Reviewer #1 (Public Review):

The authors have examined different pathways of B cell differentiation in patients with SARS-CoV-2 infection who did or did not have HIV-1 infection. They conclude that B cell responses to SARS-CoV-2 infection occur via an extra-follicular (EF) pathway to a greater extent in people with HIV-1 infection compared with people who do not have HIV-1 infection.

The data are important and generally robust but there are deficiencies related to presentation and interpretation of data, as indicated below:

1. There are concerns about nomenclature of cell populations defined by tSNE plots (figure 2A). For example, the population defined as "CSM/marginal zone" does not express IgD or IgM, as would be expected for class-switched memory B cells but not marginal zone B cells. In addition, while tissue homing and GC homing CSM B cells express expected amounts of CXCR4 and CXCR5, both express high amounts of CXCR3, which would be unexpected for GC homing cells. Finally, in line 144, the authors should clarify what is meant by "class switched, IgMhi B cells (highlighted in blue)". The population highlighted in blue in figure 2A, referred to as "IgM++ GC homing B cells", has the immunophenotype IgDlow, IgMhigh, CD27-. Aren't these cells at one end of a naïve B cell spectrum ranging from IgD+/IgM- to IgD+/IgM+ to IgDlow /IgMhigh? There are also other populations that have unconventional names and/or appear to be intermediary populations.

2. IgM switched memory B cells (lines 201-207) are referred to as IgM-only memory B cells by some investigators (for example, see - Bautista D et al. Front Immunol. 2020; 11:736). It would help the reader if this were indicated.

3. The authors have defined DN2 B cells based on expression of the activation marker CD95 (Fas) (see Figure 4) but the original definition of DN2 B cells in patients with SLE was based on expression of CD11c and lack of expression of CXCR5 (see - Jenks SA et al. Immunity. 2020; 52:203). These cells also express T-bet and therefore, have many characteristics in common with CD11c+/T-bet+ memory B cells (also known as age-associated B cells or atypical memory B cells). It would be informative if data on CXCR5- DN B cells were in analysed in addition to, or instead of, CD95+ DN B cells.

4. It might also be informative to discuss the extra-follicular (EF) response pathway in more detail. Recently published data from studies undertaken in mice indicate that CD11c+/T-bet+ MBCs interact with T follicular helper cells in lymphoid follicles but not in germinal centres (Song W et al. Immunity 2022; 55:290-307.e5), so it could be argued that the differentiation pathway is extra-GC rather than extra-follicular, at least in some situations. Also, in people with HIV-1 infection, HIV-1 gp140-specific B cells expressing T-bet are produced outside of GCs (Austin JW et al. Sci Transl Med. 2019; 11:eaax0904. Is the EF response pathway different to the extra-GC differentiation pathway? Where does it occur?

5. Similarly, in lines 288-290, the authors should re-consider the statement that "Both DN2 and activated naïve B cells mature via an EF pathway, independent of T cell help and in response to pro-inflammatory cytokines IFNγ, TNFa, and IL-21; and TLR 7 and 9 stimulation". There are data indicating that differentiation of DN2 B cells is T-cell-dependent (Keller B et al. Sci. Immunol. 2021; 6:eabh0891).

6. In lines 254-60 and figure 6, the investigators should consider the possibility that the CXCR3+ and DN2 SARS-CoV-2-specific MBCs that are increased in people with HIV infection are the same population of cells. CD11c+/T-bet+ MBCs (ie. DN2 B cells, age-associated B cells or atypical memory B cells) usually express high levels of CXCR3.

Reviewer #1 (Public Review):

The manuscript shows that bone is resorbed during the early steps of limb regeneration in urodeles, and osteoclasts are required for this process. In case of impaired resorption, integration of newly-formed tissue with the original bone shaft is compromised. The manuscript further shows that wound epithelium is required for bone resorption and suggests that it induces osteoclastogenesis or migration of osteoclasts. Furthermore, the authors showed that the formation of novel skeletal elements is initiated while the resorption of the old one is still actively ongoing.

The study is well designed, conclusions are relatively well supported, and data are presented in a clear way. Two new models of transgenic axolotls have been created. The strongest and most important finding is that partial bone resorption is required for tissue reintegration. My main concern is the novelty of this study, which is quite limited in my opinion. Specifically, resorption of bone stump during limb regeneration has been shown before in various model organisms. The role of osteoclasts in this process has not been well characterized in urodeles but has been shown during the regeneration of a mouse digit. It is reasonable to anticipate that similarly, osteoclasts are resorbing bone in salamanders, especially since this is the only cell type known for bone resorption. Thus, this observation, despite being nicely and thoroughly done, is of limited interest. The role of wound epithelium in bone histolysis is well demonstrated via skin flap experiments in this manuscript. However, upon skin flap surgery no limb regeneration occurs, implying wound epithelium is a key tissue triggering all the processes of limb regeneration. Accordingly, the absence of bone histolysis in such conditions can be secondary to the absence of any other part of the regenerative process, e.g., blastema formation, macrophage M1 to M2 transition, reinnervation, etc. The proposed link between wound epithelium and osteoclastogenesis (i.e., Sphk1, Ccl4, Mdka) is very superficial and very suggestive. No functional evidence was provided to confirm these connections. Finally, the authors showed that new bone formation occurs while resorption of the bone stump is still ongoing. This is a nice observation, but again, rather indirect as it is based on the dynamics of bone resorption and bone formation in different animals. Due to high variability among animals, direct evidence, like double staining for osteoclasts and blastema markers would address this point more precisely.

Reviewer #1 (Public Review):

The authors are trying to show that transitions between ring-like structures and clusters are driven by the balance between 2 main forces: filament treadmilling and motor protein-driven contractility. The results obtained in computer simulations are always compared with properly set experiments, making the story very convincing. In addition, the possible microscopic picture of the mechanisms is provided, although at a more phenomenological level. But given the complexity of the system, I find it very appropriate.

One of the most important achievements of this work is that the authors clearly identified and proved the factors that lead to a very non-trivial behavior. This should stimulate more work on understanding what biological regulation mechanisms might be involved in these phenomena.

I believe that this work will have a strong impact in the field. I am especially impressed by the successful combination of advanced computational and experimental methods.

Reviewer #1 (Public Review):

The aim of the present study was to develop and validate a novel mouse model that allows to determine the proteome of defined sub-cellular compartments, and to use this model in order to elucidate the molecular processes that govern the establishment of synaptic contacts between cortical and striatal neurons in the brain. Given that knowledge of the protein composition of defined sub-cellular compartments is of key importance for the characterisation of protein machines that mediate defined cellular functionalities, the establishment of corresponding mouse models to study such issues is of major general interest. The same is true for the development and function of cortico-striatal connectivity in the brain, which plays key roles in multiple major brain processes and is perturbed in many neuropsychiatric disorders.

The major strength of the present paper is that it presents a novel mouse line that promises to serve as a very helpful tool in this context. The authors generated a KI mouse line that expresses APEX2 under the control of a Cre-activatable promoter from the ROSA26 locus, and they show convincingly that this new mouse line, upon crossing with corresponding Cre-expressing driver lines, allows the identification of cell-sub-compartment specific proteomes and phosphoproteomes - via APEX2-mediated proximity biotinylation, tissue dissection, protein affinity purification, and mass spectrometric analysis.

The biological context of the present study is less convincingly established. Focussing on neuronal connections between the cerebral cortex and the striatum, bioinformatic analyses of corresponding datasets pinpoint a selection of axon guidance systems and protein kinase cascades to play roles in the development of cortico-striatal connectivity. The corresponding data partially align with the published record, but potentially new biological insights deduced from bioinformatic analyses of proteomic data were not followed up by experimental validation.

In sum, the new APEX2 reporter mouse line reported in the present paper will likely be of substantial interest to researchers in many fields of mammalian biology, but the extent of 'new biology' provided in the present study is very limited.

Reviewer #1 (Public Review):

The study by Tu and Zhang is very strong, from its technical implementation, the interesting question being addressed, and a clear presentation of the results. Indeed, the visual guides in the figures allow for easy navigation of the results and help the readers make his/her own inferences seamlessly. The quality of the MRI combined with electrophysiological recordings is excellent, as far as I can tell without looking at the data made available by the authors. The experiments and analysis follow a logical progression that makes sense. If any weakness is to be found, perhaps the authors overstep their inferences of respiration -> neuronal signal causality in the discussion.

Reviewer #1 (Public Review):

The paper by Campell et al., describes the isolation and characterization of Designed Ankyrin Repeat Proteins (DARP) that recognize distinct forms of gephryin. Gephyrin is a key determinant for postsynaptic accumulation of both glycine and GABAAR at synapses and thereby determines the efficacy of fast synaptic inhibition. In addition to this gephyrin regulates the synthesis of molybedum-cofactor, an essential co-factor for a number of metabolic enzymes.

The authors create DARPs that recognize specific splice forms of gephyrin and versions that discriminate between phosphorylated and dephosphorylated forms of gephyrin. These new tools reveal the differential recruitment of gephyrin isoforms to axo-axonic and somatodendritic synapses. In addition to these new tools allow the efficient one-step purification of differing gephyrin isoforms and their respective binding partners.

Reviewer #1 (Public Review):

The authors have previously reported the identification of a series of cell-cell junctional proteins as pTyr protein targets for the receptor-like PTPRK tyrosine phosphatase (PTP), including Afadin, a junctional plaque protein that links cell surface adhesion proteins to the cytoskeleton. They identified Afadin pY1230 as a target for PTPRK-mediated dephosphorylation, in keeping with the known role of tyrosine phosphorylation in regulating Afadin function in adherens junctions. They also showed that Afadin/PTPRK interaction did not require its tyrosine phosphorylation, and that the whole PTPRK cytoplasmic domain (ICD) was needed for in vitro dephosphorylation of pY1230 Afadin in vitro.

Here, they used two approaches to define a predicted 63-residue coiled-coil (CC) region (residues 1393-1455) in Afadin as being sufficient to bind the PTPRK intracellular domain (ICD). However, this region behaved as a monomer suggesting it is not a typical CC region. The CC bound the PTPRK ICD with low μM affinity and interacted selectively with the PTPRK D2 pseudophosphatase domain in vitro. Based on a predicted AlfaFold2/Multimer Afadin CC/D2 domain structure, they biochemically defined the key D2/CC interactions showing that a conserved core charged region, residues 1408-1448, in Afadin was essential, which then allowed them to refine the AlfaFold2 model. Their new model places the Afadin CC core region folded as an α-helix bound across the backside (?) of the D2 domain. They had shown previously that the ICD of the related PTPRU also bound Afadin whereas that of the PTPRM did not, and using the structural model showed that the key contact sites in PTPRK with the Afadin CC helix were conserved in PTPRU but not in PTPRM. When the residues in the G1273/L1335 "acidic" pocket of the D2 domain involved in Afadin helix binding were simultaneously mutated to His and Arg respectively, the basic residues found in PTPRM D2, both the double G1273H/L1335R mutant (DM) D2 alone and the entire PTPRK DM ICD failed to bind Afadin or to dephosphorylate (how much less that WT?) pY1230 in Afadin in lysates of pervanadate-treated cells, as assayed using a pY1230 specific antiserum they generated, even though both the WT and DM PTPRK ICD could dephosphorylate pTyr p120-catenin, another PTPRK substrate. On this basis the authors suggest that the D2 pseudophosphatase domain of PTPRK can act as a substrate recruitment domain that allows the active D1 domain to dephosphorylate a distant pTyr residue, in this case pY1230 ~150 residues away.

In this interesting study, the authors present evidence for the novel concept that the D2 pseudophosphatase domain of PTPRK can serve as a recruitment platform for a subset of PTPRK substrates, such as Afadin. Their evidence for this conclusion is strong, and by extension, their findings suggest that the D2 pseudophosphatase domains of other RPTPs may have a similar general function in substrate recruitment and selectivity.

1. While the AF2-Multimer prediction is quite compelling and supported by the properties of the RPTPK D2 DM mutant, this story would have been even more convincing if they had generated a co-crystal structure (perhaps using a PTPRK D2-Afadin aa 1393-1455 fusion with a long linker). In the absence of a true structure, some additional mutational validation of the proposed Afadin-D2 interaction would strengthen their conclusions.

2. The DM mutant data in Figure 4 show that the D2 domain interaction is important for Afadin pTyr dephosphorylation in vitro, but one would also like evidence that the DM PTPRK mutant lacks Afadin pY1230 dephosphorylating activity in cells. The authors have the PTPRK KO MCF10A cells they generated in their first paper that could be used to re-express the WT and DM PTPRK and then monitor Afadin dephosphorylation with their new anti-pY1230 antibodies.

3. If key residues in PTPRM are mutated into the equivalent PTPRK D2 residues, does this now confer on PTPRM the ability to dephosphorylate pY1230 in Afadin, i.e. a gain of function experiment?

4. It would be helpful to know whether any of the other PTPRK substrates that the authors identified previously have a similar motif that might allow them to bind to the D2 domain and be recruited for dephosphorylation.

Reviewer #1 (Public Review):

In this paper, the authors ask a key question in the field of adult plasticity, and in particular, amblyopia treatment: whether transient dark exposure followed by light re-introduction disrupts neural representation for basic stimulus attributes in a manner that could negatively impact vision. Prior work by Rose and colleagues using calcium imaging showed that closing one eye in adult mice leaves the responsiveness of V1 neurons unchanged but alters their orientation preference and pairwise correlations; such representational drift may require downstream areas to adjust how they readout V1 signals. The question posed here is whether binocular visual deprivation in adult mice does the same. The authors use 2-photon calcium imaging in 6 awake, head-fixed [transgenic - GCaMP6f driven by the EMX1 promoter] mice before and after transient dark exposure to record ensemble responses of layer 2/3 excitatory V1 neurons to oriented gratings of varying spatial frequencies. Data were acquired twice at baseline (allowing for an assessment of representational drift during exposure to the natural [cage] environment), once immediately after 8 days of dark exposure and once about 8 days after animals were once again exposed to their natural [cage] environment.

The study appears to be generally well designed with multiple analytical approaches trained on the same questions. Major strengths include the ability to analyze a large number of neuronal responses simultaneously in the awake-behaving state using calcium imaging in transgenic mice, and the ability to record activity in the same neurons across several weeks and following different behavioral manipulations. A relative weakness was the implication of only being able to elicit relevant visual responses from a small fraction of V1 neurons for comparison purposes. This begs the question of what may have happened to the neurons that were not tracked, and whether this in fact may have been significant. For the ~30% of V1 neurons which were tracked, the findings appear to be that dark exposure of adult mice for 8 days did not significantly corrupt their orientation or SF tuning. Instead, there were increase pairwise correlations between them, interpreted as increased stability of stimulus representation. However, when the entire neuronal pool was analyzed, a decrease in decoding accuracy was noted, attributed to decreased response reliability. Nonetheless, a recovery back to baseline was noted after mice were re-exposed to light and their natural cage environments for 8 days. The study thus provides a binocular deprivation alternative to the earlier monocular deprivation findings of Rose et al. In addition, it provides some new insights, suggesting that the early visual system (i.e. V1) of adult animals normally exhibits a flexible stimulus representation for simplistic, artificial visual stimuli such as oriented gratings, and that temporary dark exposure decreases this flexibility. Importantly for therapeutic approaches however, this can be reversed upon re-introduction of the natural, complex visual environment.

Reviewer #1 (Public Review):

This study demonstrates the role of the circadian clock in spatiotemporal regulation of floral development. The authors nicely illustrated floral development patterns in domesticated sunflower. In particular, during anthesis, discrete developmental zones, namely pseudowhorls, are established, and hundreds of florets simultaneously undergo maturation in each psudowhorl in a circadian-dependent manner. Consistently, the flower development follows key features of the circadian clock, such as temperature compensation and gating of plant response to environmental stimuli. Evolutionary advantages of this regulation will add more merit to this study.

Reviewer #1 (Public Review):

In addition to canonical bacterial signaling methods, two-component systems, and serine/threonine kinases, one of the most ubiquitous signal transduction modalities in M. tuberculosis is via adenylate cyclases. This study seeks to identify new adenylate cyclases of M. tuberculosis used to sense antibiotic treatment and resist its effects. To this end, authors employed cutting-edged techniques including genetic knock-out strategy, CRISPRi knock-down strategy, LC-MS-based target metabolite quantification, and various biochemical/microbiological methods. This study provides a conceptually novel strategy to kill M. tuberculosis with conventional tuberculosis chemotherapy.

• ffff*

Why mordern tv series like to describe middle age as a "Dark Age"?

Reviewer #1 (Public Review):

The authors have modified protocols for Phage Immunoprecipitation sequencing or PhIP-seq to allow much larger throughput and have examined value of this platform for auto-antigen discovery. Overall the manuscript is technically sound. The finding of shared auto-antigens in Kawasaki Disease and MIS-C was of interest.

Reviewer #1 (Public Review):

The paper has determined a considerable number of different structures and conformations by Cryo-EM, that describes the full conformational spectrum of the KdpFABC catalytic cycle. They also show by EPR that the non-phosphorylatable variant KdpBS162A variant was indeed arrested in the state observed by Cryo-EM.

Although they have been able to validate that the Cryo-EM structure of the off-cycle state is consistent with the conformational state probed by pulsed EPR, it is unclear what protein phosphorylates and then inactivates KdpFABC at higher K+ concentrations. As such, at present, it is not possible to fully comprehend the exact physiological conditions when the arrested state is formed.

Reviewer #1 (Public Review):

A novel approach is introduced for targeting Protein-RNA interactions. The approach (presented in Figure 1) integrates computational techniques with cellular assays, and is applicable, in principle, whenever the protein-RNA complex has a druggable binding pocket. It is demonstrated with the discovery of inhibitors of YB-1's interaction with its mRNA target. Of 22 putative hits, discovered based on virtual screen, 11 come out as very strong hits. Far beyond the 5-10 percent success rate that one often sees in drug discovery.

The main strength here is the proof of concept that protein-RNA interactions are targetable.

Reviewer #1 (Public Review):

The submitted manuscript describes an optimized tissue clearing protocol with some modest advantages including better preservation of tissue volume, compatibility with traditional histology methods, and simple processing steps. By combining known advantages of organic solvent-based and aqueous-based procedures the authors were able to generate a very simple, efficient, and fast tissue clearing protocol that can preserve endogenous and synthetic fluorescent signals. The manuscript is mostly written well and the fluorescent images are very striking. However, the lack of quantification throughout the manuscript makes it is difficult to assess how robust the results are across many samples and key experimental applications are missing.

1. Immunofluorescent labeling/staining is a very common procedure in whole, cleared tissues. Given that immunofluorescent labeling works well in tissue sections from EZ Cleared brains, it appears that it should work in the whole tissues after clearing. An extended version of the EZ Clear protocol with immunofluorescent labeling procedures in whole mouse brain tissue should be included along with quantification of fluorescent intensity as a function of depth. If EZ Clear provides more uniform immunofluorescent labeling relative to other protocols, this is a significant advantage.

2. The differences in tissue volume and sample processing steps between EZ Clear and Fast 3D are important, but relatively modest. Additional quantitative comparisons between EZ Clear and Fast 3D/3Disco would considerably strengthen the manuscript. The qualitative differences shown in Figure 1G-J are striking, but it is difficult to determine how robust this effect is across multiple samples without a quantitative comparison. Similar quantitative comparisons should be made for endogenous fluorescent intensity and immunofluorescent labeling as a function of tissue depth between the various protocols.

3. It would be helpful to see how the intensity and contrast of the fluorescent labeling changes as a function of depth (e.g. Lectin-649 labeling in Figures 2E and H). There is a clear improvement with EZ View relative to RIMS, but there are still noticeable changes in the signal as a function of depth. Quantification would help determine the extent of these changes, as well as reproducibility across multiple samples.

4. LSFM imaging should be performed in some of the other mouse tissues to demonstrate sufficient clearing for quantification purposes.

Reviewer #1 (Public Review):

The authors use a model system to investigate how three classes of kinesins (1, 2 and 3) interact with the dynein-dynactin-truncated BicD2 complex when coupled via a DNA scaffold. Complexes with kinesin 1 have been shown to have a plus-end bias, but unexpectedly the authors show that this is also true for kinesins 2 and 3 despite these motors having a higher load sensitivity. The authors reconcile this finding by showing via simulations that faster reattachment kinetics compensate for faster detachment rates under load. They conclude that motor kinetics is another important feature in understanding both the velocity and directionality that cargo is transported.

