations of multi-drug combinations.

The concept overall is interesting but is relatively preliminary in its proof of concept. The authors note that varying free-diffusion of drugs out of the cell could complicate interpretation and that most of the analysis was done on a relatively short time basis and not longer evaluation periods that were more typical of chemotherapy.

Reviewer #2 (Public Review):

The authors unexpectedly found that the protein Grb2, an adaptor protein that mediates the recruitment of the Ras guanine-nucleotide exchange factor, SOS, to the EGF receptor, can be recruited to membranes by the immune cell tyrosine kinase Btk. The authors show, using total internal reflection fluorescence (TIRF) microscopy that the interaction with Grb2 is reversible, dependent on the proline-rich region of Btk, and independent of PIP3. These experiments are well performed and unambiguous.

The authors next asked whether Grb2 binding to Btk influences its kinase activity, by evaluating (i) Btk autophosphorylation and (ii) the phosphorylation of a peptide from the endogenous substrate PLC1. The readout relies on non-specific antibody-mediated detection of phosphotyrosine but nevertheless reveals a concentration-dependent increase in both Btk autophosphorylation and PLCy1 phosphorylation. The experiments, however, have only been performed in duplicate and, particularly in the case of PLCy1 phosphorylation, exhibit enormous variability which is not reflected in the example blot the authors have chosen to display in Figure 3C. Comparison of the same, duplicate experiment presented in Figure 3 Supplement 2 paints a very different picture.

The authors next sought to determine which domains of Grb2 are required for activation of Btk. Again, these experiments were only performed in duplicates, and the authors' claims that Grb2 can moderately stimulate the SH3-SH2-kinase module of Grb2 are not well supported by their data (Figure 4C-D).

The authors next asked whether Grb2 stimulates Btk by promoting its dimerization and trans-autophosphorylation. The authors measured the diffusion coefficient of Btk on PIP3-containing supported lipid bilayers in the presence and absence of Grb2. They noted that the diffusion coefficient of individual Btk particles decreases with increasing unlabeled Btk, which they interpret as Btk dimerization. Grb2 does not appear to influence the diffusion of Btk on the membrane (Figure 5A). Presumably, the diffusion coefficient reported here is the average of a number of single-molecule tracks, which should result in error bars. It is unclear why these have not been reported. Next, the authors assessed the ability of Grb2 to stimulate a mutant of Btk that is impaired in its ability to dimerize on PIP3-containing membranes. In contrast to wild-type Btk, autophosphorylation of dimerization-deficient Btk is not enhanced by Grb2. Whilst the data are consistent with this conclusion, again, the experiment has only been repeated once and the western blot presented in Figure 5 Supplement 2 is unreadable. It is also puzzling why Grb2 gets phosphorylated in this experiment, but not in the same experiment reported in Figure 3 Supplement 2.

Finally, the authors argue that Grb2 facilitates the recruitment of Btk to molecular condensates of adaptor and scaffold proteins immobilized on a supported lipid bilayer (SLB) (Figure 6). This is a highly complex series of experiments in which various components are added to supported lipid bilayers and the diffusion of labelled Btk is measured. When Btk is added to SLBs containing the LAT adaptor protein (phosphorylated in situ by Hck immobilized on the membrane via its His tag), it exhibits similar mobility to LAT alone, and its mobility is decreased by the addition of Grb2. The addition of the proline-rich region (PRR) of SOS further decreases this mobility. In this final condition, the authors incubate the reactions for 1 h until LAT undergoes a phase transition, forming gel-like, protein-rich domains on the membrane, shown in Figure 6B. The authors' conclusion that Btk is recruited into these phase-separated domains based on a slow-down in its diffusion is not well supported by the data, which rather indicates that Btk is excluded from these domains (Figure 6B - Btk punctae (green) are almost exclusively found in between the LAT condensates (red)). As such, the restricted mobility of Btk that the authors report may simply reflect the influence of barriers to diffusion on the membrane that result from LAT condensation into phase-separated domains. The authors also present data in Figure 6 Supplement 1 indicating that Grb2 recruitment to Btk is out-competed by SOS-PRR and that Btk does not support the co-recruitment of Grb2 and SOS-PRR to the membrane. These data would appear to suggest that the authors' interpretation of the decreased mobility of Btk on the membrane may not be correct.

Alternative explanations from Walfram Math world: here.

In brief, you can use an isometry map from a banach space to another subspace in a different Banach space such that it's dense.

Reviewer #2 (Public Review):

This is an exceptional study that provides conclusive evidence for the existence of a descending pathway from the brain that inhibits nociceptive behavioral outputs in larvae of Drosophila melanogaster. The authors identify molecular both molecular and neuronal/cellular components of this pathway. Converging lines of evidence and conclusive genetic experiments indicate that the neuropeptide, drosulfakinin (DSK), and its receptors (CCK1 and CCK2) function to inhibit nociception behaviors. Interestingly, the authors show that the relevant DSK neurons have cell bodies that are in the larval brain and that these neurons send projections into the thoracic ganglion and ventral nerve cord. Several lines of evidence support the hypothesis that fourth-order nociceptive neurons called Goro, are one relevant target for these outputs. RNAi knockdown of the CCK1 receptor in these cells sensitizes behavioral and physiological responses to noxious heat. Second, the axons of DSK neurons form physical contact with processes of Goro neurons as revealed by GRASP analysis. However, the authors' careful experiments indicate that the contacts between axons and Goro neurites might not be indicative of direct synapses and instead might operate through the bulk transmission of the peptidergic signals. The study raises many interesting questions for future study such as what behavioral contexts might depend on this pathway. Using the CAMPARI approach, the authors do not find that the DSK neurons are activated in response to nociceptive input but instead suggest that these cells may be tonically active in gating nociception. Future studies may find contexts in which the output of the DSK neurons is inhibited to facilitate nociception, or contexts in which the cells are more active to inhibit nociception.

Reviewer #2 (Public Review):

The voltage-gated potassium channel KCNQ1/KCNE1 (IKs) plays important physiological functions, for instance in the repolarization phase of the cardiac action potential. Loss-of-function of KCNQ1/KCNE1 is linked to disease. Hence, KCNQ1/KCNE1 is a highlighted pharmacological target and mechanistic insights into how channel modulators enhance the function of the channel is of great interest. The authors have through several previous studies provided mechanistic insights into how small-molecule activators like ML277 act on KCNQ1. However, less is known about the binding site and mechanism of action of other type of channel activators, which require KCNE1 for their effect. In this study, Chan and co-workers use molecular dynamics approaches, mutagenesis and electrophysiology to propose an overall similar binding site for the KCNQ1/KCNE1 activators mefenamic acid and DIDS, located at the extracellular interface of KCNQ1 and KCNE1. The authors propose an induced-fit model for the binding site, which critically engages residues in the N-terminus of KCNE1. Moreover, the authors discuss possible mechanisms of action of how drug binding to this site may enhance channel function.

The authors address an important question, of broad relevance to researchers in the field. The manuscript is generally well written and the text easy to follow. A strength of the work is the parallel use of experimental and simulation approaches, which enables both functional testing and mechanistic predictions and interpretations. For instance, the authors have experimentally assessed the putative relevance of a large set of residues based on simulation predictions. A limitation is that several methods need to be described in more detail to allow for evaluation of the presented data. Also, a more extensive presentation of representative data would be useful, along with discussions on the putative impact on drug effects of the diverse intrinsic properties of tested mutants.

