Reviewer #3 (Public Review):

This is a highly interesting paper that comprehensively investigates the electrophysiological properties of granule cells in the dentate gyrus at different developmental stages. Using state-of-the-art in vitro electrophysiological techniques, the authors record granule cell responses to fluctuating current injections to study how they encode stimuli. The authors find that while immature granule cells produce less reliable stimulus responses and worse stimulus representations than mature cells (8wks and older), cell populations containing neurons of mixed ages improve overall stimulus reconstruction. These data suggest that the cellular diversity contributed by immature granule cells could be beneficial for transmitting distinct properties of stimuli with rich temporal structure, potentially improving the cellular process of pattern separation.<br /> Major strengths of the paper lie in the precise age determination of immature neurons in Ascl1-CreERT2-Tom mice, recordings of immature neurons, which are rare in in vivo and in vitro studies, precise control over cell-intrinsic properties by blocking excitatory and inhibitory inputs in vitro, and characterization of encoding properties using a spike response model (SRM).

The conclusions drawn are supported by the data, and the results are likely of great interest to a specialist community of hippocampal electrophysiologists.

Reviewer #3 (Public Review):

The manuscript by JY Toshima et al. is an excellent and important study that demonstrates very clearly the existence of an endosomal compartment in yeast, distinct from the trans-Golgi network, to which endocytic vesicles fuse upon internalization. They show that this compartment is enriched in the SNARE protein Tlg2, a yeast homologue of syntaxin, and is segregated from the Golgi-localized Sec7-containing compartment, indicating that the organization of the endocytic system in yeast is similar to that of animal cells. Furthermore, they demonstrate the trafficking machinery required for maturation of this compartment, and that it is also a station on the pathway back to the plasma membrane. Because there have been conflicting reports in the literature as to the existence of an endosomal compartment in yeast distinct from the trans-Golgi network, this paper is of great importance for the cell biology community.

Major strengths of this study are the cutting-edge imaging technology used, and the careful, quantitative analyses carried out. The authors use a super-resolution live cell imaging approach that allows them to discriminate to a high resolution different compartments and membrane domains of highly dynamic yeast organelles, and to follow an internalizing cargo over time. With their manuscript, they have provided a full set of movies, along with quantifications, to support their conclusions.

The authors use fluorescent-protein-labelled endocytic cargo (alpha-factor) and florescent-protein-labelled compartment markers, assaying them in high resolution and super-resolution live cell imaging microscopy systems. In this way, they are able to follow trafficking of cargo through compartments in real time. The authors first demonstrate that the alpha-factor cargo substantially colocalized with the SNARE protein Tlg2, a marker of early endosomes, but very little with Sec7. They also show that Tlg2 marks a sub-compartment distinct from the Sec7 compartment, but adjacent to it. Furthermore, they demonstrate using super-resolution microscopy and triple color 4D imaging that endocytosed alpha-factor cargo structures make contact with the Tlg2 compartment, adjacent to the Sec7 compartment, then disappear, supporting the conclusion that endocytic vesicles first fuse with the Tlg2 compartment. Next the authors show that alpha factor is transported from the Tlg2 compartment to the Vps21 compartment, a process that requires the GGA adaptors Gga1 and Gga2. Finally, the authors show that recycling of the endocytic R-SNARE Snc1 also occurs by passage through the Tlg2 compartment.

The use of mutants that affect different stages of endosomal trafficking is a strength of the manuscript, as it allows elucidation of the mechanism of transport through successive compartments. Importantly, using a gga1-delta gga2-delta mutant, the authors demonstrate convincingly that the GGA adaptors Gga1 and Gga2 are required for alpha factor transport from the Tlg2 compartment to the Vps21 compartment.

Throughout this study, the authors use fluorescent protein-labelled cargo and compartment markers (EGFP, mCherry, iRFP), but don't explicitly state to what extent these fusion proteins are functional compared to the endogenous proteins. They could cite previous publications or their results describing the functionality of the fusion proteins used.

Reviewer #3 (Public Review):

One key finding of this work is the identification of Xanthomonas oryzae pv. oryzae (Xoo) strains in Africa, based on their genomes sequence and their TALE repertoires, have high similarity with Asian strains. Asian Xoo strains typically overcome NLR-mediated recognition of TALEs in rice by so-called iTALEs. Moreover, some Asian strains contain a TALE resembling PthXo1, a TALE protein that was shown to overcome xa5 resistance.