This is the first study directly comparing three classes of constitutively active kinesin motors versus DDB in a controlled fashion, which is a strength of this study. The caveat is that these results may require modification when dynein and kinesin are coupled via an activating adaptor rather than DNA. However, the studies in the current manuscript are a required prerequisite, as different activating adaptors would be needed for the different classes of kinesin, thus introducing another variable into how the two classes of motors interact. Moreover, results from these studies can be used as a platform for further investigation of the effect of MAPs, regulatory proteins, and PTMs of the MT on model bidirectional complexes.

Reviewer #1 (Public Review):

GCaMP indicators have become common, almost ubiquitous tools used by many neuroscientists. As calcium buffers, calcium indicators have the potential to perturb calcium dynamics and thereby alter neuronal physiology. With so many labs using GCaMPs across a variety of applications and brain regions, it's remarkable how few have documented GCaMP-related perturbations of physiology, but there are two main contexts in which perturbations have been observed: after prolonged expression of a high GCaMP concentration (common several weeks after infection with a virus using a strong promoter); and when cytoplasmic GCaMP is present during neuronal development. As a result, GCaMP studies are often designed to avoid these two conditions.

Here, Xiaodong Liu and colleagues ask whether GCaMP-X series indicators are less toxic that GCaMPs. GCaMP-X indicators are modified GCaMPs with an additional N-terminal calmodulin binding domain that reduces interactions of the calmodulin moiety of GCaMP with other cellular proteins. Xiaodong Liu and colleagues document effects of GCaMP expression on neuronal morphology in vitro, calcium oscillations in vitro, and sensory responses in vivo, in each case showing that GCaMP-X indicators are less toxic. Their results are compelling.

Unfortunately, the paper suffers two main weaknesses. Firstly, the results demonstrate that GCaMP is toxic during development, after prolonged expression via viruses in vivo, and in cell culture where maturation of the culture likely recapitulates key steps in development. GCaMPs are known to be toxic in these circumstances, such toxicity is readily circumvented by driving expression in the adult, and there are countless examples of studies in which adequate GCaMP expression was achieved without toxicity. These new results are of little relevance to the majority of GCaMP experiments. That GCaMP-X indicators are less toxic during development is a new result and may be of interest to those who wish to deploy calcium indicators during development, but this is a relatively small number of neuroscientists.

Secondly, the authors extend their claims to conclude that GCaMP indicators are toxic under other circumstances, claims supported by neither their results nor the literature. To provide one example, at the end of the introduction is the statement, 'chronic GCaMP-X imaging has been successfully implemented in vitro and in vivo, featured with long-term overexpression (free of CaM-interference), high spatiotemporal contents (multiple weeks and intact neuronal network) and subcellular resolution (cytosolic versus nuclear), all of which are nearly infeasible if using conventional GCaMP.' The statement's inaccurate: there are many chronic imaging studies in vitro and in vivo using GCaMP indicators without nuclear accumulation of GCaMP or perturbed sensory responses. There are more examples throughout the paper where the conclusions overreach the results and are inaccurate. The results are simply insufficient to support many of the strong statements in the paper.

Reviewer #1 (Public Review):

This study attempts to understand the source of problems in allocentric navigation in older adults and children compared to young adults. Using a simple and elegant Y-maze design with extensive behavioral analyses, the authors convincingly show that older adults and children are impaired with respect to the ability to use landmark cues, but not geometric cues, in order to orient in the environment. Their testing further shows that this results from a problem of remembering spatial relations between landmarks and using those to navigate, and not an issue of encoding the landmarks themselves or attending to them. The findings are important in two respects: 1) understanding the navigational problems of older adults, 2) understanding the cognitive systems underlying allocentric navigation. With regard to the first point, the authors' results from the map drawing task demonstrate that the problem is specifically with remembering the relative configuration of the landmarks with respect to one another and to the start and goal location. With regard to the second point, the paper is exciting in that it demonstrates a dissociation between two systems of allocentric navigation - landmark-based and geometry-based. As the authors point out, most papers refer to "allocentric navigation" as a process where subjects use either the geometry or landmarks interchangeably as reference points in their mental map, but these findings suggest that those systems might be dissociable. Overall, I think that the study is well-designed, the analyses are adequate, and the research questions are addressed appropriately. The authors took care to exclude other sources of difference between groups by having both physical and virtual reality mazes, using a walking VR paradigm to eliminate computer use proficiency differences, and testing visual attention and gaze as well as an array of other variables.

Reviewer #1 (Public Review):

Redox signaling is a dynamic and concerted orchestra of inter-connected cellular pathways. There is always a debate whether ROS (reactive oxygen species) could be a friend or foe. Continued research is needed to dissect out how ROS generation and progression could diverge in physiological versus pathophysiological states. Similarly, there are several paradoxical studies (both animal and human) wherein exercise health benefits were reported to be accompanied by increases in ROS generation. It is in this context, that the present manuscript deserves attention.

Utilizing the in-vitro studies as well as mice model work, this manuscript illustrates the different regulatory mechanisms of exercise and antioxidant intervention on redox balance and blood glucose level in diabetes. The manuscript does have some limitations and might need additional experiments and explanation.

The authors should consider addressing the following comments with additional experiments.

1. Although hepatic AMPK activation appears to be a central signaling element for the benefits of moderate exercise and glucose control, additional signals (on hepatic tissue) related to hepatic gluconeogenesis such as Forkhead box O1 (FoxO1), phosphoenolpyruvate carboxykinase (PEPCK), and GLUT2 needs to be profiled to present a holistic approach. Authors should consider this and revise the manuscript.<br /> 2. Very recently sestrin2 signaling is assumed significant attention in relation to exercise and antioxidant responses. Therefore, authors should profile the sestrin2 levels as it is linked to several targets such as mTOR, AMPK and Sirt1. Additionally, the levels of Nrf2 should be reported as this is the central regulator of the threshold mechanisms of oxidative stress and ROS generation.<br /> 3. Authors should discuss the exercise-associated hormesis curve. They should discuss whether moderate exercise could decrease the sensitivity to oxidative stress by altering the bell-shaped dose-response curve.<br /> 4. It would not be ideal to single-out AMPK as a sole biomarker in this manuscript. Instead, authors should consider AMPK activation and associated signaling in relation to redox balance. This should also be presented in Fig 7.

Reviewer #1 (Public Review):

Klein and colleagues have developed a new setup to artificially activate genetically targeted neurons in temporal precise correlation with specific behaviors in larva of Drosophila melanogaster. The work explores how the activation of specific sets of reward and punishment coding neurons during the execution of side-specific bending alters the occurrence of this behavior. Indeed, activating serotonergic neurons during specific bending in a training phase, biases bending direction in the test. Since altering behavior as a consequence of its rewarding or punishing outcome is considered operant learning the authors conclude that the targeted neurons mediate operant conditioning. Below I will point out the strength and my criticisms concerning the presented work.

The newly developed closed-loop set-up is impressive and will pave the way for many exciting studies on learned behavior and beyond. To validate the set up the authors induce rolling behavior by thermo- or optogenetically activating two sets of previously described neurons in individual larva. Both approaches show convincing induction of the behavior per se. However, it is worth pointing out that there seems to be an interaction of the different tools used (thermo and opto-genetic) and the targeted neurons: the authors observe different dynamics of the behavior across the three stimulation cycles depending on stimulation method and labeled neurons. These findings make it difficult to understand why the authors choose only the optogenetic activation to investigate operant conditioning. The strength of the setup is that individual animals can be targeted. Though the presented data show that behavior can be reliably induced in stimulated animals, it lacks the information about the behavior of non-targeted larva during the stimulation. Thus, it would strengthen the work if the authors could show the behavior of the non-targeted larva during the time when targeted larva receive light or heat.

The authors use their setup to investigate operant conditioning. In operant conditioning an animal learns to associate its action with the consequences. Their new setup allows the authors to artificially induce consequences, the activation of reward or punishment coding neurons, upon side-specific bending behavior. The experiments show that side specific bending in the test is slightly biased towards the side previously paired with the neuronal stimulation. Interestingly, the data suggest that this effect requires the activity of serotonergic neurons outside of the brain (in the VNC) and that it is not mediated by dopamine signaling in the brain. Though, the effects seem to be reproducible with Ddc- and the Tph-GAL4 the reported differences are small, and the origin of the relative difference between left and right bending in the paired group is not entirely clear. Thus, it will be important to strengthen the work by additional experiments and extend the analysis of the presented data. Given the novelty of the method and the differences between the tools in the proof of principle experiments the authors should repeat the key experiments (Figure 3 b and e) with the thermogenetic stimulation. Further it would strengthen the investigation on operant conditioning if the authors would explore the temporal relationship between the CS and US, especially since the effect might be a reduction of the unreinforced behavior (see below). Concerning the analysis, the authors should consider that given the small effect they observe, they want to be sure that it originates from training. Though they show the pretraining results for one of the experiments (Figure 3b, the trained group), the pretraining bending is very relevant for each of the operant learning experiments. In fact, training induced effects should not only be measured by looking at the left vs right bending in the final test but as a change between pre versus post or between a trained and a mock control group. This is done for one group (Ddc-GAL4) in Figure 3b but will be mandatory for all operant learning experiments. It would improve the accessibility of the learning induced change of behavior if the authors could show the pre vs post training results for each run (10-12 larva in a plate). Further, they should plot the numbers of reinforced behaviors in each of the training protocols and relate it to the test performance. The presented data clearly suggests a decrease of the unstimulated bending rather than a change in the reinforced behavior. Though the authors mention it, they do not explain or discuss it. It will be very important for the logic of the manuscript that the authors explain this phenomenon and how it relates to operant conditioning.

Lastly, though the manuscript discusses most of the data carefully, in my view the authors miss an important issue: it remains to be shown if fly larva are capable of operant learning using external reward or punishment. The presented evidence is based on artificial activation of neurons, which arguably is a hint but not a prove that operant conditioning is withing the repertoire of a fly larva, an issue the authors should mention and discuss.

Reviewer #1 (Public Review):

The goal of the current study is to determine the impact of sleep on resilience to social stress. The research team accomplished their goals using male mice that underwent social defeat stress by a larger conspecific. The team found that sleep is necessary and sufficient for promoting stress resilience to social defeat stress. They also identified the prefrontal cortex as a major player in the link between sleep and stress resilience.

Overall, this is a well-written manuscript that is strengthened by the translational relevance and significance, the well-executed study design, and the robustness of the data.

Reviewer #1 (Public Review):

In this manuscript, the authors leverage novel computational tools to detect, classify and extract information underlying sharp-wave ripples, and synchronous events related to memory. They validate the applicability of their method to several datasets and compare it with a filtering method. In summary, they found that their convolutional neural network detection captures more events than the commonly used filter method. This particular capability of capturing additional events which traditional methods don't detect is very powerful and could open important new avenues worth further investigation. The manuscript in general will be very useful for the community as it will increase the attention towards new tools that can be used to solve ongoing questions in hippocampal physiology.

Additional minor points that could improve the interpretation of this work are listed below:

- Spectral methods could also be used to capture the variability of events if used properly or run several times through a dataset. I think adjusting the statements where the authors compare CNN with traditional filter detections could be useful as it can be misleading to state otherwise.

- The authors show that their novel method is able to detect "physiological relevant processes" but no further analysis is provided to show that this is indeed the case. I suggest adjusting the statement to "the method is able to detect new processes (or events)".

- In Fig.1 the authors show how they tune the parameters that work best for their CNN method and from there they compare it with a filter method. In order to offer a more fair comparison analogous tuning of the filter parameters should be tested alongside to show that filters can also be tuned to improve the detection of "ground truth" data.

- Showing a manual score of the performance of their CNN method detection with false positive and false negative flags (and plots) would be clarifying in order to get an idea of the type of events that the method is able to detect and fails to detect.

- In fig 2E the authors show the differences between CNN with different precision and the filter method, while the performance is better the trends are extremely similar and the numbers are very close for all comparisons (except for the recall where the filter clearly performs worse than CNN).

- The authors acknowledge that various forms of SWRs not consistent with their common definition could be captured by their method. But theoretically, it could also be the case that, due to the spectral continuum of the LFP signals, noisy features of the LFP could also be passed as "relevant events"? Discussing this point in the manuscript could help with the context of where the method might be applied in the future.

- In fig. 5 the authors claim that there are striking differences in firing rate and timings of pyramidal cells when comparing events detected in different layers (compare to SP layer). This is not very clear from the figure as the plots 5G and 5H show that the main differences are when compare with SO and SLM.

- Could the above differences be related to the fact that the performance of the CNN could have different percentages of false-positive when applied to different layers? Alternatively, could the variability be related to the occurrence (and detection) of similar events in neighboring spectral bands (i.e., gamma events)? Discussion of this point in the manuscript would be helpful for the readers.

Overall, I think the method is interesting and could be very useful to detect more nuance within hippocampal LFPs and offer new insights into the underlying mechanisms of hippocampal firing and how they organize in various forms of network events related to memory.

Reviewer #1 (Public Review):

The manuscript by Arnason et al. reports a careful in-depth analysis of genomic patterns of diversity of the Atlantic codfishes, sampled twice near the Icelandic coast. The manuscript is scientifically sound and provides thorough details of the statistical analysis and of the underlying models. In essence, the analysis demonstrates that recurrent selective sweeps are the most compatible scenario to explain the data. The analysis is extremely detailed, well constructed, and very convincing. It also advertises the family of Multiple-Merger Coalescents (MMCs) as good models for standard population genetics analyses. Overall, I found this article very interesting and extremely well-documented.

Reviewer #1 (Public Review):

The experiments presented in this extensive study by Ronzano et al. are a tour-de-force investigating the spatial organization of premotor interneurons in the mouse spinal cord to re-examine the fundamental question of whether there is spatial segregation of interneurons with monosynaptic connections to motoneurons innervating functionally antagonistic (flexor and extensor) pairs of limb muscles. Such segregation has been proposed from earlier studies utilizing strategies for retrograde trans-synaptic tracing of spinal premotoneurons with rabies virus (RabV) following muscle injection. This spatial organization has been suggested to provide an anatomical substrate for labeled line inputs from proprioceptive afferents to motor neurons with possibly organization advantages for motor control. The present premotor circuit mapping experiments, involving four different collaborating laboratories applying an extensive set of complementary RabV-based trans-synaptic circuit tracing techniques, convincingly demonstrate complete spatial overlap among flexor and extensor premotor interneurons, contradicting the previous mapping results that suggest spatial segregation. The present results revise our understanding of the spatial organization of spinal premotor circuits and provide an alternative view of the role of interneuron positioning in sensory input connectivity without specific spatial patterning of output connectivity to motoneurons, with fundamental implications for understanding motor circuit function.

Strengths of these studies include:

1. The investigators systematically tested and directly compared most of the available premotor circuit tracing strategies utilizing genetically modified mouse strains and viruses, as well as the previous approaches, with all tests replicating the spatial overlap of flexor and extensor premotor interneurons.

2. The authors utilized a mouse genetic strategy combining a Cre conditional allele expressing RabV glycoprotein G from the rosa locus with either the ChAT::Cre or Olig2::Cre mouse lines, which in contrast to previous RabV-based approaches, enables selective and potentially high levels of G expression in all motoneurons at the time of RabV muscle injection and likely robust transsynaptic transfer for premotor neuron labeling.

3. The authors present a very useful instructive exposition of the currently available techniques for labeling premotor interneurons outlining experimental strategies and indicating advantages and disadvantages for interpretation of results by illustrating RabV trans-synaptic transfer pathways that could confound experimental results.

4. The authors also used transgenic strategies in combination with their other approaches to differentiate inhibitory or putative excitatory premotor interneurons controlling the activity of flexor and extensor muscles and demonstrated from technically elegant spatial analyses that flexor and extensor premotor neurons were always spatially intermingled regardless of their neurotransmitter identity.

5. The authors further confirmed the lack of spatial segregation by pooling together all the results obtained with the different circuit tracing methods.

6. The authors thoroughly discuss the limitations of their mouse genetic strategy for circuit tracing including off-target G complementation in cells other than the targeted cholinergic motoneurons with the possibility of labeling disynaptic pathways via cholinergic spinal interneurons. Also considered is the problem in identifying the number of motoneurons with G complementation, which is a main determinant of reproducibility in RabV tracing experiments and a key parameter for comparing results from different circuit tracing approaches.

7. Overall the experiments are rigorously performed with a design that reduces biases associated with the various RabV-based circuit tracing methods, and the data although very extensive with numerous data source files and supplemental illustrations, are clearly presented.

Reviewer #1 (Public Review):

This project by Li et al. describes a colony morphology of P. aeruginosa that arises on agar plates and is especially pronounced in mutants lacking flagella, which were used for the majority of experiments in the paper. The paper documents the formation of large channels in the projections of swarming colonies, and within these channels, the rapid transport of fluid, cells, and extracellular vesicles. This transport is measured with great care and supported with additional support modeling.

- By and large, the project looks to have been executed with strong methodology and attention to detail in describing the channel formation effect found among colonies of P. aeruginosa flgK mutants. The authors have done very well in pushing known imaging methods to document transport within the colony channels and to make a case for how this transport is being driven physically. I think the aims of the detailed description of the physical phenomenon of colony growth in this environmental condition have been accomplished.

- A limitation here is that this colony morphology only seems to manifest strongly in mutants lacking flagella, which I don't think is common among wild P. aeruginosa isolates. To the extent that groups of P. aeruginosa cells have been imaged in situ, e.g. in the sputum of CF patients, this kind of channel formation does not occur in more realistic conditions. See DePas et al. (2015) https://journals.asm.org/doi/epub/10.1128/mBio.00796-16. I think it's more likely that this colony morphology is idiosyncratic to the agar growth substrate on which the cells are growing in this case, so the more interesting thing here is the physics of the system rather than its applications to clinical or ecological settings.

- The authors have established that flgK-null P. aeruginosa forms colonies with channels in this agar growth and incubation environment, and made a strong case for the physics underlying the spontaneous formation of this morphology. The idea that this morphology reflects a multicellular developmental program for P. aeruginosa is not strong, though, as this morphology is not found in the wild. In general, the idea that groups of microbes on agar are analogous to multicellular organisms with circulatory systems has little support from in-situ imaging experiments, or from fundamental evolutionary theory. So, I would advise shifting the introduction and discussion away from the multicellular organism focus toward a greater focus on the physics of the system and its potential for synthetic systems. See for example Yan et al. (2019) https://elifesciences.org/articles/43920

Reviewer #1 (Public Review):

The study presented by AL Seufert et al. follows the trajectory of trained immunity research in the context of sterile inflammatory diseases such as gout, cardiovascular disease and obesity. Previous studies in mice have shown that a 4 week Western-type diet is sufficient to induce systemic trained immunity, with gross reorganization of the bone marrow to support a potentiated inflammatory response [PMID: 29328911]. The current study demonstrates that mice on a Western-type diet (WD) and the more extreme Ketogenic diet (KD; where carbohydrates are essentially eliminated from the diet) for 2 weeks results in a state of increased monocyte-driven immune responsiveness when compared to standard chow diets (SC). This increased immune responsiveness after high-fat diet resulted in a deadly hyper-inflammatory in the mice in response to endotoxin (LPS) challenge in vivo. These initial findings as displayed in Figure 1 are made difficult to interpret because the authors use a mix of male and female mice coupled with very small sample sizes ( n = 5 - 9). Male and female mice are shown to have dimorphic responses to LPS exposure in vivo, with males having elevated cytokine levels (TNF, IL-6, IL-1β, and also interesting IL-10) increased rates severe outcomes to LPS challenge [PMID: 27631979]. As a reader it is impossible to discern from their methodological description what the proportion of the sexes were in each group, and therefore cannot determine if their data are skewed or biased due to sexual dimorphic responses to LPS rather than diet. Additionally due to the very small sample sizes, the authors can't perform a stratified analysis based on sex to determine whether the diets are having the greatest effects in accordance with LPS induce inflammation.