Reviewer #2 (Public Review):

The authors provide comprehensive results showing that pharmacological inhibition of PI3K negatively affects heart tube formation via misoriented and slower cardiac movements. They used several cellular and molecular assays to demonstrate the potential mechanisms involved in PI3K-dependent cardiac fusion defects. Moreover, they use several imaging techniques and quantitative assessments to support their findings. Although the manuscript is well-written and most of their results support their conclusions, the manuscript and its findings heavily rely on high concentrations of PI3K small-molecule inhibitors, which will have off-target effects. The off-targets of PI3K pharmacological inhibition should be interpreted with caution and further evaluated. The authors suggest PI3K inhibition mediates heart tube formation throughout PI3K-mediated migration defects rather than PI3K-mediated proliferative defects. However, the authors did not further evaluate this later point; it should be considered carefully.

Reviewer #2 (Public Review):

In this study, Lamire et al. use a calcium imaging approach, behavioural tests, and pharmacological manipulations to identify the molecular mechanisms behind visual habituation. Overall, the manuscript is well-written but difficult to follow at times. They show a valuable new drug screen paradigm to assess the impact of pharmacological compounds on the behaviour of larval zebrafish, the results are convincing, but the description of the work is sometimes confusing and lacking details.

The volumetric calcium imaging of habituation to dark flashes is valuable, but the mix of responses to visual cues that are not relevant to the dark flash escape, such as the slow increase back to baseline luminosity, lowers the clarity of the results. The link between the calcium imaging results and free-swimming behaviour is not especially convincing, however, that is a common issue of head-restrained imaging with larval zebrafish.

The strong focus on GABA seems unwarranted based on the pharmacological results, as only Picrotoxinin gives clear results, but the other antagonists do not give a consistent results. On the other hand, the melatonin receptor agonists, and oestrogen receptor agonists give more consistent results, including more convincing dose effects.

The pharmacological manipulation of the habituation circuits mapped in the first part does not arrive at any satisfying conclusion, which is acknowledged by the authors. These results do reinforce the disconnect between the calcium imaging and the behavioural experiments and undercut somewhat the proposed circuit-level model.

Overall, the authors did identify interesting new molecular pathways that may be involved in habituation to dark flashes. Their screening approach, while not novel, will be a powerful way to interrogate other behavioural profiles. The authors identified circuit loci apparently involved in habituation to dark flashes, and the potentiation and no adaptation clusters have not been previously observed as far as I know.

The data will be useful to guide follow-up experiments by the community on the new pathway candidates that this screen has uncovered, including behaviours beyond dark flash habituation.

Reviewer #2 (Public Review):

The authors sequenced a clinical pathogen, Klebsiella FK688, and definitively establish the genetic basis of the carbapenem-resistance phenotype of this strain. They also show that the causal mutations confer reduced fitness under laboratory conditions, and that carbapenem sensitivity readily re-evolves in the lab due to the fitness costs associated with the resistance mutations in the clinical isolate. They also establish that subinhibitory concentrations of ceftazidime select for the otherwise deleterious blaDHA-1 gene. Based on this finding the authors speculate that prior beta-lactam selection faced by the ancestors of Klebsiella FK688 potentiated the evolution of the carbapenem-resistance phenotype of this strain. If this hypothesis is true, then prior history of beta-lactam exposure may generally potentiate the evolution of carbapenem resistance.

Strengths:

From a technical perspective, the findings in this paper are solid. In addition, the authors establish a simple genetic basis for carbapenem resistance in a clinical strain, which is a valuable and non-trivial finding (i.e. they show that the CRE phenotype in this strain is not an omnigenic trait distributed over hundreds of loci).

Weaknesses:

The main weakness of this paper is that the authors draw overly broad conclusions of a conceptual nature from narrow experimental findings. This could be addressed by drawing more modest and narrow implications from the findings.

1) The title of this paper is "Treatment history shapes the evolution of complex carbapenem-resistant phenotypes in Klebsiella spp." But they provide no data on the treatment history of the patient from whom this strain was isolated from. Therefore, the authors have no evidence to support their central claim. Indeed, it is completely possible that this strain never faced beta-lactam selection in the past, or that the patient's hypothetical history of betalactamase was irrelevant for the evolution of FK688. First, it is completely possible that this is a hospital-acquired infection, such that the history of this strain is due to selection in other contexts in the hospital that have little to do with the patient's treatment history. Second, it is completely possible that this strain (the chromosome anyway) has no prior history of beta-lactamase selection, and that it acquired the megaplasmid containing blaDHA-1 via conjugation from some other strain. In this second hypothetical scenario, it is possible that the fitness cost of the blaDHA-1 gene is not particularly high in a different source strain, but that it has some cost in the FK688 strain that it was isolated from. And of course, fitness costs in the human host could be very different than fitness costs in the laboratory, where strains are evolving under strong selection for fast growth. And given the benefit of resistance, it's clear that this strain clearly has a strong fitness advantage over faster-growing sensitive strains in the context of the source patient under antibiotic treatment.

My general point here is that the broad claims made about patient history or prior history shaping the evolution of this strain are largely indefensible because there is no data here to make solid inferences about *how* prior history shaped the evolution of this strain.

2) Historical contingency. The authors claim that their work shows how historical contingency shapes the evolution of resistance. One problem with this claim is that it is trivial- this is only a significant claim if the reader believes that prior history is not important in the evolution of antibiotic resistance, which is a straw-man null hypothesis, to mix a couple metaphors. To be more concrete, clearly strain background (prior history) matters-eliminating the plasmid with the resistance gene eliminates resistance. But that is not particularly surprising, given the past 50 years of evolutionary microbiology literature on plasmids and resistance. By contrast to this work, the major contribution of papers that examine the role of historical contingency in evolution (i.e. various Lenski papers) is that those works *quantitatively* measure the role of history in comparison to other factors (chance, adaptation). Since this work is a deep dive into a single clinical isolate, the data presented here do not and cannot shed light on the role of historical contingency in the emergence of this strain. The authors' claims about the prior history that led to the CRE phenotype are reasonable- but are fundamentally speculative. I have nothing against speculation, as long as it is clear what claims are speculative, and what are concrete implications. But the authors frame these speculative claims as concrete implications of their findings.

3) The authors claim that "[This work] suggests that the strategic combinations of antibiotics could direct the evolution of low-fitness, drug-resistant genotypes". I suppose this is true, but I also think this is a stretch of an implication given these findings. To be blunt, while I suppose it's better to have costly resistance variants that re-evolve sensitivity than to have low-cost high-resistance strains circulating, I think the patient's family would probably disagree that the evolution of a low-fitness drug-resistant genotype was good or strategic in the clinical context, even if better from a public health perspective. Low-fitness drug-resistant strains are just as lethal under clinical antibiotic concentrations!

The authors do show the plausibility of their hypothesis/model that prior beta-lactam selection is sufficient to potentiate the evolution of carbapenem-resistance (by the additional ompK loss-of-function mutation). I think those findings are very nice. But the authors undermine their results by extrapolating too far from their data. Hence, I think narrowing the scope of the implications would improve this paper.

In addition to narrowing the scope of the implications as written, I also would like to add that there may be other ways of framing this paper (other than historical contingency) that may make the significance of this work more apparent to a broader audience. This may be worth considering during the revision process.

Reviewer #2 (Public Review):

Toxoplasma gondii (Tg) and Plasmodium falciparum (Pf) are two protozoan parasites that both present threats to global human health as the causal agents of toxoplasmosis and malaria, respectively. In absence of effective vaccines, disease control relies heavily on the use of drugs aimed at treating infected patients to inhibit parasitic growth and eventually kill parasites to interrupt the parasitic lifecycle. These obligate intracellular vacuole-dwelling parasites quickly attach to their host cells before actively pinching through their plasma membranes and completing their complex respective lifecycles.