The authors now found that some of the newly identified African strains have iTALEs and PthXo1-like TALEs. Such newly evolved African strains were found to be fully virulent on the African rice elite variety Komboka, which is resistant to a broad panel of African Xoo strains.

Previous studies have shown that TALEs bind to effector binding elements (EBEs) present in promoters of rice SWEET genes to promote disease. Work from the lab of the authors and other labs has shown that TALEs can no longer promote the disease if matching EBEs are changed or deleted by CRISPR or TALEN-mediated mutagenesis. In fact, pioneering work by Bing Yang, one of the authors of this article published about ten years ago a Nature Biotechnology article where he showed that rice plants with mutated EBEs are resistant to Xoo. Recently, a combined effort of the Yang and Frommer labs resulted in two further Nature Biotechnology publications (2019), in which they described along with other useful tools rice lines where multiple EBEs were mutagenized in parallel and that provide broad spectrum resistance.

The work under review describes now CRISPR mutagenesis of an African elite cultivar resulting in a line that mediates resistance to Asian and newly evolved African strains.

Overall, the work is technically sound. Yet, the approach that has been described - mutagenesis of multiple EBEs - has been used before and is a routine procedure for labs that are focused on such undertakings. While such approaches do not provide new insights for fundamental research, they nevertheless are certainly important and useful in translational research, as demonstrated here.

Reviewer #3 (Public Review):

Huff et.al further characterise the anatomy and function of a population of excitatory medullary neurons, the Post-inspiratory Complex (PiCo), which they first described in 2016 as the origin of the laryngeal adduction that occurs in the post-inspiratory phase of quiet breathing. They propose an additional role for the glutamatergic and cholinergic PiCo interneurons in coordinating swallowing and protective airway reflexes with breathing, a critical function of the central respiratory apparatus, the neural mechanics of which have remained enigmatic. Using single allelic and intersectional allelic recombinase transgenic approaches, Huff et al. selectively excited choline acetyltransferase (ChAT) and vesicular glutamate transporter-2 (VGluT2) expressing neurons in the intermediate reticular nucleus of anesthetised mice using an optogenetic approach, evoking a stereotyped swallowing motor pattern (indistinguishable from a water-induced swallow) during the early phase of the breathing cycle (within the first 10% of the cycle) or tonic laryngeal adduction (which tracked tetanically with stimulus length) during the later phase of the breathing cycle (after 70% of the cycle).

They further refine the anatomical demarcation of the PiCo using a combination of ChAT immunohistochemistry and an intersectional transgenic strategy by which fluorescent reporter expression (tdTomato) is regulated by a combinatorial flippase and cre recombinase-dependent cassette in triple allelic mice (Vglut2-ires2-FLPO; ChAT-ires-cre; Ai65).

Lastly, they demonstrate that the PiCo is anatomically positioned to influence the induction of swallowing through a series of neuroanatomical experiments in which the retrograde tracer Cholera Toxin B (CTB) was transported from the proposed location of the putative swallowing pattern generator within the caudal nucleus of the solitary tract (NTS) to glutamatergic ChAT neurons located within the PiCo.

Methods and Results<br /> The experimental approach is appropriate and at the cutting edge for the field: advanced neuroscience techniques for neuronal stimulation (virally driven opsin expression within a genetically intersecting subset of neurons) applied within a sophisticated in vivo preparation in the anaesthetized mouse with electrophysiological recordings from functionally discrete respiratory and swallowing muscles. This approach permits selective stimulation of target cell types and simultaneous assessment of gain-of-function on multiple respiratory and swallowing outputs. This intersectional method ensures PiCo activation occurs in isolation from surrounding glutamatergic IRt interneurons, which serve a diverse range of homeostatic and locomotor functions, and immediately adjacent cholinergic laryngeal motor neurons within the nucleus ambiguous (seen by some as a limitation of the original study that first described the PiCo and its roll in post-I rhythm generation Anderson et al., 2016 Nature 536, 76-80). These experiments are technically demanding and have been expertly performed.

The supplemental tracing experiments are of a lower standard. CTB is a robust retrograde tracer with some inherent limitations, paramount of which is the inadvertent labelling of neurons whose axons pass through the site of tracer deposition, commonly leading to false positives. In the context of labelling promiscuity by CTB, the small number of PiCo neurons labelled from the NTS (maybe 5 or 6 at most in an optical plane that features 20 or more PiCo neurons) is a concern. Even assuming that only a small subset of PiCo neurons makes this connection with the presumed swallowing CPG within the cNTS, interpretation is not helped by the low contrast of the tracer labelling (relative to the background) and the poor quality of the image itself. The connection the authors are trying to demonstrate between PiCo and the cNTS could be solidified using anterograde tracing data the authors should already have at hand (i.e. EYFP labelling driven by the con-fon AAV vectors from PiCo neurons (shown in Fig5), which should robustly label any projections to the cNTS).