When comparing SC to the KD, the authors identify large changes in fatty acid distribution circulating in the blood. The majority of the fatty acids were shown to relate to saturated fatty acids (SFA). Although Lauric, Myristic, and Myristovaccenic acid where the most altered after KD, the authors focus their research on the more thoroughly studied palmitic acid (PA). PA was shown to increase the expression of inflammatory cytokines gene expression and protein production of TNF, IL-6 and IL-1β in bone marrow derived macrophages (BMDMs). The authors tie these effects to ceramide synthesis through a pharmacological blockade as well as the use of oleic acid, which allegedly sequesters ceramide synthesis. The author's claim that oleic acid supplementation reverses the inflammatory signaling induced by PA is invalid, as oleic acid was shown to induce a high level of cytokines in their model. When PA was added along with oleic acid, the cytokine levels returned to the levels produced by BMDM's stimulated with PA alone (see Figure 4 panels D- F).

Finally the authors test whether injection of PA into mice can recapitulate the systemic inflammatory response seen by WD and KD feeding followed by LPS exposure. They were able to demonstrate that injecting 1 mM of PA, waiting for 12h, and then exposing the mice to LPS for 24h could similarly result in a hyper-inflammatory state resulting in greater mortality. The reviewer is skeptical that 1 mM of PA truly represents post-prandial PA levels as one would expect to see after a single fatty meal, and whether this injection is generally well tolerated by mice. Looking into the paper cited by Eguchi et al. to inform their methods, it's shown that the earlier study continuously infused an emulsified ethyl palmitate solution (which contained 600 mM) at a rate of 0.2 uL/min. As far as I can read by Eguchi, they only managed to reach a serum PA concentration of 0.5 mM. This is hardly the same thing as a single i.p. injection of 1 mM PA. and reflects a single bolus injection of double the serum concentration of PA achieved by Eguchi et al.

PA is known to induce inflammation in monocytes and macrophages, therefore the findings certainly make sense in the context of previously published literature. However the authors have made some poor methodological decisions in their mouse studies, namely haphazardly switching between groups of young and old mice (4-6 weeks, 8-9 weeks, and 14-23 weeks), using different LPS injection protocols (6, 10, and 50 mg/ml of LPS), and including multiple sexes of mice. All of which are drastically alter the interpretation of the data, and preventing solid conclusions from being drawn.

Reviewer #1 (Public Review):

In this manuscript, the authors generated a zbtb14 mutant zebrafish strain via CRISPR-Cas9. In the mutant fish, they found an abnormal expansion of primitive macrophages during early development and adult macrophages in the kidney marrow. The abnormal expansion of macrophages in the mutants was confirmed to be caused by the loss of zbtb14 function, as over-expressing either zebrafish zbtb14 or human ZBTB14 could rescue the phenotype. To explore the underlying molecular mechanism, the authors performed RNA-seq analysis and found that the expression of pu.1 was up-regulation in the mutant macrophages. They further showed that the injection of mpeg1.1:pu.1-DBD construct into the mutant embryos to suppress the Pu.1 activity was able to rescue the mutant phenotype. The authors then went on to show that the SUMOylation of Zbtb14 plays an essential role in the transcriptional repression activity of the proteins. Finally, the authors documented that over-expressing the S8F mutant form of human ZBTB14, an AML associated mutation, failed to rescue the macrophage phenotype in zbtb14 mutants, suggesting that the loss of ZBTB14 function may be associated with the development of AML. Overall, the findings are interesting in developmental biology and gene regulation, especially in normal and malignant myelopoiesis.

Reviewer #1 (Public Review):

The authors have used many cleverly chosen mouse models (periodontitis models; various models that lead to an on-switch of genes) and methods (immune localizations of high quality; single cell RNA sequencing) for the quest of elucidating a role for telocytes. They describe that more telocytes are present around teeth in mice that had periodontitis. These cells proliferated, and they expressed a pattern of genes that allowed macrophages to differentiate into a different direction. In particular, they showed that telocytes in periodontitis express HGF, a molecule that steers macrophage differentiation towards a less inflammatory cell type, paving the way for recovery. As a weakness, one could state that an attempt to extrapolate to human cells is missing.

Reviewer #1 (Public Review):

It was previously shown that HGF and Met controls development of the diaphragm muscle. In particular, the signal induces delamination and migration of muscle progenitor cells that colonize the diaphragm. The present manuscript by Sefton and coworkers confirms and extends these observations using (i) conditional mouse lines in which the HGF gene was targeted by Cre/loxP recombination in the pleuroperitoneal folds (Prx1-cre) and at other sites PdgfraCreERT2, and of (ii) Met inhibitors. Overall, the technical quality of the data on diaphragm muscle development is excellent; the conceptual advance over previous work is not exceptional; the evidence for Met/HGF-dependent development of the phrenic nerve is marginal and needs to be strengthened.

The data show that fibroblasts provide HGF signals received by Met in muscle progenitor cells that is essential for diaphragm development. The PdgfraCreERT2 line was used to demonstrate that HGF produced by fibroblasts but not by muscle progenitors is essential for diaphragm development. Moreover, development of dorsal and ventral regions of diaphragm muscle requires continuous MET signaling. Thus, HGF is not only required for the delamination of progenitors, but also for proliferation and survival of those muscle progenitors that reached the anlage of the diaphragm.

My major concern is the limited data on the HGF-dependent development of the phrenic nerve (defasciculation). While it is well documented that HGF acts as a trophic factor for motor neurons in culture, its role in development of motor neurons was highly debated due to the fact that some changes observed in Met or HGF mutant mice in vivo are also present in other mutants that lack the muscle groups derived from migrating muscle progenitors. Moreover, careful genetic analyses previously demonstrated indirect mechanisms of Met during motor neuron development, i.e. a non-cell-autonomous function of Met during the recruitment of motor neurons to PEA3-positive motor pools (Helmbacher et al., Neuron 2003).

Sefton et al. provide an analysis of a single time point, one histological picture (3G, magnified in 3H) that indicate that in Met+/- animals defasciculation of the phrenic nerve does not occur correctly. This is accompanied by a quantification that barely reaches significance (Fig. 3K). Data shown in Fig. 7 using Met inhibitors show a major change in phrenic nerve branching, which is presumably due to the major change in diaphragm development, as conceded by the authors.

Despite this weakness on the experimental side, the role of HGF/Met in phrenic nerve development is strongly emphasized in abstract /intro/discussion (e.g. line 414: However, PPF-derived HGF is crucial for the defasciculation and primary branching of the nerve, independent of muscle). The data need to be strengthened in order to conclude that HGF coordinates both, diaphragm muscle and phrenic development.

סיימתי!

DOI: 10.1016/j.isci.2022.105028

Resource: (ZFIN Cat# ZDB-ALT-130506-1,RRID:ZFIN_ZDB-ALT-130506-1)

Curator: @evieth

SciCrunch record: RRID:ZFIN_ZDB-ALT-130506-1

What is this?

Reviewer #1 (Public Review):

This Methods paper explores methods of assaying the balance between muscle cell quiescence and activation. If successful, it offers a miniaturized assay that will permit systematic investigations of long-standing queries in key areas of muscle function such as regulation of adult stem cell pool size and functional heterogeneity. It could also be used to discover regulators of quiescence.

Reviewer #1 (Public Review):

This paper represents the first spatio-temporal functional parcellation derived from infant multimodal imaging data. The parcellations are generated from the longitudinally collected baby connectome project, and clearly benefit from incorporating repeat samples from individuals. Analyses demonstrate that parcellations estimated for different age groups (3, 6, 9, 12, 18 and 24 months) are fairly consistent and that repeat generation of the parcellations, using shuffled 'generating' and 'repeating' groups is robust.

In general, I think the paper does an extremely good job of robustly testing its claims and therefore I have relatively few suggestions for improvement. However, I do have some concerns that the differences in network clustering reported in Fig 6 may be due to noise and I think the comparisons against the HCP parcellation could be more robust.

Specifically, with regard to the network clustering in Fig 6. The authors use a clustering algorithm (which is not explained) to cluster the parcels into different functional networks. They achieve this by estimating the mean time series for each parcel in each individual, which they then correlate between the n regions, to generate an nxn connectivity matrix. This they then binarise, before averaging across individuals within an age group. It strikes me that binarising before averaging will artificially reduce connections for which only a subset of individuals are set to zero. Therefore averaging should really occur before binarising. Then I think the stability of these clusters should be explored by creating random repeat and generation groups (as done for the original parcells) or just by bootstrapping the process. I would be interested to see whether after all this the observation that the posterior frontoparietal expands to include the parahippocampal gryus from 3-6 months and then disappears at 9 months - remains.

Then with regard to the comparison against the HCP parcellation, this is only qualitative. The authors should see whether the comparison is quantitatively better relative to the null clusterings that they produce.

While it's clear from the results that the template achieves some good degree of spatio-temporal coherence, from the considerable benefit of the longitudinal imaging, not all individuals appear (from Fig 8) to be acquired exactly at the desired timepoints, so maybe the authors might comment on why they decided not to apply any kernel weighted or smoothing to their averaging? Pg. 8 'and parcel numbers show slight changes that follow a multi-peak fluctuation, with inflection ages of 9 and 18 months' explain - the parcels per age group vary - with age with peaks at 9 and 18 - could this be due to differences in the subject numbers, or the subjects that were scanned at that point?

I also have some residual concerns over the number of parcels reported, specifically as to whether all of this represents fine grained functional organisation, or whether some of it represents noise. The number of parcels reported is very high. While Glasser et al 2016 reports 360 as a lower bound, it seems unlikely that the number of parcels estimated by that method would greatly exceed 400. This would align with the previous work of Van Essen et al (which the authors cite as 53) which suggests a high bound of 400 regions. While accepting Eickhoff's argument that a more modular view of parcellation might be appropriate, these are infants with underdeveloped brain function. Further comparisons across different subjects based on small parcels increases the chances of downstream analyses incorporating image registration noise, since as Glasser et al 2016 noted, there are many examples of topographic variation, which diffeomorphic registration cannot match. Therefore averaging across individuals would likely lose this granularity. I'm not sure how to test this beyond showing that the networks work well for downstream analyses but I think these issues should be discussed.

Finally, I feel the methods lack clarity in some areas and that many key references are missing. In general I don't think that key methods should be described only through references to other papers. And there are many references, particular to FSL papers, that are missing.

Reviewer #1 (Public Review):

In this paper, Abadchi et al. investigate neocortical activity patterns surrounding sharp-wave ripples in awake head-fixed mice. To do so, the authors combine multiple approaches, including wide-field voltage and glutamate imaging, 2-photon single-cell calcium imaging, and electrophysiology, used to monitor the hippocampal LFP and MUA. The authors' previous findings in anaesthetized and head-fixed sleeping mice indicated that the majority of cortical areas were strongly activated by ripples. In contrast, they now show that ripple-related neocortical patterns in the awake brain show predominantly suppression of activity. Interestingly, this deactivation seems to be most pronounced and to occur earliest in the agranular retrosplenial cortex (aRSC). To gain a better understanding of the internal dynamics underlying ripple modulation in the RSC the authors perform 2-photon calcium imaging and find that similar proportions of superficial excitatory cells are activated and suppressed during ripples.

Ripple oscillations have been implicated in multiple cognitive processes including memory consolidation, memory retrieval, and planning, and there is causal evidence suggesting that awake and sleep ripples are differentially involved in those functions. Consequently, understanding the physiological mechanisms underlying hippocampal-neocortical communication during both brain states is of pivotal importance. Many studies investigated the modulation of various cortical areas by ripples during sleep and wakefulness, but the majority of those studies focused on one or few areas. The author's previous study (Abadchi et al., 2020) was an exception in this regard, as it provided a rich characterization of activity surrounding sleep ripples in multiple neocortical areas, including latency to response and direction of propagation. The present study purports to be complementary to those published results, although it lacks many of the analyses used for the sleep paper, which is a missed opportunity. The stark sleep/wake differences in cortical peri-ripple activity reported by the authors are surprising, interesting, and potentially of substantial importance for understanding the functions of ripples in the awake vs. sleep state. However, many of the results presented in the paper are insufficiently analyzed and their statistical significance is unclear, demanding further quantification and clarifications. Moreover, while the paper's major strength lies in the combination of multiple large-scale approaches, it could do better in combining those observations into a coherent conclusion.

Major points:

1) There is affluent evidence that the cortical activity in the waking brain, even in head restrained mice, is not uniform but represents a spectrum of states ranging from complete desynchronization to strong synchronization, reminiscent of the up and down states observed during sleep (Luczak et al., 2013; McGinley et al., 2015; Petersen et al., 2003). Moreover, awake synchronization can be local, affecting selective cortical areas but not others (Vyazovskiy et al., 2011). State fluctuations can be estimated using multiple criteria (e.g., pupil diameter). The authors consider reduced glutamatergic drive or long-range inhibition as potential sources of the voltage decrease but do not attempt to address this cortical state continuum, which is also likely to play a role. For example: does the voltage inactivation following ripples reflect a local downstate? The authors could start by detecting peaks and troughs in the voltage signal and investigate how ripple power is modulated around those events.

2) Ripples are known to be heterogeneous in multiple parameters (e.g., power, duration, isolated events/ ripple bursts, etc.), and this heterogeneity was shown to have functional significance on multiple occasions (e.g. Fernandez-Ruiz et al., 2019 for long-duration ripples; Nitzan et al., 2022 for ripple magnitude; Ramirez-Villegas et al., 2015 for different ripple sharp-wave alignments). It is possible that the small effect size shown here (e.g. 0.3 SD in Fig. 2a) is because ripples with different properties and downstream effects are averaged together? The authors should attempt to investigate whether ripples of different properties differ in their effects on the cortical signals.

3) The differences between the voltage and glutamate signals are puzzling, especially in light of the fact that in the sleep state they went hand in hand (Abadchi et al., 2020, Fig. 2). It is also somewhat puzzling that the aRSC is the first area to show voltage inactivation but the last area to display an increase in glutamate signal, despite its anatomical proximity to hippocampal output (two synapses away). The SVD analysis hints that the glutamate signal is potentially multiplexed (although this analysis also requires more attention, see below), but does not provide a physiologically meaningful explanation. The authors speculate that feed-forward inhibition via the gRSC could be involved, but I note that the aRSC is among the two major targets of the gRSC pyramidal cells (the other being homotypical projections) (Van Groen and Wyss, 2003), i.e., glutamatergic signals are also at play. To meaningfully interpret the results in this paper, it would be instrumental to solve this discrepancy, e.g., by adding experiments monitoring the activity of inhibitory cells.

4) I am puzzled by the ensemble-wise correlation analysis of the voltage imaging data: the authors point to a period of enhanced positive correlation between cortex and hippocampus 0-100 ms after the ripple center but here the correlation is across ripple events, not in time. This analysis hints that there is a positive relationship between CA1 MUA (an indicator for ripple power) and the respective cortical voltage (again an incentive to separate ripples by power), i.e. the stronger the ripple the less negative the cortical voltage is, but this conclusion is contradictory to the statements made by the authors about inhibition.<br /> 5) Following my previous point, it is difficult to interpret the ensemble-wise correlation analysis in the absence of rigorous significance testing. The increased correlation between the HPC and RSC following ripples is equal in magnitude to the correlation between pre-ripple HPC MUA and post-ripple cortical activity. How should those results be interpreted? The authors could, for example, use cluster-based analysis (Pernet et al., 2015) with temporal shuffling to obtain significant regions in those plots. In addition, the authors should mark the diagonal of those plots, or even better compute the asymmetry in correlation (see Steinmetz et al., 2019 Extended Fig. 8 as an example), to make it easier for the reader to discern lead/lag relationships.

6) For the single cell 2-photon responses presented in Fig. 3, how should the reader interpret a modulation that is at most 1/20 of a standard deviation? Was there any attempt to test for the significance of modulation (e.g., by comparing to shuffle)? If yes, what is the proportion of non-modulated units? In addition, it is not clear from the averages whether those cells represent bona fide distinct groups or whether, for instance, some cells can be upmodulated by some ripples but downmodulated by others. Again, separation of ripples based on objective criteria would be useful to answer this question.

7) Fig. 3: The decomposition-based analysis of glutamate imaging using SVD needs to be improved. First, it is not clear how much of the variance is captured by each component, and it seems like no attempt has been made to determine the number of significant components or to use a cross-validated approach. Second, the authors imply that reconstructing the glutamate imaging data using the 2nd-100th components 'matches' the voltage signal but this statement holds true only in the case of the aRSC and not for other regions, without providing an explanation, raising questions as to whether this similarity is genuine or merely incidental.

8) The estimation of deep pyramidal cells' glutamate activity by subtracting the Ras group (Fig. 4d) is not very convincing. First, the efficiency of transgene expression can vary substantially across different mouse lines. Second, it is not clear to what extent the wide field signal reflects deep cells' somatic vs. dendritic activity due to non-linear scattering (Ma et al., 2016), and it is questionable whether a simple linear subtraction is appropriate. The quality of the manuscript would improve substantially if the authors probe this question directly, either by using deep layer specific line/ 2-P imaging of deep cells or employing available public datasets.

Cited literature<br /> Abadchi, J.K., Nazari-Ahangarkolaee, M., Gattas, S., Bermudez-Contreras, E., Luczak, A., McNaughton, B.L., and Mohajerani, M.H. (2020). Spatiotemporal patterns of neocortical activity around hippocampal sharp-wave ripples. Elife 9, 1-26.<br /> Fernandez-Ruiz, A., Oliva, A., Oliveira, E.F. De, Rocha-Almeida, F., Tingley, D., and Buzsáki, G. (2019). Long-duration hippocampal sharp wave ripples improve memory. Science (80-. ). 364, 1082-1086.<br /> Van Groen, T., and Wyss, J.M. (2003). Connections of the Retrosplenial Granular b Cortex in the Rat. J. Comp. Neurol. 463, 249-263.<br /> Luczak, A., Bartho, P., and Harris, K.D. (2013). Gating of Sensory Input by Spontaneous Cortical Activity. J. Neurosci. 33, 1684-1695.<br /> Ma, Y., Shaik, M.A., Kim, S.H., Kozberg, M.G., Thibodeaux, D.N., Zhao, H.T., Yu, H., and Hillman, E.M.C. (2016). Wide-field optical mapping of neural activity and brain haemodynamics: considerations and novel approaches. Philos. Trans. R. Soc. B Biol. Sci. 371.<br /> McGinley, M.J., David, S. V., and McCormick, D.A. (2015). Cortical Membrane Potential Signature of Optimal States for Sensory Signal Detection. Neuron 87, 179-192.<br /> Nitzan, N., Swanson, R., Schmitz, D., and Buzsáki, G. (2022). Brain-wide interactions during hippocampal sharp wave ripples. Proc. Natl. Acad. Sci. 119.<br /> Pernet, C.R., Latinus, M., Nichols, T.E., and Rousselet, G.A. (2015). Cluster-based computational methods for mass univariate analyses of event-related brain potentials/fields: A simulation study. J. Neurosci. Methods 250, 85-93.<br /> Petersen, C.C.H., Hahn, T.T.G., Sakmann, B., Grinvald, A., and Mehta, M. (2003). Interaction of sensory responses with spontaneous depolarization in layer 2/3 barrel cortex. Proc. Natl. Acad. Sci. 100, 13638-13643.<br /> Ramirez-Villegas, J.F., Logothetis, N.K., and Besserve, M. (2015). Diversity of sharp-wave-ripple LFP signatures reveals differentiated brain-wide dynamical events. Proc. Natl. Acad. Sci. 112, E6379-E6387.<br /> Steinmetz, N.A., Zatka-Haas, P., Carandini, M., and Harris, K.D. (2019). Distributed coding of choice, action and engagement across the mouse brain. Nature 1-8.<br /> Vyazovskiy, V. V, Olcese, U., Hanlon, E.C., Nir, Y., Cirelli, C., and Tononi, G. (2011). Local sleep in awake rats. Nature 472, 443-447.

Reviewer #1 (Public Review):

The Cretaceous dinosaur Spinosaurus has recently drawn significant attention as it was hypothesized as the first aquatic dinosaur. The authors provide additional lines of evidence including the CT-based skeletal restoration of Spinosaurus and biomechanical tests to challenge the 'aquatic hypothesis'. The key claims of the manuscript are supported by the new data and the new analyses are important for the further clarification of the Spinosaurus lifestyle.