Kumar et al. seek to understand the complex process of host cell recognition, attachment, and invasion in order to devise possible strategies to possibly interfere and/or block to prevent invasion of the host cell or compromise egress from the infected cell. Characterizing the 3D structure at atomic resolution and dynamics of the glideosome molecular machinery involved in parasite attachment and invasion/egress provides grounds for the future rational design of novel anti-parasitic therapies targeting novel molecular targets and phylum-specific biological processes. Toxoplasma belongs to the same large family of obligate intracellular parasites such as the malaria parasite Plasmodium. These protozoa actively attach and glide at the surface of their target host cell before invading it. Such motility and propulsion at the surface of the host cell are powered by a large protein complex, the glideosome.

The article elegantly combines structural, biophysical, biochemical, computational, and cell biology approaches to dissect the structure and mechanism of action of TgGAC (and PfGAC).

The crystal structure of TgGAC was solved at an apparent 2.7A resolution by se-mad and although it is overall well described it requires further polishing in terms of model quality and accuracy. This is a very large protein, so it represents a considerable amount of work to build and refine. We note deficiencies in the way refinement (atomic displacement parameters and model building in general) and phasing statistics description were carried out or presented. This warrants further inspection and requires significant improvement and corrections to meet the usual standards expected from this field of research.

Solution scattering data while supporting the model of a conformational change between a compact (closed) conformation observed in the crystal obtained at pH 5 and an extended monomeric conformation observed at pH 8 more amenable to interactions with other cellular partners in the context of a functional glideosome needs some clarification. Because of the way proteins seem to be prepared for the SAXS analysis, I have some objections to the interpretation of some of the data.

The biochemical analysis of lipid binding specificity of the small c-terminal pleckstrin-like domain of TgGAC and PfGAC (full-length or c-terminal domain) using liposome binding assays, elegant NMR relaxation methods but also molecular dynamics on full-length GAC models are extremely convincing and support all authors claim.

The fact that however the CTD lipid binding activity is not required in vivo is a bit surprising although CTD seems required to stabilize the protein in vitro.

The section describing the hydrogen-deuterium exchange analysis of TgGAC conformation is confusing as it stands and requires clarification. It fails to be compelling in my personal opinion.

Reviewer #2 (Public Review):

The study aimed to provide information on the extent to which the COVID-19 pandemic impacted cervical cancer (CC) screening and treatment in 3 Canadian provinces. The survey methodology is appropriate, and the results provide detailed descriptive statistics by province and type of practice. The results support the authors' conclusions. This evidence together with data gathered from other national surveys may provide baseline data on the impact of the pandemic on CC outcomes such as late-stage diagnoses and CC treatment outcomes due to these delays.

Reviewer #2 (Public Review):

OTOP channels are relatively newly discovered and their physiology is poorly understood. Zn activation appears to be a differentiating feature of OTOP function and Zn is a pharmacological tool for research. The Zn potentiation of OTOP3 is a curious phenomenon that is studied very carefully here. The language in this manuscript is appropriately nuanced in the interpretation of results and is delightfully agnostic with regards to function vs binding. The major strengths of this work are the very thorough characterization of the zinc effect and the identification of the 11-12 loop as necessary and sufficient for the zinc effect.

Reviewer #2 (Public Review):

The authors investigated patterns of fMRI activation for familiar words in two groups of deaf people. One "language rich" group received exposure to sign from birth, whereas the "language poor" group included kids born to hearing parents who had limited exposure to language during the first few years of life. The primary findings involved group differences in BOLD activation patterns across different areas of interest within the semantic network when participants made intermittent 1-back category judgments for words appearing in succession.

There was much to be liked about this study, including the rigor of the methods and the novel contrasts of two deaf samples. These strengths were balanced by a number of questions about the assumptions and theoretical interpretations underlying the data. I will elaborate on the major points in the paragraphs to follow, but briefly, the ways in which the authors are framing critical period constraints in language fundamentally differ from the standard nativist perspectives (e.g., Chomsky, Lenneberg). The assumptions of what constitutes a deprivation model require further justification and perhaps recasting to avoid unnecessary stigma (i.e., this reviewer was uncomfortable with the assertion that being born deaf to hearing parents by default constitutes deprivation). The introduction lacked principled hypotheses that motivated the choice of comparing abstract and concrete words, and potential accounts of group differences were underdeveloped (e.g., how do parents in China typically react to having a deaf child, and what supports are in place for preventing language deprivation? Are newborn infants universally screened for hearing loss in China? The answers to these questions might help the readers to understand why/how deaf children in this circumstance might experience deprivation).

References to critical periods require a bit more elaboration with respect to lexical-semantic vs. semantic acquisition. The nature of the critical period in language acquisition remains controversial with respect to its constraints. Lenneberg and Chomsky speculated that the limit of the critical period for language acquisition was about puberty (13ish years of age). This is much older than the deaf sample tested here so arguments about aging out of the critical period at least for language acquisition need more nuance. Another issue relates to learning semantic mappings vs. learning language as falling under the same critical period umbrella. This seems highly unlikely as semantic acquisition in early childhood is aided by linguistic labeling but would likely occur in parallel even in the context of language deprivation. Much of the prior literature on critical periods and nativist approaches to language development has focused on syntactic acquisition and elements such as recursion rather than a mapping of symbols to conceptual referents. This makes the critical period group comparison somewhat tenuous because what you are really interested in is a critical period for word meaning acquisition not the more general case of syntactic competency.

The point above is highlighted in the following statement underlying one of the primary assumptions of the study:<br /> Pg. 3, "Here, we take advantage of a special early-life language-deprivation human model: individuals who were born profoundly deaf in hearing families and thus had very limited natural language exposure (speech or sign) during the critical period of language acquisition in early childhood"

"hypofunction of the language system as a result of missing the critical period of language acquisition" (pg 3), same critique as previous - the critical period window is thought to be 13ish years old.

There are a couple of problems with this assertion/assumption. Although it is true that most children who are born deaf have hearing parents, it is not justifiable to label this condition an early-life deprivation model. Hearing parents who are extremely motivated to learn sign language and pursue related language enrichment strategies can successfully offset many of these effects. Similarly, it is not inconceivable that a deaf child born to a deaf parent might be neglected or abandoned without the benefit of early sign exposure. My argument here is that classifying deaf children born to hearing parents as automatically 'language deprived' is potentially both stigmatizing and scientifically unjustified.

Pg. 6 "It should be noted that the neural semantic abstractness effect does not equate with language-derived semantic knowledge, as it might arise from some nonverbal cognitive processes that are more engaged in abstract word processing (Binder et al., 2016)." - I had great difficulty understanding what this meant.

Reviewer #2 (Public Review):

This paper describes a relatively unbiased and sensitive method for identifying the contributions of different behavioral parameters to neural activity. Their approach addresses, in an elegant way, several difficulties that arise in modeling of neuronal responses in population imaging data, namely variations in temporal filtering and latency, the effects of calcium indicator kinetics, interactions between different variables, and non-linear computations. Typical approaches to solving these problems require the introduction of prior knowledge or assumptions that bias the output, or involve a trade-off between model complexity and interpretability. The authors fit individual neuron's responses using neural network models that allow for complex non-linear relationships between behavioral variables and outputs, but combine this with analysis, based on Taylor series approximations of the network function, that gives insight into how different variables are contributing to the model.

The authors have thoroughly validated their method using simulated data as well as showing its applicability to example state of the art data sets from mouse and zebrafish. They provide evidence that it can outperform current approaches based on linear regression for the identification of neurons carrying behaviorally relevant signals. They also demonstrate use cases showing how their approach can be used to classify neurons based on computational features. They have provided Python code for the implementation and have explained the methods well, so it will be easy for other groups to replicate their work. The method could be applied productively to many types of experiments in behavioral and systems neuroscience across different model systems. Overall, the paper is clearly written and the experiments are well designed and analysed, and represent a useful contribution to the neuroscience field.