The retrograde labelling from laryngeal muscles seems unnecessary: the laryngeal motor pool is well established (within the nAmb and ventral medulla), and it would be unprecedented for a population of glutamatergic neurons to form direct connections with muscles (beyond the sensory pool).

The authors support their claim that PiCo neurons gate laryngeal activity with breathing through the demonstration that selective activation of glutamatergic and cholinergic PiCo neurons is sufficient to drive oral/pharyngeal/laryngeal motor responses under anaesthesia and that such responses are qualitatively shaped by the phase of the respiratory cycle within which stimulation occurs. Optical stimulation within the first 10% of the respiratory cycle was sufficient to evoke a complete, stereotyped swallow that reset the breathing cycle, while stimuli within the later 70% of the cycle, evoked discharge of the laryngeal muscles in a stimulus length-dependent manner. Induced swallows were qualitatively indistinguishable from naturalistic swallow induced by the introduction of water into the oral cavity. The authors note that a detailed interpretation of induced laryngeal activity is probably beyond the technical limits of their recordings, but they speculate that this activity may represent the laryngeal adductor reflex. This seems like a reasonable conclusion.

The authors propose a model whereby the PiCo impinges upon the swallowing CPG (itself a poorly resolved structure) to explain their physiological data. The anatomical data presented in this study (retrograde transport of CTB from cNTS to PiCo) are insufficient to support this claim. As suggested above, complementary, high-quality, anterograde tracing data demonstrating connectivity between these structures as well as other brain regions would help to support this claim and broaden the impact of the study.

This study proposes that the PiCo in addition to serving as the site of generation of the post-I rhythm also gates swallowing and respiration. The scope of the study is small, and limited to the subfields of swallowing and respiratory neuroscience, however, this is an important basic biological question within these fields. The basic biological mechanisms that link these two behaviors, breathing and swallowing, are elusive and are critical in understanding how the brain achieves robust regulation of motor patterning of the aerodigestive tract, a mechanism that prevents aspiration of food and drink during ingestion. This study pushes the PiCo as a key candidate and supports this claim with solid functional data. A more comprehensive study demonstrating the necessity of the PiCo for swallow/breathing coordination through loss of function experiments (inhibitory optogenetics applied in the same transgenic context) along with robust connectivity data would solidify this claim.

Reviewer #3 (Public Review):

The relevance of Y90 phosphorylation as a regulatory mechanism is shown by the comparison of Src kinase activity, transforming potential, cell invasiveness, and lateral diffusion in membranes. Mechanistically, Y90E mutation affects the opening of the structure, estimated from FRET experiments, and the phosphorylation status of the three main downstream signaling pathways.

The effect of the Y90E mutation is very clear, although its description as "phosphomimicking" is, in my opinion, not accurate. Glutamic acid has a negative charge but is significantly different from phosphotyrosine. Maybe other polar mutants (lysine, glutamine...) would have a similar destabilizing effect on hydrophobic interactions. Erpel 1995 showed some effects of the Y90A mutants.

The effect of tyrosine phosphorylation on the SH3 domain of proteins having the conserved ALYDY motif supports the proposed role, although the evidence for in vivo Y90 phosphorylation in c-Src is scarce. The possible autophosphorylation of Y90 is suggested but the evidence is not very strong and does not rule out other kinases, especially some downstream of Src itself -as already suggested by the authors.

The authors suggest that the perturbation of Y90 reduces the interaction with the connector domain. This is a reasonable explanation, supported by the opening of the structure, but additional effects may exist: The SH3 hydrophobic region including Y90 is also the binding site for the myristoyl group (Le Roux et al. iScience, 12, 194-203) and mutations in the SH3 RT loop significantly affected lipid binding. This could contribute to the observed reduced diffusion in the lipid bilayer.