Reviewer #1 (Public Review):

Bosada et al present a study on how regulatory elements found in two atrial fibrillation-associated regions at the TBX5 locus correlate to Tbx5 expression levels and arrhythmia susceptibility. In transgenic mouse models, the investigators deleted the orthologues of these regulatory elements at the human Tbx5 locus. Tbx5 expression levels were increased in both models, and the downstream impact on epigenetic and gene expression levels was assessed. The RE(int)-/- mice had higher expression levels of Tbx5 compared to RE(down)-/- mice and this was correlated with increased atrial arrhythmia inducibility and higher numbers of transcripts impacted in the atrial gene regulatory network analysis. Multiple pathways are affected, and the authors present data on the interaction between Tbx5 and Prrx1, which encodes a cardiac transcription factor and the human ortholog harbors an atrial fibrillation-associated variant. The presented work links with the prior observation that increase in Tbx5 expression is associated with human atrial fibrillation and provides a plausible mechanistic link.

Reviewer #1 (Public Review):

The authors aim at establishing a biologically plausible learning rule for the Successor Representation (SR) to be computed by neural circuits.

The study is well designed with a strong logical flow moving from a simple example (random process on a circle) to comparison with real neural data. The manuscript is well written in all its components and figures are clear. All the results provided in the main paper are backed up by a thorough theoretical analysis outlined in the supplementary material. As it is common the theoretical analysis does not have much space in the manuscript. I would suggest summarizing with more specific statements the theoretical results that are achieved whenever there is a pointer to a supplementary note.

While the authors perform an extensive and careful review of the literature, a lot of it is confined to the Discussion. As the results of the paper strongly rely on the normalizing term in Eq.4. I would suggest potentially moving upfront part of the discussion of this term. I would suggest enlarging the paragraph that discusses the biological plausibility of this specific term. Clearly laying out, for the non-expert reader, why it is biologically plausible compared to other learning rules. And I would also consider moving the required material to establish the novelty of such term: a targeted review of the relevant literature (current lines 358-366 and 413-433). This would allow the reader to understand immediately the significance and relative novelty of such term. For example, I personally wondered while reading the paper how different was such term from the basic idea of Fiete et al. Neuron 2010 (DOI 10.1016/j.neuron.2010.02.003).

I would also suggest writing a "limitations" paragraph in the discussion clearly outlining what this learning rule couldn't achieve. For example, Stachenfeld et al Nat.Neuro. have many examples where the SR is deployed. I wonder if the learning rule suggested by the authors would always work across the board, or if there are limitations that could be highlighted where the framework suggested would not work well. I am not suggesting performing more experiments/simulations but simply sharing insight regarding the results and the capability of the proposed learning rule.

Reviewer #1 (Public Review):

This is an elegant and fascinating paper on individuality of structural covariance networks in the mouse. The core precepts are based on a series of landmark papers by the same authors that have found that individuality exists in inbred mice, and becomes entrenched when richer environments are available. Here they used structural MRI to provide whole brain analyses of differences in brain structure. They first replicated brain (mostly hippocampal) effects of enrichment. Next, they used their roaming entropy measurements to show that, after dividing their mice into two groups based on their roaming entropy, that there were no differences in brain structure between the two groups yet significant differences in brain networks as measured by structural covariance. Overall I enjoyed this paper, though am confused (and possibly concerned) about how they arrived at their two groups and have some less important methods questions.

The division of mice into two groups (down and flat) is confusing. The methods appear to suggest that k-means clustering combined with the silhouette method was used (line 380). The actual analyses used involves 2 groups of 15 mice each. The body of the manuscript suggests that 10 intermediate mice were excluded (line 100), but the methods (line 390) suggest that 8 mice were excluded, 2 for having intermediate results and 6 for having high RE slope values.

This leads to a series of questions:<br /> - How many mice were excluded and for what reasons, given the discrepancy between body and methods?<br /> - Was the k-means clustering actually used? It appears that the main division of mice was based on visual assessments.<br /> - If the clustering was used, did it result in 2 or 3 groups?<br /> - The intermediate group bothers me (if it was indeed 10 intermediate mice as indicated by the body rather than 2 as indicated in the methods): if these are indeed intermediate shouldn't they be analyzed and shown to be intermediate on the graph or other measures?<br /> - Please explain the reasoning for excluding mice for having too high of a slope (if there were indeed mice excluded for having too high of a slope).

I'd also appreciate more discussion about the structural covariance differences between flat and down mice. It is not clear what the direction of effects are - it appears that flats show mostly increases in covariance?

Reviewer #1 (Public Review):

This is a very timely and substantial advance in connectomics research that allows the fast reconstruction of selected neuronal circuits at synaptic resolution using tissue expansion and light sheet imaging. The authors describe this methodology in detail as applied to Drosophila brain, with multiple examples across different neuronal types and labeling strategies. The study is very rigorously done, methods are presented with important details, and the discussion is engaging and balanced. The paper is excellently written and very informative.

The authors begin by introducing a workflow to detect and quantify presynaptic structures of specific neuronal types. This approach takes advantage of the T-bar protein Brp ubiquitously expressed at presynapses and the widely used nc82 antibody against it, as well as the fact that presynapses are larger neurites that are readily resolvable with light microscopy. Using three distinct neuronal types, the authors show that the number of presynapses obtained with the presented light microscopy method, matches well the synapse number quantified by the gold standard, electron microscopy.

Next, the authors present two approaches to tackle a more difficult task - the quantification of the synaptic connectivity between 2 specific neuronal types. Compared to mammals, the identification of the postsynaptic site is more difficult in the Drosophila, because each presynapse contacts several different postsynaptic neurites that are right next to each other and are much smaller in size. No ubiquitous postsynaptic marker is currently available for the fly brain either. However when there is a postsynaptic marker available for specific connection, this makes the synaptic connection identification much more reliable, as shown with the example of the synaptic connections between the cholinergic SAG neurons and their postsynaptic target, the pC1 neurons, using the postsynaptic marker Drep2. Using this strategy the authors demonstrate that mated female flies have significantly less synaptic connections between SAG neurons and pC1 neurons, compared to virgin flies.

In addition to chemical synapses, this study also shows a proof of principle that electrical synapses, gap junctions, can similarly be mapped using the same approach. This is very important, because these synapses are much more difficult to identify with electron microscopy and are not currently included in the available Drosphila connectomes. Definitive mapping of gap junctions however will require further work, outside the scope of this study, because there are different gap junction proteins and individual gap junctions may be heterotypic, composed of two different proteins.

Finally, the authors extend this approach to address the important question of whether variations in behavior can be explained by differences in underlying synaptic connectivity. Using the neuronal circuit known to be responsible for the male fly courtship song, the authors show that the synaptic connectivity between pC2l and pIP10 neurons is correlated with a specific component of the optogenetically-elicited fly song.

The developed imaging and analysis pipeline includes software for visualization of multi-terabyte images, automated neuronal segmentation, detection and quantification of pre- and postsynaptic sites. As the authors point out, these tools could be useful for circuit analysis in other species as well. The different imaging and analysis pipelines are presented very well, with multiple examples that cover different scenarios, and are well validated. While with this method it is not possible to directly correlate the fluorescence signal with the underlying ultrastructure as seen with EM, and thus it cannot be confirmed that the detected synaptic connections correspond to ultrastructurally defined synapses, the authors have convincingly demonstrated that the proposed approach is precise enough to detect a similar number of synapses as EM studies of the same neurons, and that it is sensitive enough to detect changes in synapse numbers in different experimental conditions.

Reviewer #1 (Public Review):

Current generative models of protein sequences such as Potts models, Variational autoencoders, or autoregressive models must be trained on MSA data from scratch. Therefore, they cannot learn common substitution or coevolution patterns shared between families, and require a substantial number of sequences, making them less suitable for small protein families (e.g., conserved only for eukaryotes or viruses). MSA transformers are promising alternatives as they can generalize across protein families, but there is no established method to generate samples from them. Here, Sgarbossa et al. propose a simple recursive sampling procedure based on iterative masking to generate novel sequences from an input MSA. The sampling method has three hyperparameters (masking frequency, sampling temperature, and the number of iterations) which are set by rigorous benchmarking. The authors compare their approach to bmDCA, and evaluate i) single sample quality metrics ii) sample diversity and similarity to native sequences iii) similarity between original and generated sequence distribution, and iv) phylogeny/topology in sequence space of the generated distribution.

Strengths:

- The proposed sampling approach is simple.<br /> - The computational benchmarking is thorough.<br /> - The code is well organized and looks easy to use.

Weaknesses:

- There is no experimental data to back up the methodology.<br /> - It is not clear whether the sampling hyperparameter used is optimal for all protein sizes.<br /> - I am unsure that the bmDCA baseline method was trained appropriately and that the sampling method was adequate for protein design purposes (regular sampling).<br /> - Quality assessment of predicted structures is incomplete.<br /> - The proposed metrics for evaluating the diversity of generated sequences are fairly technical.

Impact assessment: The claim that MSA Transformer could be useful for protein design is supported by the computational benchmark. This work will be useful for researchers interested in applying MSA-Transformer models for protein design

Reviewer #1 (Public Review):

Our understanding of the early stages of myelination within the CNS is relatively rudimentary. In this manuscript the authors use selective cell labeling to visualize the initial interactions between individual oligodendrocytes and their target axons in the developing zebra fish with the goal of understanding the regulation of myelin sheath formation.

There are considerable strengths to the manuscript. The work extends earlier studies through the use of high spatial and temporal resolution analysis. This approach reveals a highly dynamic interaction between oligodendrocyte processes and local axons that had not previously been appreciated. The data on the initial interactions between an individual oligodendrocyte and its target axons is closely analyzed, which reveals a number of interesting traits. For example, while dorsal cells have a higher number of initial axonal interactions and ultimately myelinate more axons than ventral cells, the proportion of initial interactions that lead to a myelin sheath is similar between the two populations. To begin to examine the molecular regulation of the initial oligodendrocyte and axon interactions and subsequent formation of myelin sheaths the authors perturb selective components of the endocytic pathway and provide evidence that disruption of Rab5 selectively affects the long-term stabilization of myelin sheaths.

While there are some new advances in the current manuscript, the significance of many of the observations is unclear. For example, while the data documents extensive interactions between oligodendrocytes and axons, the nature of those interactions is not well defined. The authors describe the loss of olig/axon interactions as "ensheathment destabilization" however, it is not clear from the data that they don't represent simple oligodendrocyte process retraction.

The different interactions of dorsal and ventral cells with their target axons is interesting and may reflect different oligodendrocyte populations or environments.

Reviewer #1 (Public Review):

In this manuscript, the authors investigate the genes involved in the retention of eggs in Aedes aegypti females. They do so by identifying two candidate genes that are differentially expressed across the different reproductive phases and also show that the transcripts of those two genes are present in ovaries and in the proteome. Overall, I think this is interesting and impressive work that characterizes the function of those two specific protein-coding genes thoroughly. I also really enjoyed the figures. Although they were a bit packed, the visuals made it easy to follow the authors' arguments. I have a few concerns and suggested changes, listed below.

1. These two genes/loci are definitely rapidly evolving. However, that does not automatically imply that positive selection has occurred in these genes. Clearly, you have demonstrated that these gene sequences might be important for fitness in Aedes aegypti. However, if these happen to be disordered proteins, then they would evolve rapidly, i.e., under fewer sequence constraints. In such a scenario, dN/dS values are likely to be high. Another possibility is that as these are expressed only in one tissue and most likely not expressed constitutively, they could be under relaxed constraints relative to all other genes in the genome. For instance, we know that average expression levels of protein-coding genes are highly correlated with their rate of molecular evolution (Drummond et al., 2005). Moreover, there have clearly been genome rearrangements and/or insertion/deletions in the studied gene sequences between closely-related species (as you have nicely shown), thus again dN/dS values will naturally be high. Thus, high values of dN/dS are neither surprising nor do they directly imply positive selection in this case. If the authors really want to investigate this further, they can use the McDonald Kreitman test (McDonald and Kreitman 1991) to ask if non-synonymous divergence is higher than expected. However, this test would require population-level data. Alternatively, the authors can simply discuss adaptation as a possibility along with the others suggested above. A discussion of alternative hypotheses is extremely important and must be clearly laid out.

2. The authors show that the two genes under study are important for the retention of viable eggs. However, as these genes are close to two other conserved genes (scratch and peritrophin-like gene), it is unclear to me how it is possible to rule out the contribution of the conserved genes to the same phenotype. Is it possible that the CRISPR deletion leads to the disruption of expression of one of the other important genes nearby (i.e., in a scratch or peritrophin-like gene) as the deleted region could have included a promoter region for instance, which is causing the phenotype you observe? Since all of these genes are so close to each other, it is possible that they are co-regulated and that tweedledee and tweedledum and expressed and translated along with the scratch and peritrophin-like gene. Do we know whether their expression patterns diverge and that scratch and peritrophin-like genes do not play a role in the retention of viable eggs?

References:<br /> Drummond DA, Bloom JD, Adami C, Wilke CO, Arnold FH. 2005. Why do highly expressed proteins evolve slowly? Proc Natl Acad Sci U S A. 102:14338-14343.

McDonald JH, Kreitman M. 1991. Adaptive protein evolution at the Adh locus in Drosophila. Nature. 351:652-654. doi: 10.1038/351652a0.

Reviewer #1 (Public Review):

The rice sensor NLR protein Pik-1 carries a HMA domain to sense fungal AVR proteins. Past studies have shown that it is possible to modify the HMA domain to change new recognition specificity. However, whether this approach can generate broad-spectrum NLRs that function in rice plants remains to be shown. Prior work from the authors have shown that each of the existing Pik-1 alleles only recognizes some, but not all AVR-Pik alleles. Interestingly, they found that a natural rice target protein HIPP19 is capable of binding to all known AVR-Pik proteins. In the current study, the authors tested the idea that AVR-Pik-binding sequence in HIPP19 could be utilized to engineer Pik-1 protein with broader recognition specificity. Strikingly, the engineered Pikp-1OsHIPP19-mbl7 is capable of recognizing AVR-PikD, C, and F, whereas the original Pikp-1 is only capable of recognizing Avr-PikD. This is supported by both HR-elicitation and protein-protein interactions in N. benthamiana plants. The authors further used a structure-guided approach to identify specific amino acids responsible for expanded recognition of AVR proteins. To this end, they show that the Pikp-1SNK-EKE variant is capable of recognizing all three of the aforementioned AVR-Pik proteins. The proper interactions of the newly introduced amino acids with the Avr-Pik proteins were nicely demonstrated with structural work. Most excitingly, the Pikp-1OsHIPP19-mbl7 and Pikp-1SNK-EKE constructs were introduced in to rice plants lacking Pik-1 as stable transgenes. These lines displayed disease resistance to rice blast strains carrying any of the three AVR-Pik proteins. Overall, the study is well executed and shows how knowledge of structural and evolutionary studies can help engineering disease resistance in a major crop plant. The weakness is with the use of a strong promoter to drive the expression of the engineered Pikp-1 variants in rice and a lack of assessment of potential effects on traits.

Reviewer #1 (Public Review):

This is an awesome comprehensive manuscript. Authors start by sorting putative stromal cell-containing BM non-hematopoietic (CD235a-/CD45-) plus additional CD271+/CD235a-/CD45- populations to identify nine individual stromal identities by scRNA-seq. The dual sorting strategy is a clever trick as it enriches for rare stromal (progenitor) cell signals but may suffer a certain bias towards CD271+ stromal progenitors. The lack of readable signatures already among CD45-/CD45- sorts might argue against this fear. This reviewer would appreciate a brief discussion on number & phenotype of putative additional MSSC phenotypes in light of the fact that the majority of 'blood lineage(s)'-negative scRNA-seq signatures identified blood cell progenitor identities (glycophorin A-negative & leukocyte common antigen-negative). The nine stromal cell entities share the CXCL12, VCAN, LEPR main signature. Perhaps the authors could speculate if future studies using VCAN or LEPR-based sort strategies could identify additional stromal progenitor identities?

The authors furthermore localized CD271+, CD81+ and NCAM/CD56+ cells in BM sections in situ. Finally, referring to the strong background of the group in HSC research, in silico prediction by CellPhoneDB identified a wide range of interactions between stromal cells and hematopoietic cells. Evidence for functional interdependence of FCU-F forming cells is completing the novel and more clear bone marrow stromal cell picture.<br /> An illustrative abstract naming the top9 stromal identities in their top4 clusters by their "top10 markers" + functions would be highly appreciated.

Reviewer #1 (Public Review):

The present study used an innovative meta-analytic approach to elucidate the functional organization of the lateral prefrontal cortex (LPFC). Co-activation profiles based upon over 14,000 fMRI studies revealed a principle rostral-caudal gradient in the LPFC, as well as a secondary dorsal-ventral gradient. Rostral-ventral zones in this gradient tended to contain areas in cognitive control (Control B) and salience networks and were associated with terms involving memory and affect. Caudal-dorsal zones in the gradient tended to contain areas in cognitive control (Control A) and spatial attention networks and were associated with terms involving perception and action. Areas in-between overlapped prominently with a variety of networks including Control A and were associated with various cognitive terms associated with language, working memory, and cognitive control. Moreover, the authors found hemispheric asymmetries with the left hemisphere associated with language-related topics and the right hemisphere with response inhibition and error processing. Hemispheric differences did not show an obvious rostral-caudal topography. Collectively, the data provide quantification of the general organization of the LPFC along rostral-caudal, dorsal-ventral, and hemispheric axes. From the associations of networks and terms, the authors conclude that the rostral-caudal axis reflects an internal/external axis, with areas in the middle supporting integrative processing.

Detailing the functional organization of the LPFC has remained a challenge given the diversity of its functions and widespread involvement across various tasks. Due to the limitations of single studies in terms of what can be measured (i.e. number of tasks used), construct validity of what is measured (e.g. purity of contrasts), and the reliability and reproducibility with which things can be measured, a meta-analysis of this scale can provide a welcome synthesis.

A major challenge with meta-analyses of fMRI data is obtaining appropriate specificity. Most meta-analytic methods that have been applied to fMRI data are both spatially and functionally coarse, which hinders efforts to properly synthesize the literature. Here, the authors employ innovative techniques to maximize specificity insofar as possible. As a result, the present data can be considered our best summary to date of the functional organization of the LPFC as detailed by fMRI.

Even as the study has innovated over previous attempts, limitations of meta-analyses must still be considered. Meta-analysis will never have the spatial resolution of well-performed individual studies. Indeed, the techniques used here may cause spatial blurring given the impression of spatially ordered consistency which may not actually be present. For example, there are data to suggest that there may be multiple rostral-caudal axes along the LPFC, which can potentially be blurred together into a single axis here. So, the spatial organization detailed here may offer a gross overall picture of how the LPFC is organized, but we will naturally get more fine-grained details from carefully conducted individual studies.

Nevertheless, the approach used here is helpful not only for detailing the functional organization of the LPFC, but as a proof-of-concept that can be applied to future investigations. These techniques may be helpful for detailing the organization of other heteromodal zones of the brain such as the medial frontal wall, and parietal cortices, offering a means of distilling the thousands of fMRI studies that have been conducted into a comprehensive whole.

Reviewer #1 (Public Review):

Oppong and colleagues present a study on the association of mitochondrial DNA abundance in blood and personality traits, both of which have been linked to morbidity and mortality in aging populations. They found that mtDNAcn is negatively associated with traits related to neuroticism as well as positively with a higher personality-mortality index (PMI). The association of the PMI with mortality was attenuated by including mtDNAcn in the model, indicating that the association is mediated by mitochondrial abundance in blood.

General comments:<br /> • Previous studies have shown that mtDNAcn are potentially mediated by hormonal levels and thus menopause. Given the mean age of 57 in the SardiNIA cohort, the authors should investigate in more detail the potential confounding effects of menopause in women.<br /> • The only personality trait (out of the big five) available in the UK Biobank is neuroticism. Since the authors found that most of their associations are significant for this complex, I would strongly suggest they try to replicate their findings in patients from the UK Biobank which have both, genome-wide sequencing data as well the summary score of neuroticism (Data-Field 20127)<br /> • The amount of mtDNA varies across populations and across different haplogroups. The authors should therefore compute the major haplogroups present in Europeans and adjust/account for those variables in the correlation and mortality analyses.