Reviewer #2 (Public Review):

This important paper is a real tour de force and combines functional MRI, behaviour, and brain stimulation to characterise the effect of stimulation of the lateral habenula in a rodent model for depression. The results are stunning and the data presented seems compelling.

My only comment is I would like more discussion on the relevance of these results for the treatment of depression in humans, both in terms of the rodent model and in terms of the results shown in this study.

Reviewer #2 (Public Review):

This manuscript is clear in that it shows no/minimal weight gain in a mouse model of trisomy 21 compared to the control mouse, even under a high-calorie diet. The difference is the clear demonstration of the increased expression of sarcolipin. It is important that the expression of SERCA was also shown not different between the genotypes. Additionally, an important result is that manipulating the skeletal muscle was sufficient to promote weight loss without the need for hypermetabolism in other tissues such as adipose tissue.

- A clear explanation of why the expression of sarcolipin/hypermetabolism is different between mouse and human under the same condition would be useful.

- p.12-13 and15. The language around 'futile' cycling is not correct because Ca movement through the sarcoplasmic reticulum of the resting fiber is essential to the function of the muscle. Firstly, the cycle of Ca through the SR is through the ryanodine receptor (RyR) as well as due to slippage through the SERCA (PMID: 11306667, PMID: 35311921). This is not made clear anywhere in the manuscript. Ca leak out of the SR through RyR is an essential component to the control/setting of the resting cytoplasmic [Ca2+] via the activation of store-operated Ca2+ entry, which is in a balance with the activation of the PMCA on the t-system membrane (PMID: 35218018). The SERCA resequesters the leaked Ca2+ from the SR. It is not possible that the resting [Ca2+] is set by the reduced efficiency of the SERCA, as indicated in the ms (PMID: 20709761). It is expected that the mito [Ca2+] steady state is set by the raised resting cyto [Ca2+] (PMID: 20709761). Ca2+ transients during EC coupling will promote transient increases in mito Ca2+ (PMID: 21795684, PMID: 36121378), but not steady-state increases. Some of these problems are highlighted by the errors in the diagram Fig 5D: please change/correct (i) the invagination of the sarcolemma is called the t-system; (ii) the cycle of Ca leak through the SR starts with RyR Ca leak, where the Ca is resequestered by the SERCA, in addition to Ca slippage through the pump. Draw a RyR opposite the t-system on the SR terminal cisternae. The heat generated by SERCA is absorbed in the cytoplasm, metabolites enter the mito and the OxPhos generates heat (PMID: 31346851). (iii) Ca does not enter mito because it cannot get into the SR (the resting cyto Ca is controlled by the t-system/plasma membrane, PMID: 20709761, PMID: 35218018). Please redraw.

- The changing of the properties of the muscle towards oxidative properties is consistent with the expression of sarcolipin in mouse muscle (all of it is in type II fibers). It is important to show whether the muscles have fiber-type shifts. Please report the fiber types of the muscles that have been surveyed in this project.

- Non-shivering thermogenesis (NST) is mentioned in this manuscript as the means of hypermetabolism, as has the lengthened duration of the cyto Ca transients during EC coupling. It is not clear at all what the contribution of NST compared to the increased work of the SERCA to clear released Ca from the cyto to the hypermetabolism. What are the relative proportions? If sarcolipin is largely for NST, then hypermetabolism is about the resting muscle.

- The link that SLN is causing more ATP use at the pump but the heat generated by OxPhos in mito is important and should be made, see Barclays' work (eg. PMID: 31346851). A direct link between the SERCA function and mito function is occurring but I currently don't see one being made in the ms. This could be made clear in Fig 5D diagram.

- p.22. "The reprogramming of glycolytic...elevated Ca transients...". The language is wrong here. Oxidative fibers do not have elevated Ca transients compared to glycolytic. The amplitude of Ca release is greater in glycolytic and the duration of the transient is longer in the oxidative (eg. PMID: 12813151).

- p.22. "as less calcium is being transported into the SR due to uncoupling of the SERCA pumps". The same amount of Ca is being transported, just at the expense of more ATP than would be the case in the absence of SLN. Otherwise, the SR Ca2+ content would not be at a steady state while the SR continuously leaks Ca2+.

- p.23. Tavi & Westerblad (PMID: 21911615) show how Ca transient amplitude and frequency signal in slow and fast twitch fibres. Here, we are not concerned with what is happening in myotubes, where the SR is less developed than in adult fibres.

Reviewer #2 (Public Review):

The authors present a new method of determining the boundaries of superficial, input, and deep cortical layers from laminar multielectrode recordings in non-human primates.<br /> It is based on using the generalized phase (GP) of the LFP (filtered between 5-50Hz) in conjunction with phase coupling (to the GP) of spiking activity (from single or multi-units). They report that phase coupling differs between layers. Critically the preferred LFP phase differs between the deep layers and layers above (input/superficial layers), and this measure can be reliably used to infer input/deep layer boundaries.

Spiking on a given channel (for all channels) tended to occur at +/- pi relative to LFPs recorded at superficial/input layers, but at 0pi relative to deep-layer LFPs. This relationship can be used to estimate the input/deep layer boundary. Generally, the estimate obtained was well correlated with measures derived from traditional CSD analysis. Where discrepancies occurred between CSD and phase coupling-based depth estimates, phase coupling-based depth estimates correlated better with additional measures such as firing rates, and low/high-frequency spectral power cross-over, that have been previously reported to align with cortical depth.

These results were present in areas MT (marmoset), V4 (macaque), and PFC (marmoset), and can be performed on short sequences of data under multiple experimental conditions.

This is a novel, easier, and potentially more precise way to assign cortical depth in non-human primates, which may prove useful to the wider research community.

Reviewer #2 (Public Review):

Previous work by the same group has shown that the potassium channel TWIK2 contributes to the activation of the NLRP3 inflammasome in macrophages. In this manuscript, the authors provide new insights into the biology of TWIK2 and show that TWIK2 translocated to the plasma membrane of macrophages following stimulation with ATP. They show that ATP stimulation induced exocytosis, via a process dependent on the purinergic receptor P2X7, the presence of calcium and vesicle fusion. Genetic deletion of P2X7, depletion of calcium, and pharmacological inhibition of vesicle fusion collectively contributed to the inhibition of current changes and NLRP3 inflammasome activation. The authors also show that the endosomal protein Rab11a translocated to the plasma membrane following ATP stimulation and that Rab11a contributed to NLRP3 inflammasome activation. Depletion of Rab11a in macrophages prevented lung injuries and NLRP3 inflammasome activation in mice treated with LPS.

The major strength of the work is the use of a combination of cell culture work and a mouse model to address the cell biology of inflammasome activation.<br /> The weakness is that the current set of data is not able to fully support the conclusion that Rab11a, P2X7 and calcium influx mediate the translocation of TWIK2 to the plasma membrane. The characterisation of inflammasome activation is also partial. If these weaknesses can be addressed, the authors would have achieved their aims and increased the impact of their work in the field of inflammasome biology.

Reviewer #2 (Public Review):

Overall, I thoroughly enjoyed reading and reviewing this manuscript. I think that it contributes importantly to the literature and illustrates an appealing way to connect neural data to normative ideas, phenomenological models, and mechanic explanations. In particular, the suggestion that the retina is specifically tailored to support predictive information encoding is normatively appealing, because animals obtain ecological advantages by anticipating their environment. It would be very exciting to figure out how the retina accomplishes this task. The authors begin their analysis of this question by using spatiotemporal receptive fields to phenomenologically describe how retinal ganglion cells nonlinearly integrate visual signals presented in different regions of the visual field. This allows them to identify several spatiotemporal components of the receptive field, termed kernels, that contribute differentially to predictive information encoding. The authors then use neural circuit modeling to reproduce these receptive field properties using biologically plausible bipolar cell inputs to the retinal ganglion cells. This allows them to hypothesize how specific circuit properties may contribute to predictive information encoding. For example, the authors' current models allow them to address the roles of bipolar cell nonlinearities, spatially local coupling between bipolar cells, patchy bipolar cell to retinal ganglion cell connectivity, and activity-dependent neuronal adaptation.