Reviewer #3 (Public Review):

By using chemogenetic manipulations of direct pathway neurons in the dorsomedial part of the striatum (DMS) of anesthetized mice combined with fMRI, Markicevic et al explore changes in BOLD dynamics at local (striatum) and macro-scale (brain-wide) levels. The article is appropriately written, and the main findings are well organized and presented in 7 figures. Figures 1 and 2 schematize the techniques and document the motor effects of chemogenetic manipulations. Figures 3-7 describe neural changes induced by these manipulations. The main strength of this work is the level of specificity of the chemogenetic manipulations, which combined with brain-wide functional exploration, provide a very useful map of the consequences of activating a specific striatal subpopulation. In my opinion, the main weakness of this work is that the results are under-discussed and not appropriately contextualized in the current views of the functions of the basal ganglia. My main concerns are exposed in the following lines:

1. In the first finding the authors show that D1 activation/inactivation produces reliable changes in the infected region (DMS), but most importantly, also produced changes in adjacent areas, suggesting intra-striatal communication. The way the data is presented and discussed appears to be confirmatory of what has been previously described with electrophysiological recordings. In my opinion, the most important part of this section would be to fully describe the differences between activation and inactivation groups. Is interesting that opposite manipulations of D1 receptors produced very similar maps of discrimination (Fig. 3). Therefore, it would be necessary to discuss the meaning of obtaining similar classification accuracy indices with opposite manipulations. Perhaps, the use of SVM classifiers can be complemented with other analytical techniques to further disentangle the consequences of manipulating intrastriatal D1 receptors.

2. The second finding (Fig. 4) indicates that thalamic regions forming "closed loops" with the striatum were more affected by chemogenetic manipulations. We knew from anatomical studies that the BG are part of anatomically segregated cortico-BG-thalamic loops. Therefore, it would be expected that these anatomical boundaries would somehow limit functional connectivity maps. Here again, I consider that the manuscript would be improved with further analysis or discussion. For example, it would be interesting to perform further analysis relating the previous section (local striatal connectivity) with this one. In this section, several thalamic nuclei presented higher levels of classification accuracy, but in the previous section, the authors showed that DMS manipulation also produced the same effects in different intrastriatal regions. Therefore, it is not possible to know if the thalamic effects are related to the manipulation of D1 in the DMS or its adjacent regions.

3. In the third finding (Fig. 5) the authors show that the most "sensitive" cortical regions to the manipulations were classified as "unimodal". This is an interesting result; however, it would be necessary to at least provide further discussion on its potential meaning. It is important to consider that the cortical regions with significant changes, for example, primary sensorimotor cortices, mainly target the dorsolateral, not the dorsomedial striatum. In this context, would it be possible to establish a new analysis to characterize potential correlations between cortical regions and striatal subregions?

4. The fourth finding (Figure 6) is that thalamic but not cortical regions presented low-frequency fluctuations. What is the meaning of an increase in slow fluctuations? Why did D1 activation (and not inactivation) induced this effect? Are striatal sub-regions also presenting these slow fluctuations?

5. In the last finding (Figure 7), the authors explored potential changes in functional connectivity (FC) between the striatum and cortical and subcortical regions. Contrary to the results obtained with the SVM-based analytical tool, FC analysis revealed that D1 activation and inactivation produced opposite results, while D1 activation decreased FC in several cortical and subcortical regions, D1 inactivation increased it. While this set of data is clearly described, the implications of these relationships could be further discussed. For example, how do the authors explain that FC with SSp was not significantly changed with this analytical method, but was one of the most affected regions with the Balanced Classification Accuracy method?

6. Finally, there is no section in the discussion where the behavioral effects observed in figure 2 are contextualized in the massive set of BOLD results presented in the following sections.

Reviewer #3 (Public Review):

The manuscript by Bravo-Plaza et al. identifies and characterizes new mutations (E6K and G540S) in the Uso1 globular head domain that suppress the loss of function mutations in Rab1. Further experiments show that the combined E6K/G540S mutant restores apparent Golgi-localization of Uso1 in Rab1 deficient cells, that this mutant preferentially co-purifies with ER/Golgi SNARE proteins, that monomeric E6K/G540S globular head-domain binds more avidly to purified Bos1 SNARE protein than wild type head-domain, and that overexpression of E6K/G540S or wild type head-domain alone is sufficient for viability. Based on these findings the authors propose that long-distance tethering by Uso1 is dispensable and that the head domain provides an essential function to directly regulate ER/Golgi SNARE-dependent membrane fusion.

Strengths of the study are that an unbiased screen was used to identify new Rab1 suppresser mutations that land in the Uso1 globular head domain. Characterization of these suppressor mutants reveals that SNARE binding activity of Uso1 resides in the head domain and that elevated expression of the Uso1 head domain is sufficient for viability. Imaging experiments document the localization and dynamics of Uso1 on Golgi compartments and biochemical studies show the properties and binding activity of Uso1 domain mutants. These are new findings and the conclusion that monomeric globular head-domain interacts with specific SNAREs to maintain viability is justified.