Reviewer #1 (Public Review):

Liu et. al. applied an existing method to study the subtypes of CRC from a network perspective. In the proposed framework, the authors calculated the perturbation of expression-rank differences of predefined network edges in both tumor and normal samples. By clustering the derived perturbation scores in CRC tumors using publicly available gene expression datasets, they reported six subtypes (referred to as GINS 1-6) and then focused on the association of each subtype with clinical features and known molecular mechanisms and cell phenotypes. My recommendation is major revision.

Major concerns:

(1) While this study originates from the network-perspective, it is unclear to me if the new subtypes provide key novel insights into the gene regulatory mechanisms for the development of CRC. For example, the "Biological peculiarities of six subtypes" section is descriptive and lacks a punch point.

(2) To further demonstrate the novelty of the identified subtypes, the authors need to show the additional benefit of the GINS1-6 to patient stratification derived from existing methods, such as integrative clustering based on multiple genomic evidence (copy number alterations, gene expression and somatic mutations).

Reviewer #1 (Public Review):

The hippo signaling pathway has emerged as a key signaling pathway in cancer and many other diseases, but there is a lack of high-quality chemical tools that would enable functional studies. The developed chemical probe targeting TEAD is therefore a much-needed chemical tool enabling more functional studies on this pathway in diverse diseases. The chemical MYF-03-69 is comprehensively characterized and it, therefore, represents a high-quality probe for future studies.

Reviewer #1 (Public Review):

The manuscript "BRCA2 BRC missense variants disrupt RAD51-dependent DNA repair" by Jimenez-Sainz et al focuses on the characterization of three BRCA2 mutants that were previously classified as Variants of Uncertain Significance (VUS) with unknown functional consequences. Mutations in the BRCA2 tumor suppressor gene predispose to breast, ovarian, pancreatic, prostate, and other cancers and are responsible for nearly half of all hereditary breast cancers and ovarian cancers. Identification of truly pathogenic BRCA2 missense mutations is a challenging but very important task for early cancer diagnostics. In this study, the authors developed a methodology for the identification of pathogenic BRCA2 mutations. They performed comprehensive analyses of three BRCA2 mutations including S1221P and T1980I, which map to conserved residues in the BRC2 and BRC7 repeats, and T1346I, located within the spacer region between BRC2 and BRC3 repeats. Using an impressive array of cellular and biochemical approaches they demonstrated that the first two BRCA2 mutants have a detrimental effect on RAD52-dependent DNA repair, and therefore likely to be pathogenic. In contrast, T1980I seems to have no effect on DNA repair in various tested assays and is likely to be a passenger mutation.

Overall, I found the presented study of high quality. The developed methodology can be applied for analyses of other potentially pathogenic mutations in BRCA1, BRCA2, or other genes involved in DNA double-strand break repair. The work may have a broad impact on the biomedical field. The presentation quality is good as well.

Reviewer #1 (Public Review):

In this manuscript the authors found a direct synaptic connection between inhibitory neurons in the central nucleus of the amygdala and inhibitory and other neurons in the zona incerta. They conducted a rigorous and detailed anatomical study of both the anterograde and retrograde connections between PKCdelta CeA neurons and the zona incerta. Furthermore they conducted rigorous chemogenetic investigation of the zona incerta inhibitory neurons across pain modalities. This led to the overall conclusion that PKCdelta neurons inhibit zona incerta inhibitory neurons leading to enhanced pain processing. While the results mainly support the conclusions, there is a lack of direct support for the CeA-PKCdelta-->vGAT-ZI hypothesis.

Reviewer #1 (Public Review):

The authors use both genome-wide correlations between genetic effects on metabolite pairs ('genetic correlation') and the pleiotropic effects of individual genetic variants to build an understanding of how biochemical pathways relate to global ('genetic correlation') and local (individual variant or pathway) pleiotropy. The authors look at metabolites, which are themselves interesting and predictive of metabolic health, but also serve as a useful 'model system' for understanding genetic correlation.

The authors demonstrate that genetic variants that have 'discordant' effects on a pair of metabolites, i.e. effects whose product of signs is opposite to the sign of the genome-wide genetic correlation, tend to be variants (likely) affecting pathway-relevant enzyme or transporter genes and/or affect biochemical pathways 'between' the two metabolites.

The authors attempt to extend this further to a variant associated with coronary artery disease (CAD), which they hypothesize to act by decreasing the activity of the gene PCCB. While an interesting hypothesis, establishing such a mechanism in the etiology of CAD would require further validation.

This paper represents an advance in linking statistical genetics constructs such as 'genetic correlation' to a biochemical mechanism for an important case: metabolites. While I expect their approach to be influential in showing how to dissect genetic correlation in a way that can point to the biological mechanism, extending their method to more complex phenotypes with less well-characterized biochemical pathways may be challenging.

Reviewer #1 (Public Review):

In this study, the authors aim to analyze the functions of the motor subunit klc4 in nervous system development and function. This is an important question to address, as not much is known about the cellular functions of klc4 even though mutations in this gene cause early onset hereditary spastic paraplegia in human. The authors used CRISPR/Cas9 to generate a klc4 mutant in zebrafish and analyzed the development of sensory neurons in embryos as well as behavior in adults. The strengths of this study include the generation of a novel klc4 mutant in zebrafish, the use of high and super-resolution live microscopy over time coupled to a rigorous analysis to reveal unsuspected developmental defects in klc4 mutants, including the formation of aberrant projections by sensory neurons and an abnormal development of peripheral sensory axons that appear less branched and fail to repel each other. The behavioral assays conducted by the authors also yielded robust results supporting a role for klc4 in adult neural circuits regulating stress response. The data are very well quantified and support the key findings of the study. Although the study does not delineate the molecular mechanisms causing an abnormal development of sensory neurons, its findings have a high impact, as they suggest specific functions of Klcs in neuronal patterning and compartimentalization and identify klc4 as a novel gene associated with anxiety behavior.

Reviewer #1 (Public Review):

In the manuscript "Airway Basal Cells Show Regionally Distinct Potential to Undergo Metaplastic Differentiation" by Yizhou, Yang et al., the authors take an unbiased approach to interrogate basal cell heterogeneity in the trachea. Their single-cell RNA-seq data suggests that several sub-populations of basal cells exist. Follow-up studies support the conclusion that two major basal cell populations exist corresponding to the dorsal and ventral trachea. Strikingly, their functional data also supports that the microenvironment of the dorsal or ventral trachea, being surrounded by smooth muscle or cartilage respectively, and that loss of cartilage leads to aberrant patterning of BC1 and BC2. Overall, this is an interesting study with reasonable conclusions that are supported by the data, and, the data is clear and of high quality. One point that requires further discussion pertains to the KRT13 expression following injury, and whether calling KRT13 activation "aberrant" is appropriate if it is simply a part of the natural repair process.

Reviewer #1 (Public Review):

The present manuscript offers valuable transcriptomic data sets of manually picked adult zebrafish photoreceptors from dissociated retinas of different transgenic lines, in which rods and cones (UV, S, L, M) were marked by the fluorescent reporter proteins. This is a very valuable approach because allows for selecting "healthy cells". Whether the approach is comparable to single-cell RNA-seq as the authors do (see page 3 and discussion) is however questionable as each of their samples is composed of 20 cells.

The authors further focused on transcription factors that are differentially expressed in the five photoreceptors cell types that they analyze, identifying a large number of them with still unidentified functions. This is very valuable information. However, the idea that this analysis will help to identify new TF involved in the specification of the photoreceptors (as stressed in the title) is at odds with the experimental setup. The authors have analyzed adult photoreceptors and thus by definition cells that had been already specified. Many of the TF involved in the specification may no longer be expressed. The analysis rather offers a list of TFs that are involved in photoreceptor homeostasis, some of which had been also involved in their specification. Proof of this is the fact that none of the four TFs of yet uncharacterized function (Skor1a, Sall1a, Lrrfip1a, and Xbp1) turned out to be involved in photoreceptor specification. The F0 screen only confirmed factors that were already known to be involved in cell specification and that in adult photoreceptors likely play a different role.

The authors further investigate the activity of the two tbx2 zebrafish paralogues in photoreceptors' specification, showing a novel role for tbx2 in the repression of different opsin in specific photoreceptor cell types. This is an interesting finding, however, it is overinterpreted by the authors. Indeed, tbx2 cannot be considered as a "master regulator of photoreceptor fate" (page 7) but, at best, a TF is required to control an appropriate proportion of the different photoreceptors' subtypes.

Overall this is an interesting and well-performed study with valuable information. The conceptual framework of the study should however be re-elaborated, further avoiding overinterpretations.

Reviewer #1 (Public Review):

Kim and coauthors have performed multiple simultaneous whole cell recordings in living slices of human neocortex obtained from neurosurgical resection in order to study the properties of synaptic connections from excitatory pyramidal neurons onto various types of inhibitory interneurons. Strengths of the study include the unique ability to study biophysical properties of human synapses, and the sophisticated in situ hybridization and other approaches used to identify the class of the postsynaptic interneurons. The main finding of the study is that a key principle identified in rodent neocortex: that fast-spiking parvalbumin-positive neurons receive initially depressing synapses, whereas other categories of interneurons receive more initially facilitating synapses, is conserved in the human. The authors also make important technical contributions to our ability to study synapses in human tissue including a slice culture technique that prolongs the use of these valuable samples, and a multi-pronged approach to characterizing interneuron identity. The main weaknesses of the current version of the manuscript relate to incomplete analyses and a somewhat confusing presentation that leave in question the relative importance of interneuron identity vs. other factors in determining the degree of synaptic facilitation and depression.

Reviewer #1 (Public Review):

Ugrankar et al provide an interesting article exploring the impact of the actin network in adipocyte cell size and nutrient uptake. The manuscript is well written and presents gaps in current knowledge well. The authors use Drosophila to address their research questions, describing a specific isoform of actin, Actin 5C, as the critical mediator of lipid metabolism in the larval fat body. In support, they show that loss of a mediator of actin dynamics, twinfilin, can have similar impacts as actin 5C loss. The authors further probe for impacts of additional cytoskeletal proteins, spectrins, in this process, concluding that spectrin activity differs from Actin 5C. Last, the authors attempt to explore how actin network in the fat body impacts nutrient uptake in multiple ways. Overall, this is an interesting study that sheds light on adipocyte cytoskeletal dynamics. However, there are a number of concerns, including: a need to validate the many RNAi used, the need to add data to rule out a potential contribution from other actin isoforms, further characterization of the assays used to address nutrient uptake, and further validation of the data used to argue that actin 5C is not essential during embryogenesis.

Reviewer #1 (Public Review):

This study has some neat technological features that go a long way to reconcile contradictory data regarding functions of disease associated PTPN22 variants. These include:<br /> • Crispr/Cas9 gene editing of exon 14 of PTPN22 in primary human T cells to generate HDR for WT, and gene editing for risk and KO sequences<br /> • Use of cord blood T cells, mitigating against any variability in T cell responses that could be influenced by activation or differentiation state<br /> • Lentiviral infection of these T cells with high and low avidity TCRs that recognise the same peptide from the islet cell autoantigen IGRP, presented by HLA-DRB1*0401; the TCRs are chimeric, allowing detection of LV transgene and detection of TCRs that have not cross-paired with endogenous TCR chains<br /> • Cis-linked GFP to detect those T cells expressing TCR transgenes. Infection is undertaken using titres of virus likely to avoid high copy number TCRs and therefore variable TCR expression<br /> • Repeat experiments using multiple donors<br /> • TCR stimulations using a range of different readouts

The main findings and things to look out for are:<br /> • The HDR editing process leads to reduced expression of PTPN22 when compared to unedited/mock edited wild type T cells; thresholds of signalling are therefore different. But this is ok because expression of phosphatase in edited wild type and risk variants is equivalent, albeit at lower levels (Fig 1).<br /> • The technology inevitably leads to hemizygosity with biallelic editing events, and this needs to be born in mind when considering the homogeneity of T cell populations<br /> • The impact of the PTPN22 risk variant or phosphatase deficiency is uncovered under conditions of lower avidity/low signal strength, where loss of negative regulation leads to increased proliferation and cytokine production (IFN or IL-2)<br /> • Consistent with this PTPN22 regulates responses of T cells expressing low avidity L-TCR, but not high avidity H-TCR<br /> • Thus, the risk variant mimics the knockout, to a large extent

Additional things/experiments that might strengthen the study:<br /> • The claims of the authors might be further substantiated if they extended the range of T cell stimulatory readouts eg different cell surface markers such PD-1, OX-40, 41BB, ICOS, GPR56, whose expression is linked to TCR signalling thresholds<br /> • Additional signalling experiments such as phospho-flow using phospho-Erk specific antibodies would be a bonus; I worry a bit about only showing pS6 data<br /> • Repeat the experiments comparing wild type and ko T cells and study cytokine expression eg IFNg in non-risk edited and risk edited T cells. As it stands the only data we see comparing these genotypes is proliferation.

Reviewer #1 (Public Review):

The authors aimed at explaining the origin of the persistent activity observed in neural populations recorded from larval zebrafish, its dependence on the temperature of the water the fish was immersed in, and the effects of visual stimulation. They deploy a popular data-driven model to capture the statistical structure of large neural populations, fitting a maximum entropy model (Ising model) to the average activity and pairwise correlation of recorded neurons. Using mean field methods, they reduce this high-dimensional model to two dimensions, describing the average activities of populations in the left and right hemispheres. Both the high and low dimensional models are capable of generating the long timescale of persistent activity, even though they were only trained to learn the static mean and pairwise correlation structure. The crucial theoretical insight is that this long timescale emerges from the energy landscape of the reduced model in terms of stochastic transitions between metastable attractors following the well known Arrhenius law. The height of the barriers separating the attractors is modulated by water temperature, explaining the change in transition times and persistent activity. The model can also explain the dependence of persistent activity on the water temperature.

The major strength of the present work is that, by using a simple and well motivated statistical model (maximum entropy model) based on minimal assumptions, the authors are able to quantitatively reproduce complex spatiotemporal effects of fish behavior. The authors explain why this is the case due to the emergence of metastable dynamics based on stochastic transitions between local minima of the free energy. This classic model is very easily interpretable and of wide appeal for the neuroscience and larger life science community.

In my opinion, the current manuscript has three main weaknesses. The first one is that the model fit and its comparison to the data is not cross-validated and thus likely affected by overfitting. I strongly recommend recasting all results in terms of comparison of cross-validated observables. The second weakness is the fact that it is not explained how the water temperature appears in the model, which is the central quantity whose dependence they aim to model. There is a significant confusion on issues of water temperature vs. temperature in the model Gibbs measure. The author should make sure this point gets clarified. The third weakness is that, although the authors claim that the sign of the difference between the mean population activities of left and right hemispheres is the observables that determines whether the fish is going to change swimming directions, they don't actually provide direct evidence for this, but only compare the statistical distribution of this observable with the behavioral distribution. I recommend the authors explicitly test the predictive nature of the neural observable by showing that changes in swim directions are temporally aligned to the onset of a sign change.

If the results still stand after applying cross-validation, which I believe is a quite likely outcome, I believe this manuscript will have a strong impact in the field since they demonstrated the power of a principled and well-known approach in capturing complex spatiotemporal activity of large neural populations. This work has the potential to be widely adopted and generalized to many different directions in systems neuroscience and beyond.

Reviewer #1 (Public Review):

Carlos Serpa et al., build on prior work from their laboratory showing that the rat ventrolateral orbitofrontal cortex (OFC) is not involved in goal-directed action control per se, but is involved in the updating of such actions. Here they demonstrated that noradrenergic but not dopaminergic inputs within the OFC (and not the medial PFC) are necessary for action-updating in this manner. The conclusions are well supported by the data. Overall this is an excellent manuscript with many strengths and few weaknesses.

Strengths are as follows:<br /> 1. The manuscript is written beautifully<br /> 2. The rationale for the study is clear.<br /> 3. The data are mostly very solid. All the claims are statistically supported, not only by pairwise comparison statistics but also interactions. This is very important in ensuring robustness and replicability of effects.

Weaknesses<br /> 1. There are no major weaknesses. As a minor point, a clearer demonstration of precise anatomical placements would be helpful as the function of the OFC (and the medial PFC) can differ significantly with even small alterations in placement.

I think these data will be of interest to neuroscientists and possibly psychopharmacologists. It may also be of interest to researchers in other fields, such as clinicians, although it doesn't have extremely clear health implications, so clinician interest could be limited.

Reviewer #1 (Public Review):

The manuscript describes changes in single cell RNA sequencing signatures of dorsal root ganglion neurons over the development of neuropathic pain in the murine chronic constriction injury (CCI) model. Bioinformatic algorithms were employed to cluster neurons into sub-classes described previously, based on transcriptomic signatures. The authors report emergence of 4 new clusters, resulting from loss of cellular identity of neurons in known clusters and induction of inflammatory and hyperexcitability-associated genes. Moreover, they segregated neurons between injured and uninjured subclasses and observed induction of genes in both categories as well as differences between these categories. Some injured neurons maintained cellular identity. conversely, non-injured neurons were also found to show significant transcriptional plasticity in clusters with a prominent role in pain sensitivity. Sexual dimorphism was noted, particularly with respect to the c-LTMR class of sensory neurons.

The results of the current study are interesting, and the study is very well-performed. The fact that fluorescently labelled DRG neurons were employed here is an advantage since it led to lower representation of non-neuronal genes and better representation of neuronal genes expressed at low levels. However, it is largely descriptive and the level of advance beyond recent single cell transcriptomics studies on DRG neurons as well as older studies on bulk sequencing in models of neuropathic pain is debatable.

Reviewer #1 (Public Review):

This study analyzes the R-ISS-related plasma cell (PC) heterogeneity by 10X Genomics ScRNA sequencing and identified the two subsets of PCs(GZMA+ cytotoxic PCs and proliferating PCs). Three R-ISS-dependent gene modules in cytotoxic CD8+ T and NKT cells were also functionally analyzed. Potential immuno cell-cell communication such as SIRPA-CD47 and TIGIT-NECTIN3 were explored for the potential immunotargets which is an important direction for treating R/R MM. The work holds a promising way to study the drug resistance of R/R myeloma. However, the cost and complexity of the experimental method make it difficult to be widely used.

Reviewer #1 (Public Review):

SRSF6 is an understudied SR family member, best characterized for its role in controlling alternative splicing. Through comparative RNA-Seq analysis, the authors find that knockdown of SRSF6 results in a markedly different gene expression program than other SR proteins tested in that SRSF6 depletion leads to a dramatic increase in expression of interferon responsive genes (ISGs) and a downregulation of mitochondrial related genes. Given this correlation the authors explore the possibility that loss of SRSF6 leads to mitochondrial damage, which releases dsDNA to trigger the innate immune response through the DNA-sensor cGAS. They further propose that mitochondrial damage is due to a change in splicing of the gene BAX. The data shown in the manuscript are consistent with these conclusions, however do not rule out additional mechanisms. In particular, the mitochondrial and BAX phenotypes are much less dramatic than the interferon response. Moreover, the authors do not show that the change in BAX splicing induced by loss of SRSF6 is sufficient to lead to a change in ISG expression.

Reviewer #1 (Public Review):

This is an interesting study, addressing a timely question of the crosstalk between cancer, immune, and stromal cell populations in the tumor microenvironment, and the effect of therapy on the tumor microenvironment. The authors were aiming to show that the ratio between neutrophils and lymphocytes could predict treatment responses in pancreatic cancer. They indeed show that there is an association between the Neutrophil to lymphocyte ratio (NLR) and treatment outcome, suggesting that this could be a predictive marker. They go on to use a mouse model to perturb the NLR and combine this with treatment similar to that used in the clinic and find that targeting neutrophils affects tumor growth, suggesting a costive and not the only correlative role. Finally, they show that this could be mediated through the stromal compartment since this treatment affects the ratio of inflammatory to myofibroblastic CAFs.

The main strength of the paper is in tying together neutrophils, lymphocytes, and CAFs and showing how these populations affect each other. The correlations in human patients are promising and the regulation of CAF transitions is interesting.

While the correlation between NLR and survival is convincing and strong, the relevance of CAF transitions to this effect in human patients is weak, and shown only in mice and not in humans. Also in the mouse, the evidence for CAF transitions should be strengthened to support the authors' full conclusions.