By connecting predictive information encoding to receptive field properties and candidate circuit mechanisms, the authors hope to identify biological fingerprints of predictive information encoding that could carry over to other neural circuits in the brain. I did not find this component of the argument to be convincing. My main concern is that stimulus statistics and neuronal activity statistics dually contribute to the meaning of predictive information, but this study did not dissect the role of stimulus statistics at all. As a result, I think the paper places too much emphasis on mechanism, and not enough emphasis on natural sensory statistics. The authors do devote a figure to illustrating that their receptive field estimation procedure is insensitive to the stimulus ensemble used for fitting (Fig. 4). Indeed, perhaps the receptive field kernels would stay similar if they were fit to natural stimuli. However, it would still be the case that the pattern of predictive information encoding captured by these kernels would strongly vary as a function of stimulus ensemble. For example, here the authors use random synthetic stimuli with relatively short correlation times, which means that the temporal horizon for predictive information encoding is limited (see Liu et al., Nat Neuro, 2021). The pattern of predictive information encoding for natural stimuli may be very different, and it may be that different receptive field components and neural circuit mechanisms contribute to predictive information encoding in that context. Similarly, other sensory systems are adapted to process stimuli with other sensory statistics, and I do not think it's clear that the receptive field components and neural circuit mechanisms identified here will be universally relevant.

The manuscript uses information theoretic methods to infer multiple kernels that describe linear stimulus features that modulate spiking activity of retinal ganglion cells. A potentially interesting limitation of the study is that it assumes that "outputs of these kernels are summed prior to passing through a common nonlinearity." However, many other papers have found that neuronal activity is sometimes governed by multiple linear features that cannot be summed prior to their nonlinear action. It would be interesting to know whether these kinds of features contribute to predictive information encoding in the retina.

A major problem with the manuscript is that its methods are inadequately described. I think that a major revision will be required before readers will be able reproduce the manuscript's results. These missing methodological details also make it difficult for readers to fully assess the manuscript's conclusions, strengths, and limitations.

Reviewer #2 (Public Review):

By analyzing hundreds of genomes, authors studied the so-called elusive genes, i.e., genes present in human genome but their orthologs deleted in some other mammals. Authors showed their bioinformatic pipeline of identifying these genes (Fig. 1), the genomic or evolutionary features of these genes (e.g. high GC content, Fig. 2), conservation of these features in other vertebrates including remotely related gar or shark (Fig. 3) together with polymorphism level, transcriptional features, epigenetic features of these genes (Fig. 4-6). Finally, in the Discussion section, the authors showed the chromosomal contributions of elusive genes and argued that these genes could be derived from ancient microchromosomes (Fig. 7).

Reviewer #2 (Public Review):

Whether and how molecularly defined neuronal groups in the spinal cord process distinct modalities are of great interest. In this study, Boyle et al. characterized roles of inhibitory neurons expressing NPY in adult mice. By using chemogenetic, electrophysiological tools and behavioral measurements, the authors discovered that activating NPY+ interneurons strongly reduced pruritogen-evoked itch and reflexive behaviors (acute nociception or under inflammation / neuropathic pain states). Silencing NPY+ spinal interneurons enhanced spontaneous and chemical itch in a GRPR+ neurons dependent manner. The authors concluded that, unlike previous findings suggesting that these neurons are selective for mechanical itch, adult NPY+ interneurons play dual roles in gating various types of itch and pain.

Strengths:

The authors performed careful characterization and comparisons between development lineage and adult spinal neurons expressing NPY. This lays the foundation of the current study. The behavioral measurements were also well designed with proper controls.

Weaknesses:

There is inadequate discussion about previous studies of NPY interneurons. Specifically, the authors should address why a more restricted subset of these neurons (this study) have broader effects than seen previously.

I cannot see the reason for including results from manipulation of Dyn+ interneurons in this paper. First, the title does not reflect roles of spinal Dyn+ population. In addition, without further experiments characterizing relationships between NPY and Dyn interneurons in modulating itch and/or nociception, Dyn datasets seem to deviate from the main theme.

While the authors provided convincing evidence that GRPR+ neurons serve as a downstream effector of NPY+ neuron evoked itch, the relationship between GRPR and NPY neurons in modulating pain is not examined. Therefore, Fig. 7B is pure speculation and should be removed.

Reviewer #2 (Public Review):

The paper by Huan, Yong, et al. studies epithelial cell extrusion in MDCK monolayers grown on sinusoidally wavy surfaces in varying media osmolarities, finding that both curvature and osmolarity-mediated basal hydraulic stress spatially regulate extrusion events. The authors fabricated wavy substrates of varying periods and amplitude out of PDMS (and PA hydrogels) and monitored monolayer evolution and cell extrusion over time, by combining live-cell imaging with a convolutional network-based algorithm for automatic detection of extrusions.

In general, the study has been elegantly designed, starting with convincing evidence for enhanced extrusion rates in concave valleys with respect to convex hills. Next, the authors showed that hyper-osmotic medium reduced cell extrusion rate, which was demonstrated in a variety of different media compositions (e.g. with sucrose, DMSO, or NaCl), while hypo-osmotic medium increased cell extrusion rate. Additionally, the authors applied reflection interference contrast microscopy to reveal fluid spaces between the substrate and the basal side of the monolayer, which were found to grow when media composition was altered from hyper-osmotic to normal osmotic conditions. Using a 3D traction force microscopy approach, the authors demonstrated that cells on convex regions apply a downward pointing force on the substrate, opposite to cells on the concave regions. This was linked to a larger basal separation on the concave valleys as opposed to the convex hills. Finally, the authors focussed on the FAK-Akt pathway to explore the hypothesis that basal hydraulic stress interferes with focal adhesions, leading to differences in cell extrusion rates in media of different osmolarity and on convex or concave surfaces.

Despite the host of relevant experiments and the interesting data acquired with a variety of techniques, some aspects of the manuscript would need to be strengthened or explained in more detail to better support the claims and to provide more convincing evidence.

1) The sinusoidal wavy substrate that the authors use in their investigation is interesting and relevant, but it is important to realise that this is a single-curved surface (also known as a developable surface). This means that the Gaussian curvature is zero and that monolayers need to undergo (almost) no stretching to conform to the curvature. The authors should at least discuss other curved surfaces as an option for future research, and highlight how the observations might change. Convex and concave hemispherical surfaces, for example, might induce stronger differences than observed on the sinusoidal substrates, due to potentially higher vertical resultant forces that the monolayer would experience. The authors could discuss this geometry aspect more in their manuscript and potentially link it to some other papers exploring cell-curvature interactions in more complex environments (e.g. non-zero Gaussian curvature).

2) The discussion of the experiments on PAM gels is rather limited. The authors describe that cells on the PAM gels experience fewer extrusions than on the PDMS substrates, but this is not discussed in sufficient detail (e.g. why is this the case). Additionally, the description of the 3D traction force microscopy and its validation is quite limited and should be extended to provide more convincing evidence that the measured force differences are not an artefact of the undulations of the surface.

3) The authors show nuclear deformation on the hills and use this as evidence for a resultant downward-pointing force vector. This has, indeed, also been observed in other works referenced by the authors (e.g. Werner et al.), and could be interesting evidence to support the current observations, provided the authors also show a nuclear shape on the concave and flat regions. The authors could potentially also characterise this shape change better using higher-resolution data.

4) The U-net for extrusion detection is a central tool used within this study, though the explanation and particularly validation of the tool are somewhat lacking. More clarity in the explanation and more examples of good (or bad) detections would help establish this tool as a more robust component of the data collection (on all geometries).