Weaknesses are that it is well documented that both Rab1 and Uso1 activity can be bypassed by activation of ER/Golgi SNARE machinery either by overexpression of SNARE proteins or by the single copy SLY1-20 allele. Therefore, it was not surprising that tethering by the Uso1 coiled-coil domain is dispensable. The proposal that the E6K mutation in the head domain of Uso1 promotes membrane targeting was not well supported by experimental evidence. And while the AlphaFold modeling of Uso1 with the ER/Golgi fusion machinery was intriguing, the proposed molecular models remain speculative until further tested.

Reviewer #3 (Public Review):

The strongest aspects of this study are the structural analysis of the 90 residue KER domain. This is an important advance, discovering a founding member of a novel class of DNA binding motifs, termed a SAH-DBD (single alpha helix-DNA binding domain). Interestingly, they define a subregion of KER (termed "middle-A", residues 155-204 of Cac1) that has nearly the same DNA binding affinity and confers similar in vivo phenotypes as the full KER domain.

This study also shows that the biological role of KER partially overlaps compensatory factors in vivo, both within the same Cac1 protein subunit (e.g. the WHD domain) and also with other proteins acting in parallel (e.g. Rtt106). That is, the presence of either WHD or Rtt106 renders the drug-resistance and silencing assays employed here insensitive to loss of the KER domain.

However, the drug resistance and gene silencing phenotypes are inherently indirect measures of the most important claim of this work, that KER is a molecular ruler for DNA for the purpose of ensuring sufficiently large templates deposition of histone H3/H4 cargoes. Therefore, this study would be of greater impact if the authors more directly tested this measurement idea in assays that directly assess histone deposition. There are multiple options. Since the authors have in hand recombinant wild-type and mutant CAF-1 complexes, one could examine the number and/or spacing of nucleosomes formed during in vitro deposition reactions. Complementary in vivo experiments using the authors' existing mutant strains could be based on the finding that CAF-1 is particularly important for histone deposition onto nascent Okazaki fragments during DNA replication (Smith and Whitehouse, 2012; pmid: 22419157), and that the spacing pattern of nucleosomes on this DNA is greatly perturbed in cac1-delete cells.

Reviewer #3 (Public Review):

This study describes a descending circuit that can modulate pain perception in the drosophila larvae. While descending inhibition is a major component of mammalian pain perception, it is not known if a similar circuit design exists in fruit flies. Overall the authors use clean logic to establish a role for DSK and its receptor in regulating nociception. The following concerns still stand:

1) It's not completely clear why the authors are staining animals with an FLRFa antibody. Can the authors stain WT and DSK KO animals with a DSK antibody? Also, can the authors show in supplemental what antigen the FLRFa antibody was raised against, and what part of that peptide sequence is retained in the DSK sequence? This overall seems like a weakness in the study that could be improved on in some way by using DSK-specific tools.

2) What is the phenotype of DSK-Gal4 x UAS-TET animals? They should be hyper-reactive. If it's lethal maybe try an inducible approach.

3) Figure 9. This was not totally clear, but I think the authors were evaluating spontaneous (i.e. TRPA1-driven) rolling at 35C. The critical question is "Does activating DSK-expressing neurons suppress acute heat nociception?" and this hasn't really been addressed. The inclusion of PPK Gal4 + DSK Gal4 in the same animal clouds the overall conclusions the reader can draw. The essential experiment is to express UAS-dTRPA1 in DSK-Gal4 or GORO-Gal4 cells, heat the animals to ~29C, and then test latency to a thermal heat probe (over a range of sub and noxious temperatures). Basically, prove the model in Figure 10 showing ectopic activation or inhibition for each major step, then test heat probe responses.

4) It would also then be interesting to see how strong the descending inhibition circuit is in the context of UV burn. If this is a real descending circuit, it should presumably be able to override sensitization after injury.