Reviewer #1 (Public Review):

The manuscript by Lian et al. presents a population graph deep learning model constructed using Transformer-generated imaging features and non-imaging clinical characteristics that were proven to be effective at predicting the survival of patients with early-stage NSCLC. This study demonstrates GNN-based model significantly outperforms the TNM model and ResNet-Graph model in predicting survival in all datasets. The paper is well-written, clear for a general audience, takes nice innovations in computer vision into the medical field, and presents a usable tool for survival analysis. The strengths and limitations of the approach are brought forth in the discussion.

Reviewer #1 (Public Review):

This publication shows a strong understanding and implementation of large-scale multiprotein MD simulations. It is the first application of MD simulations to full-length membrane-bound TSHR. The authors showed that the LR is intrinsically disordered, contrasting a previously published homology model. Some simulation results are supported by cryo-EM structures. Finally, it is significant that the inclusion of TSH in the binding site altered the dynamics of the LR region, supporting a hypothesis that the LR is involved in a signaling mechanism, though the authors acknowledge this result as preliminary.

Weaknesses:<br /> The methods section lacks sufficient detail, and arbitrary choices made in the simulation setup may have biased the results. The author's finding that the LR is disordered does not provide obvious mechanistic insights, and the simulations with the bound ligand are too preliminary to make solid conclusions. Although this manuscript is technically strong, the significance of the results is often unclear.

Reviewer #1 (Public Review):

Wang et al., developed a CRISPR/Cas 9 based protocol with the aim to accurately and quickly detect bacteria in ICU patients with severe pneumonia.

The development of such a tool is important as quick and reliable identification of pathogens is extremely important. This study is innovative and aims to address an important clinical problem. The authors de novo designed an algorithm to screen species-specific . Then they used the species specific DNA tags to identify 10 pathogens.

1) It is not very clear on which epidemiological data these pathogens were selected on. Moreover, the selected pathogens are only bacteria.

2) Page 9. It is not very clear on how the primers' specificity was evaluated.

3) Page 9. Were patients on antibiotics before getting into the trial?

4) Page 10 At which timepoint the patients received different treatment based on the results of the culture or SSBD? Was this consistent?

5) Page 11. The second sentence of 3.1 section in results is not clear.

6) How were patients allocated to groups? Randomised?

7) The table describing the patient cohort is in supplementary. This shall be in the main manuscript. It seems that the control and experimental groups were not balanced.

8. The exact protocol of the study needs to be in the supplementary.

9. Were any samples poly-microbial?

10. Which was the threshold level of fluorescence (Figure 3) which was considered important?

Reviewer #1 (Public Review):

In this paper, Gao et al report that Kiaa1024L/Minar2 causes hearing loss in mice and in zebrafish. The animal studies are well executed. Mechanistically, the authors claim that Kiaa1024L/Minar2 is responsible for the enrichment of an accessible pool of cholesterol in the hair bundle membrane. Increasing cholesterol levels rescues hair cell defects whereas decreasing cholesterol aggravates the problem.

Unfortunately, the mechanistic arm of this study doesn't go beyond this correlation. The characterization of cholesterol levels and pools is not rigorous and it is unclear why cholesterol matters for hearing.

Reviewer #1 (Public Review):

The authors examined the impact of pre-gravid obesity in human mothers on the monocytes of newborns by collecting umbilical cord blood. Additionally, the authors also used a non-human primate (NHP) model of diet-induced obesity to isolate fetal macrophage and assess the impact of maternal obesity on fetal macrophage function.

The comprehensive analysis of the human umbilical cord blood monocytes by studying cytokine release, bulk RNA-seq and bulk ATAC-seq, single cell RNA-seq and single cell ATAC-seq, responses to pathogen stimulation as well as metabolic studies such as glucose uptake are major strength of the work. They present convincing evidence that the monocytes of offspring with obese mothers have epigenetic and transcriptomic profiles consistent with impaired immune responses, both during baseline conditions and upon stimulation.

However, it is not clear from the data how the epigenetic data and the transcriptomic data are related to each other. The implication that the epigenetic changes drive the downstream transcriptional differences is not clearly demonstrated. Furthermore, it is not clear which of the observed attenuations of monocyte transcriptional responses overlap with chromatin accessibility differences. Such an overlap would make a stronger case for the mechanistic link.

The increased phagocytosis of E.coli in umbilical cord monocytes of newborns with obese mothers appear counter-intuitive because it implies greater host defense capacity.

One of the most remarkable aspects of the manuscript is the analysis of the fetal macrophages in a non-human primate (NHP) model of diet induced obesity because of the challenge of studying fetal macrophages in humans. The cytokine assays nicely show that the fetal macrophages in the obesity model show impaired cytokine production, consistent with what was seen in the umbilical cord blood monocytes of human newborns. This is especially important because circulating monocytes or monocyte progenitors seed the fetal tissues and give rise to fetal macrophages, thus elegantly linking the human work on circulating umbilical cord blood monocytes to the tissue macrophages in the NHP model.

However, the NHP studies do not show any additional macrophage characterization beyond the cytokine assays. Flow cytometry analysis of the macrophage phenotype and functional assays would strengthen the conclusions regarding macrophage dysregulation.

Reviewer #1 (Public Review):

The authors endeavored to determine molecular pathways that could enhance the viability and function of MSCs. The authors identified the master anti-oxidant regulator NRF2 as a direct regulator of DKK1, a Wnt pathway inhibitor. Moreover, the authors demonstrate over expression of NRF2 and DKK1 ameliorates liver regeneration in a model of acute on chronic liver failure. The strengths of this study are their multi-tier approach utilizing molecular biology, genetic interventions and in vitro and vivo models. These findings have uncovered a novel signaling loop with the potential for enhancing MSC function in vivo.

Reviewer #1 (Public Review):

Bacterial carboxysomes are compartments that enable the efficient fixation of carbon dioxide in certain types of bacteria. A focus of the current work is on two protein components that provide spatial regulation over carboxysomes. The McdA system is an ATPase that drives the positioning of carboxysomes. The McdB system is essential for maintaining carboxysome homeostasis, although how this role is achieved is unclear. Previous studies, by the lead author's lab, showed that the McdB system is a driver of phase separation in vitro and in cells. They proposed a putative connection between McdB phase separation and carboxysome homeostasis. The central premise of the current work is as follows: In order to understand if and how phase separation of McdB impacts carboxysome homeostasis, it is important to know how the driving forces for phase separation are encoded in the sequence and architecture of McdB. This is the central focus of the current work. The picture that emerges is of a protein that forms hexamers, which appears to be a trimer of dimers. The domains that drive that the dimerziation and trimerization appear to be essential for driving phase separation under the conditions interrogated by the authors. The N-terminal disordered region regulates the driving forces for phase separation - referred to as the solubility of McdB by the authors. To converge upon the molecular dissections, the authors use a combination of computational and biophysical methods. The work highlights the connection between oligomerization via specific interactions and emergent phase behavior that presumably derives from the concentration (and solution condition) dependent networking transitions of oligomerized McdB molecules.

Having failed to obtain specific structural resolution for the full-length McdB as a monomer or oligomer, the authors leverage a combination of computational tools, the primary one being iTASSER. This, in conjunction with disorder predictors, is used to identify / predict the domain structure of McdB. The domain structure predictions are tested using a limited proteolysis approach and, for the most part, the predictions stand up to scrutiny affirming the PONDR predictions. SEC-MALS data are used to pin down the oligomerization states of McdB and the consensus that emerges, through the investigations that are targeted toward a series of deletion constructs, is the picture summarized above.

Is the characterization of the oligomerization landscape complete and likely perfect? Quite possibly, the answer is no. Deletion constructs pose numerous challenges because they delete interactions and inevitably impose a modularity to the interpretation of the totality of the data. Accordingly, we are led to believe that the N-terminal IDR plays no role whatsoever in the oligomerization. Close scrutiny, driven by the puzzling choice of nomenclature and the Lys to Gln titrations in the N-terminal IDR raise certain unresolved issues. First, the central dimerization domain is referred to as being Q-rich. This does not square with the compositional biases of this region. If anything is Q/L or just L-rich. This in fact makes more sense because the region does have the architecture of canonical Leu-zippers, which do often feature Gln residues. However, there is nothing about the sequence features that mandates the designation of being Q-rich nor are there any meaningful connections to proteins with Q-rich or polyQ tracts. This aspect of the analysis and discussion is a serious and erroneous distraction. Back to the middle region that drives dimerization, the missing piece of the puzzle is the orientation of the dimers. One presumes these are canonical, antiparallel dimers. However, this issue is not addressed even though it is directly relevant to the topic of how the trimer of dimers is assembled. If the trimer is such that all binding sites are fully satisfied (with the binding sites presumably being on the C-terminal pseudo-IDR), then the hexamer should be a network terminating structure, which it does not seem to be based on the data. Instead, we find that only the full-length protein can undergo phase separation (albeit at rather high concentrations) in the absence of crowder. We also find that the driving forces for phase separation are pH dependent, with pH values above 8.5 being sufficient to dissolve condensates. Substitution of Lys to Gln in the N-terminal IDR leads to a graded weakening of the driving forces for phase separation. The totality of these data suggest a more complex interplay of the regions than is being advocated by the authors. Almost certainly, there are complementary electrostatic interactions among the N-terminal IDR and C-terminal pseudo IDR that are important and responsible for the networking transition that drives phase separation, even if these interactions do not contribute to hexamer formation. The net charge per residue of the 18-residue N-terminal IDR is +0.22 and the NCPR of the remainder is ≈ -0.1. To understand how the N-terminal IDR is essential, in the context of the full-length protein, to enable phase separation (in the absence of crowder), it is imperative that a model be constructed for the topology of the hexamer. It is also likely that the oligomer does not have a fixed stoichiometry.

Therefore, the central weakness of the current work is that it is too preliminary. A set of interesting findings are emerging but by fixating on Lys to Gln titrations within the N-terminal IDR and referring to these titrations as impacting solubility, a premature modular and confused picture emerges from the narrative that leaves too many questions unanswered.

The work itself is very important given the growing interest in bacterial condensates. However, given that the focus is on understanding the molecular interactions that govern McdB phase behavior - a necessary pre-requisite in the authors minds for understanding if and how phase separation impacts carboxysome homeostasis - it becomes imperative that the model that emerges be reasonably robust and complete. At this juncture, the model raises far too many questions. The MoRF analysis is distraction away from the central focus.

The problem, as I see it, is that the authors have gone down the wrong road in terms of how they have interpreted the preliminary set of results. Further, the methods used do not have the resolution to answer all the questions that need to be answered. Another issue is that a lot of standard tropes are erected and they become a distraction. For example, it is simply not true that in a protein featuring folded domains and IDRs it almost always is the case that the IDR is the driver of phase transitions. This depends on the context, the sequence details of the IDRs, and whether the interactions that contribute to the driving forces for phase separation are localized within the IDR or distributed throughout the sequence. In McdB it appears to be the latter, and much of the nuance is lost through the use of specific types of deletion constructs.

Overall, the work represents a good beginning but the data do not permit a clear denouement that allows one to connect the molecular and mesoscales to fully describe McdB phase behavior. Significantly more work needs to be done for such a picture to emerge.

Reviewer #1 (Public Review):

Drosophila ovarian follicle cells have been utilized as a model system to study organogenesis and tumorigenesis of epithelia. Studies have found that lack of proper cell polarity causes invasive delamination of cells and formation of multilayered epithelia, reminiscent of Epithelial-Mesenchymal Transition (EMT). Using this system, the authors analyzed the single-cell transcriptome of follicle cells and show that distinct cell populations emerge shortly after induction of polarity loss. Authors identified dynamic activation of Keap1-Nrf2 pathway Finally, subpopulation classification and analysis of regulon activity identified that Keap1-Nrf2 pathway is responsible for epithelial multilayering caused by polarity loss.

Strengths: The authors characterized the single-cell transcriptome of follicle cell subpopulations after induction of polarity loss. Using temperature-inducible driver, they can induce the polarity loss in a short period of time, which enables detection of epithelial populations in various transition stages. Detected cell-heterogeneity could be caused intrinsically or by environmental cues within in vivo tissue. Therefore, it is likely well recapitulating tumorigenesis in vivo.

Weaknesses:<br /> 1) Authors should show cells corresponding to identified key cell clusters within the tissue by immunostaining, GFP-trap, or RNA FISH.<br /> 2) Images are low magnification and difficult to see individual cells.<br /> 3) Manuscript is written weighted toward the technical aspect and more biology behind this study has to be discussed.

Reviewer #1 (Public Review):

The authors' results revolutionize our understanding of the mechanism of arrestin-mediated GPCR internalization. They identified previously unknown elements on the non-receptor-binding side of arrestins participating in the process. The findings are ground-breaking and very important to the large field of GPCR signaling.

Reviewer #1 (Public Review):

Previous studies have linked several lifestyle-related factors, such as body mass index and smoking, alcohol use with accelerated biological aging measured using epigenetic clocks, however, most of them focused on single lifestyle factors based on cross-sectional data from older adults. The current study has a couple of major strengths: it has a decent sample size, lifestyle was measured longitudinally during puberty and adolescence, it looked at the effect of multiple lifestyle measures collectively, it looked at multiple epigenetic clocks, and due to the data from twins, it could examine the contribution of genetic and environmental influences to the outcomes. I have a couple of comments that are mainly aimed at improving the clarity of the methods (e.g. how was multiple testing correction done, how did the association model account for the clustering of twin data, how many samples were measured on 450k vs EPIC and were raw or pre-QC'd data supplied to the online epigenetic age calculator), and interpretation of findings (why were 2 measures of Dunedin PACE of aging used, how much are results driven by BMI versus the other lifestyle factors, and the discussion on shared genetic influences should be more nuanced; it includes both pleiotropic effects and causal effects among lifestyle and biological ageing).

Reviewer #1 (Public Review):

This manuscript reports a systematic study of the cortical propagation patterns of human beta bursts (~13-35Hz) generated around simple finger movements (index and middle finger button presses).

The authors deployed a sophisticated and original methodology to measure the anatomical and dynamical characteristics of the cortical propagation of these transient events. MEG data from another study (visual discrimination task) was repurposed for the present investigation. The data sample is small (8 participants). However, beta bursts were extracted over a +/- 2s time window about each button press, from single trials, yielding the detection and analysis of hundreds of such events of interest. The main finding consists of the demonstration that the cortical activity at the source of movement related beta bursts follows two main propagation patterns: one along an anteroposterior directions (predominantly originating from pre central motor regions), and the other along a medio-lateral (i.e., dorso lateral) direction (predominantly originating from post central sensory regions). Some differences are reported, post-hoc, in terms of amplitude/cortical spread/propagation velocity between pre and post-movement beta bursts.

Several control tests are conducted to ascertain the veracity of those findings, accounting for expected variations of signal-to-noise ration across participants and sessions, cortical mesh characteristics and signal leakage expected from MEG source imaging.

One major perceived weakness is the purely descriptive nature of the reported findings: no meaningful difference was found between bursts traveling along the two different principal modes of propagation, and importantly, no relation with behavior (response time) was found. The same stands for pre vs. post motor bursts, except for the expected finding that post-motor bursts are more frequent and tend to be of greater amplitude (yielding the observation of a so-called beta rebound, on average across trials).

Overall, and despite substantial methodological explorations and the description of two modes of propagation, the study falls short of advancing our understanding of the functional role of movement related beta bursts.

For these reasons, the expected impact of the study on the field may be limited. The data is also relatively limited (simple button presses), in terms of behavioral features that could be related to the neurophysiological observations. One missed opportunity to explain the functional role of the distinct propagation patterns reports would have been, for instance, to measure the cortical "destination" of their respective trajectories.

Reviewer #1 (Public Review):

The transcriptome of the cells of the human meniscus have been studied in bulk or superficially via single cell methods. In this study, the authors profile the types of cells present in the normal/healthy human meniscus as well as samples from degenerative menisci using single cell RNA seq. Using pre-existing analysis packages for single cell RNA seq data, they infer the roll of the various cell type clusters that they have identified and posit which cells interact with which cells as part of the healthy meniscus and in disease. They have developed an on-line viewer to facilitate use of these data by other research groups.

Strengths: The data has been rigorously collected and appropriate quality control steps have been implemented to ensure the veracity of the data. The result is a robust data set. This is coupled with the on line viewer portal they have created, allowing the data to be available in the public domain. Further, having this tool is a huge resource as it means that the end user does not need to have advanced programing skills to be able to use it. Some of the RNA seq results have been validated via in situ and immunofluorescence. The authors have compared their results to data already published and discuss disagreements.

Weaknesses: Some of the conclusions are very over reaching. The function of clusters, the role of cells and the interactions between cells are all inferred results based on data analyses. These results gave not been experimentally validated.

Reviewer #1 (Public Review):

In this manuscript, Siepe et al. developed a high-throughput screen designed to identify novel protein-protein interactions in the extracellular human proteome. Their CRISPRa-based method induced the expression of transmembrane receptors such that they could be screened for binding to proteins of interest. Major strengths of this approach include the ability to screen multiple ligands in parallel, the ability to identify low-affinity interactions, and the availability of custom single- and multi-pass transmembrane protein libraries for selective target screening. A potential weakness is that low-affinity binders and non-specific interactions can be difficult to distinguish in certain cases, and these scenarios require more complex statistical analysis. The authors also note that the CRISPRa strategy cannot induce the expression of multi-subunit receptors that may be required for some ligands. The screen was tested against a curated set of ligand candidates and identified more than twenty novel interactions with intriguing biological implications. Both the method and newly discovered interactions will be of immediate scientific interest given the growing need to identify receptors for orphan ligands. Overall, this technology should function as a powerful new tool for ligand deorphanization in the extracellular space.

Reviewer #1 (Public Review):

The authors provide insight into which regions of the ribozymes are involved in pairings including some tertiary interactions. Overall, the data support known structures and give insight into the roles of bases as pairs, catalytic residues, and extensions. The epistasis analysis is novel and gives deeper insight than previous mutational analyses of ribozymes. However, more can be extracted from this data. This study will impact the field by helping classify the roles of possible bases. There are also numerous technical issues that must be addressed. The authors should consider why short and long pairings show different epistasis and discuss the robustness of pairings from an evolutionary point of view. The effect of the primer binding site on ribozyme activity needs to be discussed.

Reviewer #1 (Public Review):

In the current manuscript, Bolte et al., examine how a single TBI alters the heterogeneity of dorsal meningeal immune cell responses and whether age at the time of injury affects long-term transcriptional profiles of this immune compartment of the brain. Multiple complementary approaches were undertaken to achieve high resolution of meningeal transcriptional response(s) to TBI including single-cell sequencing and bulk tissue sequencing. Several innate and adaptive immune phenotypes were quantified at the protein level, demonstrating these disease-associated responses are not solely relegated to transcriptional responses. The majority of the methods and analyses are robust, which is a notable strength of the manuscript. In its current iteration, a weakness is a lack of integration between gene sets that define meningeal immune cell subsets in the single cell data (e.g. Macrophages, Tcells, Bcells, Fibroblasts, etc.) and quantifying these DEGs (up or down-regulated) to examine whether the transcripts are altered in the chronic TBI/aging bulk sequencing data. A more thorough integration of these two datasets and their discussion would significantly bolster the main premise of the manuscript related to the resolution of inflammatory responses to TBI in the young versus the aged condition, chronically.

Reviewer #1 (Public Review):

In this study, Menjivar et al. examine the specific role of the enzyme arginase 1 (Arg1), which is expressed in immunosuppressive macrophages and catabolizes arginine to ornithine, in pancreatic cancer. They use an elegant genetic approach that leverages a dual recombinase-based genetically engineered mouse model of pancreatic cancer, which efficiently deletes Arg1 and recovers extracellular arginine in cultured macrophages. Within the pancreas, macrophage Arg1 deletion increased T cell infiltration and fewer mice developed invasive pancreatic cancer. Interestingly, when tumors did develop, the authors observed that compensatory mechanisms of arginine depletion were induced, including Arg1 overexpression in epithelial cells identified as tuft cells or Arg2 overexpression in macrophages. To overcome these compensatory mechanisms, pharmacological targeting of arginase was tested and found to increase T cell infiltration and sensitize to immune checkpoint blockade, suggesting this is a promising approach for pancreatic cancer.

Strengths:

This is a very rigorous, well-designed study and the findings are broadly interesting for the metabolism, immunometabolism, and pancreatic cancer communities. The methods are comprehensive and the experimental details in the legends are complete.