5) The authors study the involvement of FAK in the observed curvature-dependent and hydraulic stress-dependent spatial regulation of cell extrusion. In one of the experiments, the authors supplement the cell medium with FAK inhibitors, though only in a hyper-osmotic medium. They show that FAK inhibition counteracts the extrusion-suppressing effect of a hyper-osmotic medium. However, no data is shown on the effect of FAK inhibitors within the control medium. Would the extrusion rates be even higher then?

Reviewer #2 (Public Review): 

The authors are trying to distinguish between four models of the role of glypicans (HSPGs) on the Dpp/BMP gradient in the Drosophila wing, schematized in Fig. 1: (1) "Restricted diffusion" (HSPGs transport Dpp via repetitive interaction of HS chains with Dpp); (2) "Hindered diffusion" (HSPGs hinder Dpp spreading via reversible interaction of HS chains with Dpp); (3) "Stabilization" (HSPGs stabilize Dpp on the cell surface via reversible interaction of HS chains with Dpp that antagonizes Tkv-mediated Dpp internalization); and (4) "Recycling" (HSPGs internalize and recycle Dpp). 

To distinguish between these models, the authors generate new alleles for the glypicans Dally and Dally-like protein (Dlp) and for Dpp: a Dally knock-out allele, a Dally YFP-tagged allele, a Dally knock-out allele with 3HA-Dlp, a Dlp knock-out allele, a Dlp allele containing 3-HA tags, and a Dpp lacking the HS-interacting domain. Additionally, they use an OLLAS-tag Dpp (OLLAS being an epitope tag against which extremely high affinity antibodies exist). They examine OLLAS-Dpp or HA-Dpp distribution, phospho-Mad staining, adult wing size. 

They find that over-expressed Dally - but not Dlp - expands Dpp distribution in the larval wing disc. They find that the Dally[KO] allele behaves like a Dally strong hypomorph Dally[MH32]. The Dally[KO] - but not the Dlp[KO] - caused reduced pMad in both anterior and posterior domains and reduced adult wing size (particularly in the Anterior-Posterior axis). These defects can be substantially corrected by supplying an endogenously tagged YFP-tagged Dally. By contrast, they were not rescued when a 3xHA Dlp was inserted in the Dally locus. These results support their conclusion that Dpp interacts with Dally but not Dlp. 

They next wanted to determine the relative contributions of the Dally core or the HS chains to the Dpp distribution. To test this, they over-expressed UAS-Dally or UAS-Dally[deltaHS] (lacking the HS chains) in the dorsal wing. Dally[deltaHS] over-expression increased the distribution of OLLAS-Dpp but caused a reduction in pMad. Then they write that after they normalize for expression levels, they find that Dally[deltaHS] only mildly reduces pMad and this result indicates a major contribution of the Dally core protein to Dpp stability. The "normalization" is a key part of this model and is not mentioned how the normalization was done. When they do the critical experiment, making the Dally[deltaHS] allele, they find that loss of the HS chains is nearly as severe as total loss of Dally (i.e., Dally[KO]). Additionally, experimental approaches are needed here to prove the role of the Dally core.

Prior work has shown that a stretch of 7 amino acids in the Dpp N-terminal domain is required to interact with heparin but not with Dpp receptors (Akiyama, 2008). The authors generated an HA-tagged Dpp allele lacking these residues (HA-dpp[deltaN]). It is an embryonic lethal allele, but they can get some animals to survive to larval stages if they also supply a transgene called “JAX” containing dpp regulatory sequences. In the JAX; HA-dpp[deltaN] mutant background, they find that the distribution and signaling of this Dpp molecule is largely normal. While over-expressed Dally can increase the distribution of HA-dpp[deltaN], over-expression of Dally[deltaHS] cannot. These latter results support the model that the HS chains in Dally are required for Dpp function but not because of a direct interaction with Dpp. 

In the last part of the results, they attempt to determine if the Dpp receptor Thickveins (Tkv) is required for Dally-HS chains interaction. The 2008 (Akiyama) model posits that Tkv activates pMad downstream of Dpp and also internalizes and degrades Dpp. A 2022 (Romanova-Michaelides) model proposes that Dally (not Tkv) internalizes Dpp.  

To distinguish between these models, the authors deplete Tkv from the dorsal compartment of the wing disc and found that extracellular Dpp increased and expanded in that domain. These results support the model that Tkv is required to internalize Dpp. They then tested the model that Dally antagonizes Tkv-mediated Dpp internalization by determining whether the defective extracellular Dpp distribution in Dally[KO] mutants could be rescued by depleting Tkv. Extracellular Dpp did increase in the D vs V compartment, potentially providing some support for their model. However, there are no statistics performed, which is needed for full confidence in the results. The lack of statistics is particularly problematic (1) when they state that extracellular Dpp does not rise in ap>tkv RNAi vs ap>tkv RNAi, dally[KO] wing discs (Fig. 6E) or (2) when they state that extracellular Dpp gradient expanded in the dorsal compartment when tkv was dorsally depleted in dally[deltaHS] mutants (Fig. 6I). These last two experiments are important for their model but the differences are assessed only visually. In fact, extracellular Dpp in ap>tkv RNAi, dally[KO] (Fig. 6B) appears to be lower than extracellular Dpp in ap>tkv RNAi (Fig. 6A) and the histogram of Dpp in ap>tkv RNAi, dally[KO] is actually a bit lower than Dpp in ap>tkv RNAi, But the author claim that there is no difference between the two. Their conclusion would be strengthened by statistical analyses of the two lines. 

Strengths: 

1. New genomically-engineered alleles

A considerable strength of the study is the generation and characterization of new Dally, Dlp and Dpp alleles. These reagents will be of great use to the field.

2. Surveying multiple phenotypes

The authors survey numerous parameters (Dpp distribution, Dpp signaling (pMad) and adult wing phenotypes) which provides many points of analysis.

Weaknesses: 

1. Confusing discussion regarding the Dally core vs HS in Dpp stability. They don't provide any measurements or information on how they "normalize" for the level of Dally vs Dally[deltaHS]? This is important part of their model that currently is not supported by any measurements.

2. Lacking quantifications and statistical analyses: 

a. Why are statistical significance for histograms (pMad and Dpp distribution) not supplied? These histograms provide the key results supporting the authors' conclusions but no statistical tests/results are presented. This is a pervasive shortcoming in the current study. 

b. dpp[deltaN] with JAX transgene - it would strengthen the study to supply quantitative data on the percent survival/lethal stage of dpp[deltaN] mutants with or without the JAK transgene <br /> c. The graphs on wing size etc should start at zero. <br /> d. The sizes of histograms and graphs in each figure should be increased so that the reader can properly assess them. Currently, they are very small. 

The authors' model is that Dally (not Dlp) is required for Dpp distribution and signaling but that this is not due to a direct interaction with Dpp. Rather, they posit that Dally-HS antagonize Tkv-mediated Dpp internalization. Currently the results of the experiments could be considered consistent with their model, but as noted above, the lack of statistical analyses of some parameters is a weakness. One problematic part of their result for me is the role of the Dally core protein (Fig. 7B). There is a mis-match between the over-expression results and Dally allele lacking HS (but containing the core). Finally, their results support the idea that one or more as-yet unidentified proteins interact with Dally-HS chains to control Dpp distribution and signaling in the wing disc. 

There is much debate and controversy in the Dpp morphogen field. The generation of new, high quality alleles in this study will be useful to Drosophila community, and the results of this study support the concept that Tkv but not Dally regulate Dpp internalization. Thus the work could be impactful and fuel new debates among morphogen researchers. <br />

The manuscript is currently written in a manner that really is only accessible to researchers who work on the Dpp gradient. It would be very helpful for the authors to re-write the manuscript and carefully explain in each section of the results (1) the exact question that will be asked, (2) the prior work on the topic, (3) the precise experiment that will be done, and (4) the predicted results. This would make the study more accessible to developmental biologists outside of the morphogen gradient and Drosophila communities.