Reviewer #3 (Public Review):

Lauterbur et al. present an expansion of the whole-genome evolution simulation software "stdpopsim", which includes new features of the simulator itself, and 15 new species in their catalog of demographic models and genetic parameters (which previously had 6 species). The list of new species includes mostly animals (12), but also one species of plant, one of algae, and one of bacteria. While only five of the new animal species (and none of the other organisms) have a demographic model described in the catalog, those species showcase a variety of demographic models (e.g. extreme inbreeding of cattle). The authors describe in detail how to go about gathering genetic and demographic parameters from the literature, which is helpful for others aiming to add new species and demographic models to the stdpopsim catalog. This part of the paper is the most widely relevant not only for stdpopsim users but for any researcher performing population genomics simulations. This work is a concrete contribution towards increasing the number of users of population genomic simulations and improving reproducibility in research that uses this type of simulations.

Reviewer #3 (Public Review):

Dominici et al studied the effects of the type I PRMT inhibitor MS023 on skeletal muscle stem cells (MuSCs) and on muscle strength in dystrophin-deficient mdx mice. The authors observed an enhanced proliferative capacity of cultured MuSCs with an increase of Pax7+/MyoD- cells. The observations are more or less in line with previous studies of the same group, describing reduced differentiation but enhanced proliferation of MuSCs after genetic inactivation of Prmt1. scRNA-seq identified different subpopulations of MuSCs, showing a shift to increased Pax7 expression and elevated oxidative phosphorylation and glycolysis after treatment with MS023. Treatment of MuSC with MS023 during expansion in vitro enhanced engraftment of MuSCs and treatment of dystrophic mdx mice increased muscle strength.

Overall, the manuscript provides new insights into the beneficial effects of the type I PRMT inhibitor MS023 for skeletal muscle regeneration. The description of the MS023-induced transcriptional and metabolic changes in MuSC is interesting and the effects on MuSC transplantation and muscle strength are stunning. However, I have the following comments and concerns:

* Control experiments with the TP-064 inhibitor (previously shown to be specific for CARM1/PRMT4) were not done for the transplantation and muscle strength experiments, which is a clear shortcoming in my view. Since MS023 is a non-selective inhibitor of type I PRMTs with comparable IC50 values for PRMT1 and PRMT4 (CARM1), and lower IC50 values for PRMT6 and PRMT8, it is still not clear whether the enhanced transplantation efficiency and the increased muscle strength is indeed only caused by inhibition of PRMT1. The authors justify their statements by pointing out that gene expression of Prmt1 is highest among the type I PRMTs in MuSCs, which is a rather poor argument, as seen by the strong effects caused by the inactivation of PRMT4.

* Clustering of the M1-M5 subpopulations. I expressed my concern about the separation of the subclusters, which appear more or less in the same cloud. The authors answered that each cluster has some genes, which are only expressed in the respective cluster. I do not doubt this observation but apparently, the transcriptional differences are minor, otherwise one would have seen a much better separation of the subpopulations.

* The authors have not done additional experiments but simply toned-down the statements about the relevance of the proposed "metabolic reprogramming" of MuSC by the type I PRMT inhibitor MS023, which was a major conclusion in the original submission. Again, the changes in the expression of metabolically relevant genes upon MS023 treatment are interesting and should be analyzed in respect to causality. It is not a solution to more or less disabandon the original hypothesis by changing the wording.

* I specifically asked the authors to check whether the dramatic six-fold increase of MuSC engraftment after MS023 treatment really goes along with the incorporation of transplanted MuSC into the MuSC niche, raising concerns that a huge share of the transplanted cells may linger around in the interstitium. It should be very easy to identify and quantify transplanted MuSC outside and inside the basal lamina. Instead of doing the requested experiment, the authors argue about suppression of endogenous MuSC competition by irradiation, at the same time admitting that several GFP-negative fibers have formed.

* I expressed my doubts that a 3-day treatment with MS023 is sufficient to dramatically enhance muscle function in mdx mice via "improvement" of the MuSC population, as reported by the authors, even 30 days after administration of MS023. It seems much more likely that MS023 exerts additional effects that are responsible for the dramatic improvement of muscle function in mdx mice. I maintain my view that this needs to be interrogated more carefully since the improvement of muscle function of dystrophic mice is a central point of the study. It has to be made clear whether this is really due to "improved" functions of MuSC. Many other processes might be involved or responsible for the effect (e.g. impact on inflammation?).