Weaknesses:

The claim that Arg1 deletion in macrophages delayed the formation of invasive disease is not completely justified by the data presented. Only a small number of mice are analyzed, and no statistics are included. Moreover, the abstract does not comprehensively summarize the findings. Many findings, including compensatory upregulation of ARG1 in tuft cells and ARG2 in myeloid cells, are not mentioned, nor was the rationale for the pharmacological approach. Finally, the claim that their data demonstrate that Arg1 is more than simply a marker of macrophage function. While this is the first time this has been examined in pancreatic cancer, a general role for Arg1 and arginine metabolism by myeloid cells in immunosuppression has already been established by multiple studies, including those cited by the authors, in multiple tumor types. This is an overstatement of the findings.

Reviewer #1 (Public Review):

The article by Solvi and colleagues aims to investigate what type and degree of information (either absolute, relative, or a weighted combination of both) is used by bumblebees when retrieving the value of an item. The authors reported recent evidence in humans and birds that suggest they seem to use a combination of absolute memories and remembering of subjective ranking, and an absence of relevant studies for other species, including invertebrates.

Thus, the authors conducted four different experiments to study what type of information is guiding the decision of bumblebees when facing different qualitative and quantitative comparisons.

In the first two experiments, the authors reported the use of relative ranking of stimuli instead of a memory of their absolute value. According to the authors, these results are confirmed by experiment three, where bees were presented with two equally-ranked choices which, in fact, were not treated as different by bees. In the last experiment, bumblebees showed a preference for the highest rank item.

Despite the presentation of well-designed experiments, the conclusions that bumblebees are using only memories of ordinal comparisons, thus showing a different strategy with respect to humans and birds, seems to not be fully supported by the results. The behaviour on the first two experiments, for instance, could be explained by a recency effect, where the higher item of the last comparison is better retrieved (the work of Giurfa on transitive inferences in bees was not mentioned, though is relevant here). Furthermore, in the last experiment, bumblebees could not have used an ordinal ranking; their choice for the higher-ranking item could be based on its higher absolute quantitative value in terms of sucrose solution.

The different behaviours and strategies used by bees here could be better explained by differences in the experimental task proposed, rather than supporting a general statement about the evolution of different strategies in comparison to other species.

Reviewer #1 (Public Review):

Li et al. use biochemical binding analysis combined with deletions/mutations to demonstrate that the bottom helix of the Rph3A C2B domain directly interacts with the first 10 residues (N-peptide region) on SNAP25, and this interaction is amplified by the intramolecular interaction of the C2B domain with RAB-binding domain. They establish the functional relevance of this interaction using live-cell imaging of dense-core vesicle exocytosis in neuroendocrine PC12 cells and in vitro SNARE assembly assay. They propose that the Rph3A binding to SNAP25 pre-structures the protein to efficiently assemble with Syntaxin and VAMP2, and thus, promoting the vesicle docking and priming process. This is a systematic analysis that clarifies the role of Rph3A in regulated exocytosis and provides novel insight into the underlying molecular mechanisms.

Manfred wants to be left alone

No one knows where this comically large helmet came from

The Prince of otranto has a father, manfred and the queen his mother and was preparing to get married to matilda then the day of the wedding he is killed by a comically large helmet

Reviewer #1 (Public Review):

This work focuses on the mechanisms that underlie a previous observation by the authors that the type VI secretion system (T6SS) of a Pseudomonas chlororaphis (Pchl) strain can induce sporulation in Bacillus subtilis (Bsub). The authors bioinformatically characterize the T6SS system in Pchl and identify all the core components of the T6SS, as well as 8 putative effectors and their domain structures. They then show that the Pchl T6SS, and in particular its effector Tse1, is necessary to induce sporulation in Bsub. They demonstrate that Tse1 has peptidoglycan hydrolase activity and causes cell wall and cell membrane defects in Bsub. Finally, the authors also study the signaling pathway in Bsub that leads to the induction of sporulation, and their data suggest that cell wall damage may lead to the degradation of the anti-sigma factor RsiW, leading to activation of the extracellular sigma factor σW that causes increased levels of ppGpp. Sensing of high ppGpp levels by the kinases KinA and KinB may lead to phosphorylation of Spo0F, and induction of the sporulation cascade.

The findings add to the field's understanding of how competitive bacterial interactions work mechanistically and provide a detailed example of how bacteria may antagonize their neighbors, how this antagonism may be sensed, and the resulting defensive measures initiated.

While several of the conclusions of this paper are supported by the data, additional controls would bolster some aspects of the data, and some of the final interpretations are not substantiated by the current data.

- The Bsub signaling pathway that is proposed is intricate and extensive as shown in Fig 5A. However, the data supporting that is very sparse:<br /> a) The authors show no data showing that the proteases PrsW and/or RasP, or the extracellular sigma factor σW are necessary, or that the cleavage of RsiW is needed, for induction of sporulation - this could presumably be tested using mutants of those genes.<br /> b) Similarly, they don't demonstrate that the levels of ppGpp increase in the cell upon exposure to Pchl.<br /> c) There is some data showing that kinA and kinB mutants don't induce sporulation (Fig supplement 7A), but that is lacking the 'no attacker' control that would demonstrate an induction.<br /> d) There is some data showing that RsiW may be cleaved (Fig 5C, D), but that data would benefit from a positive control showing that the lack of YFP foci is seen in a condition where RsiW is known to be cleaved, as well as from a time-course showing that the foci are present prior to the addition of Tse1, and then disappear. As it is shown now, it is possible that the addition of Tse1 just blocks the production of RsiW or its insertion into the membrane (especially given the membrane damage seen). Further, there is no data that the disappearance of the YFP loci requires the proteases PrsW and /or RasP - such data would also support the idea that the disappearance is due to cleavage of RsiW.<br /> - The entire manuscript suggests that T6SS is solely responsible for the induction of sporulation. While T6SS does appear to play a major part in explaining the sporulation induction seen, in the absence of 'no attacker' controls for Fig. 2A, it is impossible to see this. From the data shown in Fig. 2C, and figure supplement 2A, the 'no attacker' sporulation rate seems to be ~20%, while the rate is ~40% with Pchl strains lacking T6SS, suggesting that an additional factor may be playing a role.

Reviewer #1 (Public Review):

The authors have determined the structure of OmcZ cytochrome nanowires of Geobacter sulfurreducens by Cryo-EM.

OmcZ represents the third cytochrome nanowire of Geobacter to be structurally resolved. The structure reveals an octaheme cytochrome which oligomerizes to form an extended filament which scaffolds a continuous chain of hemes which serves to support long-range electron transport to terminal electron acceptors.

Previously identified nanowires which have been structurally resolved consisted of oligomers of OmcS and OmcE which, although lacking significant sequence identity, shared a common heme arrangement along the filament/nanowire.

OmcZ differs structurally from OmcS and OmcE, possessing a notably different heme chain configuration. OmcZ also differs from OmcS/OmcE in the nature of the interactions at the interface between subunits. Whilst in OmcS/OmcE a terminal heme is ligated by a histidine from the adjacent subunit of the wire, in OmcZ the terminal heme is ligated by a histidine within the same subunit, highlighting yet another difference between OmcZ and OmcS/E.

Based upon these observations, the authors suggest that OmcS and OmcE evolved from a common ancestor and that OmcZ evolved independently of OmcS/E. This is significant as it not only reveals the diversity of cytochrome nanowires which support long range electron transfer in Geobacter but also demonstrates that this mechanism of EET has potentially evolved multiple times and is likely to be exploited by other environmental microbes which utilize extracellular electron transport to support respiration.

Manuscript Strengths:<br /> The manuscript presents a solid detailed structural analysis of OmcZ providing new insight into the diverse range of electron transfer pathways utilized by Geobacter. By comparing OmcZ with other cytochrome nanowires of Geobacter (OmcS/OmcE) and with other electron transfer proteins such as the MtrABC complex, additional insight is gained into potential electron transfer properties of this cytochrome nanowire.

Manuscript Weaknesses:<br /> The manuscript compares previous characterisations of OmcZ filaments by X-ray scattering/IR nanospectroscopy prepared at pH 2 and pH 7 which indicated a higher percentage of alpha-helices and beta-sheets than what was observed by Cryo-EM from filaments prepared at pH 10.5 (this study). Due to the differences observed, it is suggested these previously utilized techniques are unreliable. Although there is a substantial difference in the proportion of beta-sheet that is observed/indicated between different methods, without a direct comparison available at the same pH it is perhaps not possible to attribute differences to the techniques alone.

Manuscript Impact:<br /> Through this work, the authors have made a significant contribution to the knowledge surrounding the electron transfer processes of Geobacter. Based on the structure obtained, they have sought to rationalise observed phenotypes associated with the different cytochrome nanowires and intriguingly propose how OmcZ may allow for more conductive biofilms through the formation of meshes of OmcZ filaments capable of exchanging electrons at solvent exposed hemes.

This manuscript will be of interest to scientists working across a range of disciplines including environmental microbiologists studying microbially driven redox processes in the subsurface, biochemists studying electron transfer proteins/pathways and in particular those working on extracellular electron transfer, and biotechnologists seeking to exploit bacterial electron transfer processes for biotechnological applications.

Reviewer #1 (Public Review):

This is an interesting paper, which has used cutting-edge approaches (DMS and ML) to probe an important phenomenon in protein function, namely allostery. The paper managed to acquire a large volume of data and to use this data efficiently to train ML models, which are then used to probe the question of why are some regions "allosteric" hot spots. The results are interesting and novel and suggest that despite structural homology, hotspot regions can differ among relatively close relatives, nevertheless, there are common mechanisms underpinning the allosteric mechanisms, likely linked to the conformational sampling of the proteins.

Strengths - To me, the strengths of the paper are predominantly in the experimental work, there's a huge amount of data generated through mutagenesis, screening, and DMS. This is likely to constitute a valuable dataset for future work. The experimental data allows mapping of the hotspots and much of the paper would be the same in terms of analysis without the ML, I think the experimental work with structural and sequence analysis would probably constitute a complete and impactful study alone, such is the quality. The ML obviously adds another layer of insight into the project. What is shown is that training on one homolog can allow the prediction of hotspots on related homologs. To some degree, this is as expected given these proteins share a common fold and function, yet the fact it is possible (albeit imperfect) despite quite a low sequence identity is notable.

Weaknesses - it is hard to describe this as a weakness, but the ML is obviously not perfect in the predictions, yet is still interesting. I don't have any major suggestions for revisions or changes - it is what it is and I think serves as a nice benchmark for follow-up studies with new methods and approaches. I think this reiterates the importance that the raw data is made available so that it can be used to benchmark alternative approaches and help advance the field. Scientifically, I think what is perhaps missing, and I don't want this to be misconstrued as a request for additional work, is a deeper analysis of the structural and dynamic molecular basis for the observations. In some ways, the ML is used to replace this and I think it doesn't do as good a job. It is clear for example that there are common mechanisms underpinning the allostery between these proteins, but they are left hanging to some degree. It should be possible to work out what these are with further biophysical analysis. To me, it is clear what we see here is likely some conservation in the dynamics of these proteins across the superfamily, and the allosteric mechanism involves modulation of the conformational sampling - which can happen through mutations/binding at different regions. Actually testing that hypothesis experimentally/computationally would be nice (rather than relying on inference from ML).

Achievement of aims: I think the aims are achieved, with the caveat as mentioned above, that the molecular basis for the observations is not really investigated or tested. The results support many of the conclusions, but without biophysical analysis, there is unavoidably some speculation in the discussion (which is reasonable and fine).

Impact: I think this will be impactful. I am sure others will love to get their hands on the data to run their own ML studies on, and the conclusions are interesting and impactful (seeing "deep" shared allostery across a fold). I think it is consistent with our understanding that protein folds have deep shared conformational tendencies, and that conformational sampling is at the core of much of what we term allostery.

Reviewer #1 (Public Review):

Pašukonis et al. sought to differentiate the explanatory power of two major hypotheses for sex differences in navigational ability: the adaptive specialization hypothesis, which links home range size and navigational ability, and the androgen spillover hypothesis, which links testosterone in males to navigational ability. To examine these alternative hypotheses, the authors quantify home range size, testosterone levels, and successful homing following translocation using three species of poison frog. Of particular interest, the authors were able to contrast species that vary in which sex has the larger home range, potentially disambiguating the relationship with androgens versus home range size, a feature that is lacking in many prior studies of sex differences in spatial ability. [While the authors cite one notable exception (Guigueno et al., 2014 on spatial ability in female cowbirds), they did not give this prior study as much weight as they probably should have.]

In many ways, this present study is a tour-de-force of field biology. Particular strengths include:

1) The combination of field-based observations with experimental intervention. Using intensive monitoring of individuals in the rainforest, the experimenters were able to delineate the size of home ranges, the maximum extent of movement, as well as specific behaviors (e.g., mating, parental transport of tadpoles) associated with different movement distances. This is particularly astonishing when extended to three different species.

2) The use of a natural navigational task. To assess navigational ability, the authors translocated individuals from their home ranges and determined the accuracy of, and success in, homing. While translocation is not exactly a natural experience (except for the rare occurrence, e.g., during an unusual flood), homing certainly is. Therefore, the author's assay tests wild animals in a real-world navigation problem. While the need for studying "cognition in nature" is widely recognized, it is often difficult to achieve.

3) The inclusion of multiple species that, while closely related, vary in sex roles. The authors include two species in which the male is predicted to have larger home ranges and one in which the female is predicted to do so. The potential strength of this feature is that it allows the authors to contrast the explanatory power of the adaptive specialization hypothesis - which would predict the sex with the larger home range will be more accurate and successful in homing - with the androgen spillover hypothesis - which would predict males (with their higher androgen levels) to be more accurate and successful in homing, regardless of home range size.

While the study offers a thorough and complex view of space-use and navigation in poison frogs, the study is held back by some weaknesses:

1) The comparison of accurate/successful homing across species is hampered by the application of discrete displacement distances that are not scaled to the species' natural movements. The three study species, chosen for their differences in reproductive sex roles, also differ considerably in their natural range of movements. Exploratory movements, whether near or far, give individuals the necessary experiences that familiarize them with areas so that later they can successfully/accurately return home from those areas. As a consequence, displacing O. sylvatica by 50 meters - a distance that may well be outside the range of prior experience - is unlikely to have the same significance as displacing A. femoralis by 50 meters - a species that regularly move tens of meters in a day. Species differences in accuracy/success in homing may simply reflect differences in experience, but not differences in spatial ability.

2) The authors' main conclusion is that their results contradict the adaptive specialization hypothesis for sex differences, but their results are more complex. Oophaga sylvatica is the one study species that provides the best test of this hypothesis, as the females have larger home range sizes and lower androgens. Yet, their results with O. sylvatica, in which males and females perform similarly in homing (i.e., there is a high p-value for the effect of sex), invite us to suspend judgement as to whether the sexes differ, rather than contradicting the adaptive specialization hypothesis. Not supporting one hypothesis does not necessarily lend strong support to the alternative hypothesis. Combined with the potential methodological shortcoming of using displacement distances that are not scaled to movement distances in O. sylvatica, caution is warranted.

The relationship between androgens and exploratory behaviors is an important addition to our understanding of the complexity of sex differences in spatial ability and these results do indeed provide indirect support for the androgen spillover hypothesis. Yet, more work needs to be done to disambiguate these two hypotheses in this group. Further, the authors may want to consider that both hypotheses are simultaneously at play, contributing to different features of navigation in the two sexes, and/or that the different species won't necessarily follow the same rules.

Reviewer #1 (Public Review):

This is an interesting manuscript that uses cell culture models to demonstrate the activation of mTORC1 by GPCR (GLP1R) PKA signaling and then goes on to use a PKA-insensitive mutant raptor-expressing mouse like to imply the importance of this PKA-dependent mTORC1 signaling for GLP1R agonist-dependent weight loss.

There are some important weaknesses in the manuscript as it currently stands, however:<br /> 1. There is no information on the mouse model, other than statements about the model expressing the mutant raptor in all cells and tissues. It is impossible to evaluate the results of this manuscript without some information on the genetics of the model, and some data showing the expression of the mutant, however.

2. The in vivo (mouse data) doesn't show the specificity of the weight-loss effects of GPCR-PKA signaling.

3. The cell culture data appear not to permit the direct comparison of results among conditions - is there no attenuation of Insulin-mediated pS6 by KT and no attenuation of Lira-mediated pS6 by MK? Relatedly, why does MK block FSK-mediated pS6?

Reviewer #1 (Public Review):

Here the authors aim to unravel the missing link between heme receptors and heme uptake into the cell and heme utilization. Previously, these authors uncovered the hemophore CSA2 and heme receptor RBT5 as the first steps in heme acquisition, but how heme is actually taken up by the cell and utilized as an Fe source was unknown. These authors identified the ferric reductase-like proteins Frp1 and Frp2 as having major roles in heme acquisition and utilization of heme as a sole Fe source. These are the first studies to demonstrate a role for members of the ferric reductase-like family in heme uptake and utilization. Although the exact mechanisms by which Frp1 and Frp2 affect the heme pathway are still unknown, these studies will inspire many new directions into microbial heme utilization at the host-pathogen interface. The paper is well written for a diverse audience, the experiments are comprehensive and the results are consistent with the conclusions.

Tracy and I would've been more interested in seeing more depictions of the negative aspects of religion which weren't addressed enough. A good example of this would've been possibly the Salem Witch Trials and the Last Judgement.

Reviewer #1 (Public Review):

Thyrring et al. provide a nice experiment testing the role of ocean acidification on the survival of two bivalve species. This novel work is fundamental in setting a more mechanistic understanding of the impacts of climate change on ocean species survival, and secondarily on their re-distribution across the globe. To me, the strength of the paper relies on the experimental setup, and on being honest about the limitations of metabolomics, fatty acids, and amino acids in explaining these results.

Reviewer #1 (Public Review):

In this collaborative and comparative modeling paper, three groups of investigators with well-validated mathematical models of the natural history of cervical cancer explored the potential impact of disruptions in screening services such as those associated with COVID-19 on cancer incidence. Given known disparities in access to regular screening in the United States, the authors were particularly interested in identifying heterogeneity of effects - would externally imposed restrictions on screening have a disproportionate effect on women already at increased risk because of access issues such as prolonged intervals between screening, or reliance on less sensitive screening tests?

Strengths:

--The authors used three existing, well-validated cervical cancer natural history to compare results. This comparative approach, used by these authors as well as other collaborators within NCI's Cancer Intervention and Surveillance Modeling Network (CISNET), improves confidence in the overall validity and robustness of the results, given qualitatively similar findings across models that differ in terms of structure and underlying assumptions.<br /> --The models have previously been used in the context of US screening policy.<br /> --The models used birth cohorts as well as screening frequency, which accounts for age-period-cohort effects on both risk of HPV and cervical cancer as well as competing risks such as other causes of mortality and hysterectomy.<br /> --Cervical cancer screening both detects pre-malignant lesions and allows prevention of cervical cancer, leading to decreased incidence, and, for those lesions which have progressed to invasive cancer, detects asymptomatic lesions, leading to decreased morbidity and improved survival. The use of "symptomatically detected cancers" as the primary outcome of interest is appropriate.<br /> --The qualitative results are consistent with previous modeling results in the context of screening program design--the effects of a short-term delay in screening are greatest for women with a longer time since the most recent screen, or for women screened with less sensitive (cytology) compared to more sensitive (HPV) modalities. These findings were true for both short- and long-term impacts.<br /> --The policy recommendation to prioritize outreach and appointment availability for catch-up when restrictions are lifted to women who do not have up-to-date screening according to guidelines is supported by the findings.

Limitations:

--The limitations are, for the most part, those inherent in any modeling exercise and are well described and discussed by the authors.<br /> --As the authors note, the models do not explicitly incorporate disparate impacts by race/ethnicity or other social determinants of health, and thus cannot explicitly highlight disparities within specific groups.<br /> --Potential effects on cervical cancer mortality are not captured. Given the high survival of stage I cervical cancer and, in most cases, the relatively slow progression of disease, it seems plausible that even an increase in symptomatically diagnosed disease will not have a detectable effect on mortality if there is not a shift in stage distribution; however, given that treatment of invasive cervical cancer has much greater risk of short- and long-term morbidity compared to treatment of preinvasive lesions, there is likely to be an impact on quality of life if not survival.<br /> --Related, if the factors affecting underscreening are ALSO associated with delays in care once symptoms develop, there is a potential for disparate effects on morbidity and mortality as well.