Reviewer #2 (Public Review):

Neutrophils are the most abundant circulating leukocytes in human. They play important roles in innate immune responses to infections and tissue injuries. Although they are dept in phagocytosis of microbes, neutrophils are not known to normally conduct efferocytosis or phagocytose host cells including apoptotic cells and play a significant role in apoptotic cell removal. In this report the authors provide evidence to suggest that neutrophils are involved in removal of apoptotic hepatocytes with certain specificity (i.e., they do not remove HEK293 or HUVEC endothelial cells). Moreover, the authors also show that neutrophils can burrow into the target cells and possibly ingest the target cells from the inside. The authors thus term this neutrophil-mediated efferocytosis process as "perforocytosis". Furthermore, evidence is provided to suggest that this neutrophil-mediated efferocytosis process keeps the number of apoptotic cells low in the livers and that defects in the processes may associate with autoimmune liver (AIL) disease phenotypes. Therefore, many of these findings are novel and the study is of important implications in our understanding of the role of neutrophils in autoimmune disease.

By examination of HE-stained, noncancerous liver tissue sections from patients with hepatocellular carcinoma and hepatic hemangioma, the authors observed that cells with neutrophil nuclear morphology were inside apoptotic hepatocytes. The authors also further characterized this observation by staining the sections with neutrophil and apoptosis markers. In addition, the authors observed the same phenomena in mouse livers using intravital microscopy, which also recorded the time course of the disappearance of a neutrophil-associated apoptotic cell. The author went on further characterization of neutrophil-mediated efferocytosis of cultured hepatic cells in vitro and demonstrated the process was specific for apoptotic hepatic cells, but not HEK293 or endothelial cells. The in vitro system was then used to characterize the molecular bases for neutrophil-mediated efferocytosis of apoptotic hepatic cells. The evidence was provided to suggest that IL1b and IL-8 released from and selectins upregulated in apoptotic hepatic cells were important. Importantly, the authors used two methods to deplete the neutrophils and showed that the neutrophil depletion increased apoptotic cells in livers. Finally, the authors showed that neutrophil depletion caused defects in liver function parameters. At the end, the authors presented evidence to suggest that AIL disease may be due to defective neutrophils that fail to perform "perforocytosis."

Although the evidence in its totality indicates that neutrophils burrow into apoptotic hepatocytes, the significance of this "perforocytosis" phenomenon and the circumstances under which it may occur remain to be better defined. In both neutrophil depletion models, the TNUEL-positive cells were not definitively identified rather than assuming they were hepatocytes. In addition, there are discrepancies in the number of neutrophils and apoptotic cells in mouse liver studies; Figure 2a WT (many neutrophils; locations unclear) vs Figure 5A Ctr (a few neutrophils that appear in or near a vessel), and Figure 2a DTR (a few apoptotic cells) vs Figure 5A Depletion (many apoptotic cells). Importantly, Figure 5a Ctrl, which is presumably a section from a mouse without any surgical treatment or without inflammation, the sole TUNNEL signal does not appear to be associated with neutrophils. Does this mean that "perforocytosis" primarily occurs in inflamed livers (Of note, human liver samples in Figure 1 are from patient with tumors. There should be inflammation in the livers of these patients). The data on human AIL patient neutrophils raises more questions: how many AIL patients have been examined? Do these AIL neutrophils lack IL1, IL8 receptors, and/or selectin ligands? Are there increases in apoptotic hepatocytes in AIL patients? Additionally, the overall numbers of apoptotic cells even in the absence of neutrophils are rare; thus, it is questionable that such rarity of apoptotic cells can cause significant AIL phenotypes.

Reviewer #2 (Public Review):

The role of the actin-binding protein palladin (PALLD) in cardiomyocyte development, growth, and function has not been defined. In order to address this question, the authors first identified that CARP and FHOD1 interact with PALLD in cardiomyocytes. They then performed cardiomyocyte selective deletion of PALLD in embryonic and adult mice and discovered that deletion of PALLD in adult mice leads to dilated cardiomyopathy (DCM) and intercalated disc ultrastructural changes. In contrast, embryonic deletion of cardiomyocyte PALLD did not cause a cardiomyopathy phenotype in neonatal or adult animals.

1. The divergent cardiac phenotypes of the embryonic deletion of cardiomyocyte PALLD (no cardiomyopathy) versus the adult deletion of cardiomyocyte PALLD (dilated cardiomyopathy(DCM)) is an interesting result. The authors speculate that embryonic deletion of PALLD induces compensatory pathways that prevent the development of adult cardiomyopathy in these mice. However, these compensatory pathways remain unexplored.<br /> 2. The authors discovered that mice with adult cardiomyocyte deletion of PALLD had significant changes in the cardiomyocyte intercalated disc (ICD) ultrastructure. They suggest these changes in ICD ultrastructure contribute to DCM formation in the adult PALLD deletion mice (line 270). However, it remains unclear if these changes in ICD ultrastructure are specific to mice with adult deletion of PALLD.<br /> 3. The different transgenic Cre mouse lines may be an alternative explanation for the divergent cardiac phenotypes in the embryonic versus adult deletion of cardiomyocyte PALLD. The tamoxifen dose administered for the inducible Myh6:MerCreMer mice was 30mg/kg/day x 5 which has been reported to lead to the induction of cardiomyocyte DNA damage response pathways (Dis Model Mech. 2013 Nov; 6(6): 1459-1469, J Cardiovasc Aging 2022;2:8). The electron micrograph experiments in Figure 5 did not include a group of Myh6:MerCreMer mice administered tamoxifen. The authors only compared PALLD fl/fl and Myh6:MerCreMer/PALLD fl/fl mice.<br /> 4. The apoptosis assessment was performed 24 weeks after administration of tamoxifen to the Myh6:MerCreMer/PALLD fl/fl mice. However, cardiomyocyte apoptosis may have occurred much earlier if it was secondary to Myh6:MerCreMer tamoxifen-induced cardiotoxicity (or related to PALLD deletion).<br /> 5. The animal studies in Fig 3D show a DCM phenotype in mice with adult deletion of cardiomyocyte 200kDa PALLD which suggests a potential loss of function mechanism for DCM formation. However, the authors then report in Fig 6 that human DCM heart tissue samples have a ~2.5fold increase in mRNA expression of the 200kDa PALLD transcript which would suggest a possible gain of function mechanism for DCM formation. How do the authors reconcile these divergent results with regard to palladin's role in cardiomyocyte homeostasis and cardiomyopathy formation?

Reviewer #2 (Public Review):

This paper addresses the topic of how T cells migrate in different tissues. The authors provide experimental evidence that T cell migration in the lung is more confined than in lymph nodes and gut villi. While prior studies have started to define the way T cells migrate during normal and pathological conditions, there is still a lot to learn about the factors that control this process. Thus, the topic is significant and timely. The authors use previously acquired data with two-photon microscopy from murine tissues. They compare multiple motility parameters of T cells in lymph nodes, gut villi, and inflamed lungs. Experiments demonstrate that T cells in the lung have a particular mode of migration characterized by low speeds, back-and-forth motions, and confinement.

Strengths:<br /> Overall, this is a very well-performed study. The data presented is of excellent quality and, for the most part, supports the authors' conclusions. The imaging techniques used to track T cells in various organs and the mouse models implemented are very relevant and robust. The functional analysis of the different migration features of T cells is compelling and should be of use to the community. The conclusion that T cells use different migration modes depending on the organ appears novel. This is considered of major significance.