Reviewer #3 (Public Review):

The authors report that the secretion of endosome-derived exosomes is enhanced by a calcium-dependent response to damage to the plasma membrane of cells. The authors present convincing evidence that in response to the influx of calcium that follows damage to the plasma membrane annexin A6 is recruited to multivesicular bodies (MVBs) and likely serves to tether the MVBs to the plasma membrane causing a concomitant release of exosomes. Although it is not directly addressed in the Discussion, I am left with the impression that the authors are hinting that exosome secretion is more a byproduct of plasma membrane repair rather than a means of intercellular communication. In other words, the cell needs the membrane material from the MVB to patch and repair holes in the plasma membrane and exosome ejection from the cell is a secondary (perhaps even irrelevant) consequence. Obviously, these two possibilities are not mutually exclusive. The authors are encouraged to speculate about which possibility they favor and how their findings might change our understanding of the cell biology of exosome secretion.

Reviewer #3 (Public Review):

In this manuscript, Li et al. examine how the expression of the chemokine receptor CCR4 impacts the movement of thymocytes within the thymus. It is currently known that the chemokine receptor CCR7 is important for developing thymocytes to migrate from the cortical region into the medullary region and CCR7 expression is therefore often used to define medullary localization. This is important because key developmental outcomes, like enforcing tolerance to self-antigens amongst others, occur in the medullary environment. The authors demonstrate that the chemokine receptor CCR4 is induced on thymocytes prior to expression of CCR7 and thymocytes exhibit responsiveness to CCR4 ligands earlier in development. Using elegant live confocal microscopy experiments, the authors demonstrate that CCR4 expression is important for the entry and accumulation of specific thymocyte subsets while CCR7 expression is needed for the accumulation of more mature thymocyte subsets. The use of cells deficient in both CCR4 and CCR7 and competitive migration/accumulation experiments provide strong support for this conclusion. The elimination of CCR4 expression results in decreases in apoptosis of thymocyte subsets that have been signalled through their antigen receptor and are responsive to CCR4 ligands. As expected, more mature thymocyte subsets show decreased apoptosis when CCR7 is absent. Distinct antigen-presenting cells in the thymus express CCR4 ligands supporting a model where CCR4 expressing thymocytes can interact with thymic antigen-presenting cells for induction of apoptosis. The absence of CCR4 results in an increase in peripheral T cells that can respond to self-antigens presented by LPS-activated antigen-presenting cells providing further support for the model. Collectively, the manuscript convincingly demonstrates a previously unappreciated role for CCR4 in directing a subset of thymocytes to the medulla.

Strengths:

Relevant model systems and elegant experimental techniques are used throughout the manuscript. The experiments are extensively replicated resulting in robust and convincing data sets. These findings represent an important conceptual advance in our understanding of the processes and cellular regulation of T cell development in the thymus.

Weaknesses:

Evidence demonstrating a direct interaction between CCR4 expressing thymocytes and CCR4-ligand expressing antigen-presenting cells is lacking. Furthermore, increased self-reactivity in the absence of CCR4 is measured using mature peripheral CD4 T cells, but altered self-reactivity of thymocytes is not evaluated similarly.

Reviewer #3 (Public Review):

The authors put together a rigorous study to model the impact of HPV vaccine programme disruptions on cervical cancer incidence and meeting WHO elimination goals in a low-income country - using India as an example. The study explores possible scenarios by varying HPV vaccination strategies for 10-year-old children between a) increasing vaccine coverage in a girls-only vaccination programme and b) vaccinating boys in addition to girls (i.e a gender-neutral vaccination programme).

The main strength of this study is the strength of the modelling methodology in helping to make predictions and in contingency planning. The study methodology is rigorous and uses models that have been validated in other settings. The study employs a high level of detail in calibrating and adapting the model to the Indian context despite poor data availability. The detailed methodology allows future studies to employ the model and techniques with locally-contextualised parameters to study the potential impact of HPV vaccine programme disruptions in other countries.

The work in this field can begin to help lower-income countries explore varying HPV vaccination strategies to reduce cervical cancer incidence, keeping in mind the potential for future supply chains or other related disruptions. However, the scenarios could be better sculpted to model potentially realistic scenarios to guide policymakers to make decisions in situations with limited vaccine supplies - in other words comparing scenario alternatives based on a fixed number of vaccines being available. Using comparative alternatives will help policymakers grapple with the decisions that need to be made regarding planning national HPV vaccination programmes. The results could afford to provide readers with a clearer measure of vaccine strategy 'resilience'.

In all, the authors are able to successfully explore the potential impact of varying HPV vaccination strategies on cervical cancer cases prevented in the context of vaccine disruptions, and make valid conclusions. The results produced are rich in information and are worthy of deeper discussion.