These results should prove useful to policy makers, clinicians, and patients, both in helping identifying women for prioritizing access to screening services when availability is constrained or restored, and for reassuring those women who do have up-to-date screening that delays are unlikely to significantly affect their risk of developing cervical cancer.

Reviewer #1 (Public Review):

This paper introduces a detailed computational model for synaptic plasticity, that is innovative in a number of ways. First, it includes the stochastic character of many of the biophysical processes. Second, it introduces a new way to readout the plasticity cascade. Third, it fits a number of experiments that previous models could not fit. It is a complicated model and presents a step forward towards a realistic model of synaptic plasticity. The readout mechanism is artificial but does the job well.

Reviewer #1 (Public Review):

The paper by Snoeck et al. addresses the evolution of the recognition of inceptin, a peptide from insect saliva, by plant immune receptor INR, a member of LRR-type receptor-like protein family. As a first step, the authors surveyed how broad inceptin recognition is among legumes and found that it likely emerged in the common ancestor of Phaseolid legumes. By considering available genomic information and supplementing it with several de novo sequenced species, the authors were able to show that all extant inceptin receptor sequences form a single phylogenetic clade, supporting a single origin for INR evolution, an event that was followed by several independent losses. The authors also describe a closely related INR-like clade that lacks inceptin recognition. By considering chimeras between INR and INR-like receptors, the authors map specificity to C1 (leucine-rich repeat) and C2 (insertion domain) regions of the protein. By testing inferred ancestral INR sequences they limit the number of amino acid residues responsible for the original ability to recognize inceptin to just a few residues.

The approach is well reasoned, the two complementary functional assays - ROS time course and ethylene accumulation time point - are qualitatively concordant, and the controls - expression level in heterologous assay - appropriate. Phylogenetic conclusions are likewise well supported. The authors have also done well to make the data on newly sequenced organisms available through NCBI.

There are two aspects of the study that could be improved. One is following up on the genomic events leading to independent INR loss events. Were there deletions, transposon insertions, point mutations leading to early stop codons, etc.? The other missing part is a structural interpretation of mutations leading to inceptin recognition. While I agree with the authors that an experimental structure of INR/peptide/co-receptor would be ideal, an AlphaFold or RoseTTaFold model of the N3/N4/N14 series might highlight where the key changes occurred leading to inceptin recognition. It could also hint at the N3 function, for example, was N3 already a likely foreign peptide receptor?

Reviewer #1 (Public Review):

Ryu V et al. performed a series of elegant studies to reveal a brain atlas for glycoprotein hormone receptors (i.e. TSHRs, LHCGRs, FSHRs) using combined coordinated methods and techniques including the RNAscope to detect mRNA at the single-transcript level. They find that these receptors and genes are differentially distributed in many brain regions, nuclei, and sub-nuclei. Generally, this is a timely and important study to reveal previously unknown but important central distributions of genes encoding anterior pituitary hormone receptors, providing a key resource for scientists to study the roles played by central anterior pituitary hormone receptor signaling in physiological and pathological conditions.

The experiments were designed and performed properly. The data were analyzed and interpreted accurately and presented logically in the manuscript. The conclusions of this paper were well supported by the data.

Reviewer #1 (Public Review):

This manuscript analyzes COVID-19 associated mortality in the pre-Omicron and Omicron eras to assess whether there is evidence of lower mortality associated with the Omicron variant in a large population spanning multiple countries. They used population-level data on variant frequency to infer the time periods when Omicron emerged in different countries. While there are weaknesses associated with this assumption which are well discussed by the authors, they provide a validation analysis with individual-level data from a smaller subsample suggesting that the categorization of pre-Omicron and Omicron periods is able to correctly discriminate between patients infected with different variants in the vast majority of cases. We can therefore have high confidence that the patients in the analysis are in most cases correctly identified as being likely to be infected with Omicron. The advantage of using the population-level definition is of course to allow using much larger sample sizes to determine the mortality risk associated with different variants.

Many of the tables presented suggest that the clinical characteristics of patients differed substantially in the pre-Omicron and Omicron periods, so that it is necessary to adjust for many of these characteristics (age, vaccination status, comorbidities) in order to compare mortality rates. The analysis also adjusts for country-level effects by including a random effect in the model, so that the odds ratios can be interpreted as being the average country-level effect on mortality of Omicron emergence. The results strongly suggest that after adjusting for country-level changes in clinical characteristics of patients, the risk of mortality was lower for patients hospitalized with COVID-19 during the Omicron era than previously.

There are reasons to be cautious about interpreting the results as being entirely due to differences in variant virulence, which I think are well discussed by the authors, including potential residual confounding, and potential increases in incidental infections in patients hospitalized for non-COVID-19 reasons, which would lead to a lower mortality rate in the Omicron era independently of changes in variant virulence. However, the consistency of the results with other sources of data suggests there is good reason to believe in my opinion that at least some of the observed differences in mortality risk can be attributed to lower virulence of Omicron.

While the analysis includes data from multiple countries, the vast majority of observations came from two countries (UK and South Africa); the study, therefore, has limited power to assess if there are differences across countries.

Reviewer #1 (Public Review):

This is a beautiful paper, which blends strong theoretical results (very well organised in the supplementary material) with intuitive descriptions of the results. The novelty of the theoretical developments in their own right is perhaps eclipsed by similar recent theoretical work in deep learning around the neural tangent kernel, but it is nevertheless great to see these ideas shed light on neural phenomena -- and this paper does this very well. We found that the study is given just the right scope: two learning tasks of increasing difficulty, both simple enough to enable mathematical analysis yet close enough to the type of tasks used in neuroscience as to enable meaningful comparisons to neural data. It is rare enough to be mentioned: the figure are beautiful and we found them of very high illustratory value (e.g. Figs 3 and 7, in particular, allowed us to understand the main results in a matter of seconds). We haven't found any issue in the analysis and the paper is in great shape already.

Reviewer #1 (Public Review):

Primordial germ cells are formed in the posterior pole of developing Drosophila embryo via taking up of maternally supplied germline determinants (a.k.a., germ plasm). PGC formation occurs approximately at the stage of 10th nuclear division cycle, located between minor and major ZGA waves which take place in somatic nuclei. Zelda and CLAMP are two key factors essential for global zygotic genome activation in soma. Since Zelda mutant retain apparently intact PGCs, Zelda has been thought to be dispensable for PGC formation. However, in this study, the authors identified slight loss of PGC number in both mutants lacking Zelda and CLAMP, which led authors propose a model in which somatic ZGA factors influence PGC specification.

The authors show that maternal or zygotic RNAi against Zelda or CLAMP caused abnormally broader distribution of germ plasm and resulted in an abnormal positioning of PGCs slightly away from posterior poles. The authors suggest that germline determinants are not efficiently captured by the cellularizing PGCs. As a result, the number of specified PGCs was slightly fewer. The Authors further show abnormal segregation of centrosomes accompanied with (and may be a cause of) an abnormal germ plasm trafficking. Moreover, authors show an aberrant pattern of gene expression both in soma and PGC, such as reduction of dpp transcript in posterior region, reduction of tll in posterior, and increased slam and sxl-pe in nascent PGCs when their global transcript is normally silent, suggesting that the germline-soma distinction is compromised in these mutants.

Strengths:

Historically, PGC specification in Drosophila has been believed to occur mainly by preformation-based mechanism. However, the authors focus on extrinsic regulations, particularly, function of centrosomes and cytoskeletons in proper transport of germ plasm components. This is a certainly important aspect to understand similarity and differences of PGC specification mechanism across species. The same group has demonstrated several mutant conditions causing aberrant extrinsic regulation of PGC specification in the past, and thus they are uniquely suited to pursue this line. The authors monitor germ plasm localization and gene expression by smFISH, which enables quantitative analyses. Detection of nascent transcript also reports zygotic transcription in a highly quantitative manner.

Weaknesses:

Overall the manuscript is descriptive and does not clearly provide functional interpretations of observed phenotypes. Specifically, the authors need to consider and discuss potential mechanism of this process.

Reviewer #1 (Public Review):

This study is a follow-up to the previous work by the authors in establishing a surprising role for the presynaptic adhesion molecules, neurexin (Nrxn) variants containing the SS4+ splice site, in differentially controlling postsynaptic NMDA and AMPA receptors by forming links through a shared system of extracellular cerebellins (Cbln) and postsynaptic GluD1. Here the authors show at CA1 to subiculum synapses, that the role for Clbn2 in mediating the effects of Nrxn1-SS4+ and Nrxn3-SS4+ in enhancing NMDAR and suppressing AMPAR, respectively, is redundant with that of Clbn1. Moreover, Clbns do not appear to play a role in synapse formation. Dai and colleagues extend their previous work also by highlighting the common function for Nrxn-Clbn signaling system across different synapses albeit with subtle differences and point to a lack of a role for Nrxn-Clbn signaling in morphological synapse development. Overall the data are solid, while the key findings are mostly incremental, and the basis for the selectivity in the observed differential regulation of AMPARs and NMDARs via the same trans-synaptic link through Clbns at various types of synapses remain to be clarified. Importantly, the authors make a definitive conclusion concerning the lack of a role for Nrxn-Cbln signaling complexes in synapse formation during development. Nevertheless, this is a contentious issue, and as such, the conclusions could be more compellingly supported with further experiments.

Equillibrium Vapor Pressure definition - rate of evaporation = rate of condensation.

The stronger the IM forces. The lower the vapor pressure.

Reviewer #1 (Public Review):

This paper considers decision-making problems when information and/or reward changes over time. It shows that the policy - the decision boundary that tells subjects when to make a decision - can have a very complicated shape; much more complicated than is typically considered. The authors use well-established techniques in reinforcement learning, but apply them in regimes where they are not normally used. Possibly the most important aspect of the paper is that it presents the relevant techniques in a reasonably accessible manner (and with a little work it could become very accessible). The paper also shows, in one non-trivial decision-making task, that normative models outperform heuristic ones by a large margin.

Reviewer #1 (Public Review):

Fibrotic change is a widespread biological phenomenon associated with both normal development and abnormal responses, often in response to pathological circumstances. In the heart, it is associated with both pump failure and arrhythmic change. This present study presents an intriguing murine genetic platform in which such processes are reduced. This used diphtheria toxin A (DTA) on a PDGFRa-CreERT2/+ mouse line. The authors report a reduction in ventricular, atrial and septal fibroblast density. However, this was surprisingly associated with relatively normal cardiac function with relatively normal histology and heart to body weight ratio, cardiomyocyte cross-sectional area, and ejection fractions, left ventricular (LV) chamber size, systolic and diastolic blood pressure, despite reduced collagen VI but not laminin and collagen IV levels. There were only minimal extracellular matrix proteomic changes. Furthermore, left anterior descending artery ligation left relatively moderated mortalities, unaltered changes in cardiac mass, measures of left-heart failure and LV chamber size, with actually better ejection fractions in fibroblast-ablated mice. Furthermore there was a reduced pathological compromise of cardiac function following profibrotic angiotensin II/phenylephrine challenge. Fibroblast ablation here did not affect cardiac mass or lung weight, sparing diastolic and slightly reducing systolic LV chamber size. Yet WT and fibroblast-ablated mice respectively showed slight decreases and fully recovered LV ejection fractions. These findings suggests the value of this platform for studies of the effect of fibrosis following normal or pathological change.

Reviewer #1 (Public Review):

The main result of the paper is a statistical dependence between the evolved size control strategy and the structure of the cell cycle, in that size control that manifests early (later) in the cell cycle tends to give adder- (weakly sizer-) like strategies. Notably, even when the final evolved network shows weak adder or weak sizer-like behaviour, they find strong sizer-like control in the evolutionary transient. Finally, they constrain the evolutionary algorithm to sense cell size only through stochastic fluctuations of protein concentrations and uncover a strategy that exhibits hallmarks of self-organised criticality.

The questions studied by the authors are both interesting and timely, and their results are intriguing and well documented. On the whole, the conclusions are convincingly argued, and the authors do an excellent job of extracting qualitative features from their evolved networks. However, the manuscript is a little difficult to read, with the figures being crowded and difficult to parse. In addition, while there is a lot of detail in some places (as in the description of one particular feedback control strategy), other results are less fleshed out (such as statistical summaries of the different simulations). The manuscript would benefit from a sharper presentation of the results.

A particularly interesting question addressed in the paper is why adders are more commonly found when sizers are believed to be better at controlling cell size. Here, the authors' simulations give two answers: first, that sizers tend to appear when cell size control is exerted later in the cycle (as in S. pombe). Second, that even when adders eventually evolve, the evolutionary transient passes through a strong sizer strategy. As the adder-vs-sizer question is repeatedly raised, it would strengthen the paper to have a longer and sharper discussion on (a) why early cell size control favours adders, and (b) why sizers appear as transients when fluctuations in cell size are large?

The final part of the paper, which describes a strategy based on sensing size through concentration fluctuations, is very interesting but brief, which is understandable given the quantity of results presented earlier in the paper. Nonetheless, it provides an excellent example of the power of the authors' approach.

Overall, the results in this paper are a compelling addition to the recent interest in cell size control.

Chadeau-Hyam, M., Wang, H., Eales, O., Haw, D., Bodinier, B., Whitaker, M., Walters, C. E., Ainslie, K. E. C., Atchison, C., Fronterre, C., Diggle, P. J., Page, A. J., Trotter, A. J., Ashby, D., Barclay, W., Taylor, G., Cooke, G., Ward, H., Darzi, A., … Elliott, P. (2022). SARS-CoV-2 infection and vaccine effectiveness in England (REACT-1): A series of cross-sectional random community surveys. The Lancet Respiratory Medicine, 0(0). https://doi.org/10.1016/S2213-2600(21)00542-7

Franklin, J., & Stein, R. (2022, February 4). 900,000 Americans have died of COVID in 2 years of the global pandemic. NPR. https://www.npr.org/sections/coronavirus-live-updates/2022/02/04/1078100069/covid19-deaths-us

Reviewer #1 (Public Review):

The cohesin ring model postulates that DNA entry and exit must occur through one of the ring's three interfaces thus leading to entrapment. The authors previously tested this model in vitro by engineering disulfide crosslinkers into the different interfaces. Here the authors further test this model by generating cohesin complexes in which the different interfaces can be covalently closed. Using these variants, the authors show that entrapment of DNA can occur through the hinge and SMC3/SCC1 interfaces. Removal of SCC2 and/or SCC3 shows that these regulatory proteins contribute to DNA entrapment through these interfaces, respectively. Sealing of the hinge interface does not prevent entrapment indicating that transport occurs through the passage between the SMC1 and SMC3 ATPase heads. Their data are consistent with the model that DNA entrapment through the SMC and kleisin compartments can lead to initial entrapment. Opening of the hinge may be required for the establishment of cohesion while an opening of the SMC3/SCC1 interface may be required for release. Overall, this information advances our understanding of the molecular basis of DNA entrapment in the cohesin complex.

Reviewer #1 (Public Review):

Mackevicius et al image CA activity in nucleus HVC of isolated singing zebra finches before and after tutor exposure. HVC is well known for its sequential activity during singing - and isolate song is known for its abnormal variability, raising two possibilities. Tutor exposure and subsequent practice may or may not be necessary for chain foundation. Because birdsong is a learned behavior but also subject to innate predispositions, the current manuscript provides a really important test of how nature vs nurture affects the development of song - at the mechanistic level. The authors discover HVC chains do exist, but they are unusually uncoupled from vocal output. More, the more immature chain formation is at the time of tutor exposure, the more copying there is. This finding that the existing HVC chain could become time-locked to new acoustic elements is an important verification of the long assumed, but never explicitly tested, idea that plasticity in the HVC-RA pathway drives phonological change during natural development. These results are really important for the songbird field - as they mechanistically link the timing of tutor exposure to HVC chain maturity to imitation quality. These results also will be useful for the general community of biologists interested in how innate predispositions for animal behavior can express at the level of signals and circuits.

Reviewer #1 (Public Review):

In this study, the authors compare computational MD simulations with functional activity data to determine if ligand activity can be predicted from simulations. As a test case, the authors use the ligand-binding domain (LBD) of an ancestral steroid receptor (AncSR2) that they and others have previously studied, providing a well-characterized system for their analyses. The studies include wild-type (WT) AncSR2 as well as four mutant proteins where a single methionine residue that contacts the steroid hormone within the pocket (Met75) was mutated (to Ala, Phe, Ile, or Leu). Computational analyses are performed to assess the stability of the complexes and determine whether the conformational ensembles generated show similarities or differences between the WT vs. mutant forms, or apo vs. ligand-bound forms (aromatic vs. 3-keto non-aromatic A-ring, EST/estrogen vs. progesterone/PROG). Simulations included conventional and accelerated methods. Clustering analysis of the accelerated simulations revealed some similarities and differences, which the authors then compare to luciferase reporter assay data (Gal4-fusion + WT vs. mutant LBDs) for the mutants where they performed dose-response experiments (up to 1 µM ligand added). One of the mutants studied did not show any activity (M75I); however, M75I and M75L both showed increased basal transcriptional activity (constitutively active) vs. WT without an exogenously added ligand. The authors developed a fluorescent ligand binding assay and showed the M75I mutant does not bind ligands (at least up to 1 µM added ligand). Next, hydrogen/deuterium exchange mass spectrometry data are provided to inform how the M75L mutant is constitutively active. The HDX results indicate that several regions display higher deuterium uptake in the M75L mutant and PROG binding has a larger destabilizing effect on WT vs. M75L. Finally, some structural snapshots from the MD simulations are shown (Fig 6A-C) that the authors claim to explain the altered transcriptional response of the M75 mutants vs. WT.

This study may be one of the first to attempt to make qualitative correlations between computational simulations of ligand-bound/free nuclear receptor LBDs and functional outcome. One could see a future where many different ligands are docked and a more quantitative, streamlined pipeline is used to predict functional outcome-this study takes the important first step in trying to determine if there are simulation-function correlations.

Reviewer #1 (Public Review):

It is a strength of the current manuscript that it provides a near-complete picture of how the metamorphosis of a higher brain centre comes about at the cellular level. The visualization of the data and analyses is a weakness.

I do not see any point where the conclusions of the authors need to be doubted, in particular as speculations are expressly defined as such whenever they are presented.

The fact that molecular or genetic analyses of how the described metamorphic processes are organized are not presented should, I think, not compromise enthusiasm about what is provided at the cellular level.

Reviewer #1 (Public Review):

In this manuscript, the authors use time-lapse microscopy in growing intestinal organdies and computational modelling to demonstrate a paradigm for the control of a pool of proliferative cells. They find strong correlations in the proliferative behaviour of sister cells. They propose a compartmentalised model, where cells in one compartment all have a high propensity to produce two proliferating daughter cells while cells in the other department produce daughter cells who both cease to proliferate.

The work establishes a previously suggested paradigm for the control of fluctuations in a pool of proliferating cells. This paradigm might be relevant for tissues other than the intestine such that this work will be of relevance to the general field of stem cell biology. I found this work to be a nice combination of modelling and the conclusions overall convincing. The authors could improve upon the precision in their wording and the discussion of the scope of their modelling results.

Reviewer #1 (Public Review):

In this study, Apiz-Saab et al. build up prior work by the Muir lab, which examined the metabolite composition in the tumor microenvironment and found that some metabolites like arginine are present in very different levels from that in our standard culture media. In this study, the authors have formulated a custom media based on the composition of the tumor interstitial fluid (TIF media or TIFM) and found that pancreatic cancer cells cultured in this media have a metabolic state more like tumors in vivo. This is primarily driven by very low levels of arginine, which induces arginine biosynthesis is the cancer cells to cope with this nutrient limited state. Using genetic and pharmacological approaches, the authors demonstrate that arginase expression within tumor-infiltrating myeloid cells drives tumor microenvironmental arginine depletion in vivo.

Strengths:

This is a very rigorous, well-designed study and the findings are broadly interesting for the metabolism, immunometabolism, and pancreatic cancer communities. The methods are comprehensive and the experimental details in the legends are complete. The discussion is particularly well developed and does an excellent job of putting the findings in the context of the field.

Weaknesses:

The claim that arginine biosynthesis is an adaptation to myeloid arginine depletion could be further supported in vivo.