Weaknesses:<br /> The main weakness of the manuscript is that the study remains descriptive and comparative. It is important to analyze and describe different migration modes depending on the organ. Still, it would have been desirable for the authors to provide information on the reason for such differences. One of the striking observations is the back-and-forth motion of T cells in the lung. Searching for mechanisms underlying this unique mode of displacement would strengthen the quality of the study.

Reviewer #2 (Public Review):

In this paper, the authors propose a system for annotating and curating scientific publications in the context of interspecies host-pathogen interactions. This system, called PHI-Canto (the Pathogen-Host Interaction Community Annotation Tool), is an extension of an existing tool (called Canto). In addition, they present the development of new concepts, controlled vocabularies, and an ontology for annotating relevant aspects in this domain, called PHIPO (Pathogen-Host Interaction Phenotype Ontology).

The approach has been empirically validated by annotating ten publications. The application's source code is available, as well as the associated ontologies and vocabularies and an example of the data resulting from the annotation process.

Reviewer #2 (Public Review):

The authors' manuscript has several strengths. First, the authors consider multiple relevant levels of biology including genomics, transcriptomics, structural and functional neuroimaging, cognitive neuroscience, and psychological/environmental factors. Such an approach is often necessary to deconvolute the complexities of psychiatric phenotypes. The authors have taken careful steps to think about potential confounds (e.g., ancestry for PRS) and to try to define their phenotypes (e.g., psychological resilience and biological aging) as best as they can, given the data they have access to from the ABCD study. The manuscript is well written overall.

My main concerns relate to core assumptions and techniques that underlie the premise of the study. First, while there is comorbidity between AD and MDD, a causal relationship between the two (in either direction) is not established. Though MDD often predates AD, this is to be expected given MDD's high lifetime prevalence (15-20% of the general population) and typical age of onset before age 65. Because AD typically presents late in life (>65 years of age), MDD will, by definition, usually predate AD. While new onset, late life MDD is often the first presenting symptom of AD/Parkinson's disease and other neurodegenerative conditions, it is also not clear that this is the same disorder as idiopathic MDD.

To this point, two genetic tools can help us determine the biological relationship between MDD/AD, genetic correlation and Mendelian Randomization. Using the data from the MDD PRS used in this analysis, the Supplementary Table 3 from the Howard et al. 2019 paper (https://doi.org/10.1038/s41593-018-0326-7) reveals a genetic correlation of -0.041 between the two. This indicates essentially no strong relationship between the MDD/AD (perhaps even a slightly inverse relationship). Mendelian Randomization studies in addition to the Howard et al paper (https://doi.org/10.1212/WNL.0000000000010463) find no causal role for MDD towards AD and vice versa. Thus, their comorbidity is likely mediated by additional factors. Additionally, while stress contributes to AD pathophysiology, AD is strongly genetic and, given its late onset, it is unclear how genetic risk for AD would meaningfully impact the psychological resilience of a 9 to 10-year-old.

My second concern is regarding the statement "adolescents at genetic risk for AD/MDD" when describing the sample. Per Howard et al 2019 out-of-sample prediction testing, the MDD PRS used by the authors explains between 1.5-3.2% of the phenotypic variance in MDD when used on a sample such as ABCD. MDD PRS is in its infancy and cannot reliably be used to identify individuals at high risk of MDD given that even individuals in the top 10th percentile of MDD PRS have an odds ratio for depression of only ~2.4. We would expect 90 or so individuals in this cohort to fall into this group leaving significant concerns about statistical power and the potential for false positive discoveries. While the AD PRS is significantly further along compared to MDD because of AD's simpler genetic architecture, the same concerns apply as, outside of APOE, the AD PRS does not capture the majority of phenotypic variance in AD.

The authors state that they wish to examine the effects of perinatal adversity directly/indirectly on biological aging and then assess the potential effects of biological aging on resilience. The authors use of pubertal age as a measure of accelerated aging is understandable given the data available, though not ideal. There are well validated measures of biological age such as Horvath's epigenetic clock. While advanced pubertal age is technically a form of accelerated aging, the majority of pubertal age as a phenotype is not likely to be explained by perinatal adversity. Rather, a combination of unmeasured variables including genetic variation, dietary factors, environmental exposures (endocrine disrupting chemicals), and obesity that play a substantial role in determining pubertal age. Childhood stress has been shown to have relatively small effects on pubertal age (d = -0.1) (10.1037/bul0000270).

Lastly, the authors employ the use of an as of yet unpublished technique to map neurotransmitters density to structural data from neuroimaging studies. While this technique is certainly interesting, its face validity is not clear given that many of the receptor-disease associations reported in the original preprint do not line up with what we know about the biology of these disorders from strong human genetics data or current FDA approved treatments. Moreover, the authors mention "Excitation/Inhibition" imbalance but the technique used appears to only include glutamate data from one receptor type, mGluR5. This may not be an adequate measure of E/I imbalance, despite there being a statistically significant finding.

Measuring both transcriptional output from GWAS loci and gene expression correlates from MRI data is a noisy and challenging prospect. Indeed, recent research has shown poor correlation between gene expression and neurotransmitter receptor density.(https://doi.org/10.1016/j.neuroimage.2022.119671).

Thus, fundamental aspects of this manuscript including the use of MDD PRS to identify "at risk" individuals, the unclear link between AD and adolescent psychological resilience, the use of prepubertal age as a measure of biological age, and the limited conclusions that can be drawn from the gene expression and receptor density technique limits confidence in the results as presented.

Reviewer #2 (Public Review):

The authors use a series of elegant methods to describe the nature of the interrelationship among CD8+ T cells and fibrocytes in the airways of COPD patients. They find an increased presence of these interactions in COPD and show that CXCL8-CXCR2 interactions are crucial for this interaction, leading to increased CD8+ T cell proliferation.

Major strengths of the work include the detailed functional experiments used to describe the nature of the CD8+ T cell - fibrocyte interaction. Another key strength is the translational approach of the work, building on clinical data and connecting back to these same clinical data. The conclusions of the authors are supported by the data. The impact of the work is significant and key to our understanding of the interrelationship between inflammation and tissue remodeling in COPD. Understanding this relationship holds strong potential for the identification of new drug targets and for the identification of patients at risk.

The derivation of the CXCL8/CXCR2 dependency is based on a limited number of COPD patients, which could be strengthened. Also, the impact of the interrelationship between CD8 cells and the fibrocytes is not fully described.

Reviewer #2 (Public Review):

The manuscript: "Metabolic consequences of various fruit-based diets in a generalist insect species" by Olazcuaga et al., addresses an interesting question. Using an untargeted metabolomics approach, the authors study how diet generalism may have evolved versus diet specialization which is generally more commonly observed, at least in drosophila species. Using the phytophagous species Drosophila suzukii, and by directly comparing the metabolomes of fruit purees and the flies that fed on them, the authors found evidence for "metabolic generalism". Metabolic generalism means that individuals of a generalist species process all types of diet in a similar way, which is in contrast to "multi-host metabolic specialism" which entails the use of specific pathways to metabolize unique compounds of different diets. The authors find strong evidence for the first hypothesis, as they could easily detect the signature of each fruit diet in the flies. The authors then go on to speculate on the evolutionary ramifications of this for how potentially diet specializations may have evolved from diet generalism. Overall, the paper is well written, the experiments well documented, and the conclusions convincing.

In my life thinking critically looks like reflecting on your past and history to guide you with your decision making in the future. There may not be an exact situation from the past that will be the answer, but there could be a scenario that is similar that you can use to help influence your future decision making skills.

Using information and ethically for me pretty much sums up to, giving credit where credit is due and using data to help you with decision making. In my job we collect data from students which helps us get funding for my program. I understand that, if we are not getting positive growth in our data then we run the risk of loosing funding. So I understand the importance of data.