Reviewer #3 (Public Review):

Using the zebrafish model system, this manuscript assessed the roles of Rif1 protein in replication timing control and transcription during early development, and successfully demonstrated the differential impact of Rif1 protein in replication timing control and transcription. Moreover, the comprehensive assessments of the impacts of mutating Rif1 on animal development (including animal survival and sexual development) were assessed. Although there are works that examined Rif1's implications in replication timing and transcription separately, this work is unique in assessing all these points at once.

The strength of this manuscript is the genomic analyses of replication timing and transcription being combined in a single model system. Consequently, this manuscript clearly demonstrates the differential impact of Rif1 in these processes during zebrafish development.

The weakness of this manuscript is, as the authors comment in the Discussion, analyses of replication timing and transcription were performed using bulk embryos. There is a possibility that tissue-specific changes could have been masked. Tissue-specific or single-cell analysis in the future will fill the gap in the knowledge.

Some of the findings presented in this manuscript are consistent with previous findings using different models such as Drosophila and mice, whereas other findings do not necessarily agree. I hope further studies will reveal more clearly what is common in these systems, and what is different.

Also, the suggestion that the Rif1 protein may be implicated in a function similar to Fanconi-Anemia genes/proteins is very intriguing.

Overall, the data presented in this manuscript sufficiently justify the authors' claims. Moreover, this manuscript provides interesting insights into Rif1's function, as well as how development could be controlled.

Reviewer #3 (Public Review):

Mullen et al present an important study describing how DHODH inhibition enhances efficacy of immune checkpoint blockade by increasing cell surface expression of MHC I in cancer cells. DHODH inhibitors have been used in the clinic for many years to treat patients with rheumatoid arthritis and there has been a growing interest in repurposing these inhibitors as anti-cancer drugs. In this manuscript, the Singh group build on their previous work defining combinatorial strategies with DHODH inhibitors to improve efficacy. The authors identify an increase in expression of genes involved in the antigen presentation pathway and MHC I after BQ treatment and they narrow the mechanism to be strictly pyrimidine and CDK9/P-TEFb dependent. The authors rationalize that increased MHC I expression induced by DHODH inhibition might favor efficacy of dual immune checkpoint blockade. This combinatorial treatment prolonged survival in an immunocompetent B16F10 melanoma model.

Previous studies have shown that DHODH inhibitors can increase expression of innate immunity-related genes but the role of DHODH and pyrimidine nucleotides in antigen presentation has not been previously reported. A strength of the manuscript is the use of multiple controls across a panel of cell lines to exclude off-target effects and to confirm that effects are exclusively dependent on pyrimidine depletion. Overall, the authors do a thorough characterization of the mechanism that mediates MHC I upregulation using multiple strategies. Furthermore, the in vivo studies provide solid evidence for combining DHODH inhibitors with immune checkpoint blockade.

However, despite the use of multiple cell lines, most experiments are only performed in one cell line, and it is hard to understand why particular gene sets, cell lines or time points are selected for each experiment. It would be beneficial to standardize experimental conditions and confirm the most relevant findings in multiple cell lines. The differential in vivo survival depending on dosing schedule is interesting. However, this section could be strengthened with a more thorough evaluation of the tumors at endpoint.

Overall, this is an interesting manuscript proposing a mechanistic link between pyrimidine depletion and MHC I expression and a novel therapeutic strategy combining DHODH inhibitors with dual checkpoint blockade. These results might be relevant for the clinical development of DHODH inhibitors in the treatment of solid tumors, a setting where these inhibitors have not shown optimal efficacy yet.

Reviewer #3 (Public Review):

Summary:

In their study the authors analyze the localization of multiple organelles and subcellular structure of blood stage malaria parasites with unprecedented detail. They use a 3D super-resolution imaging technique that has gained popularity in the protozoan field, ultrastructure expansion microscopy. Building on markers and labels established in the field they generate an appealing collection of images for all stages of the intraerythrocytic developmental stages of asexual blood stage parasites with some focus on nuclear division and cell segmentation stages.

Strengths:

The authors generated an impressive amount of imaging data that presents the most comprehensive analysis of ultrastructural organization of the parasite cell so far. This atlas can serve as a reference for researchers studying the cell biology of the intraerythrocytic development cycle. The authors achieve a nice catalogue of the reorganization of well-established markers, which together with the improved resolution allows them to highlight some novel observations and consolidate previous findings. They e.g. improve our understanding of organization, duplication and constitutive tethering of the malaria parasite centrosome to the plasma membrane. Further they provide some interesting observations on rhoptry biogenesis, cytostome morphology, and organelle fission during segmentation.

Weaknesses:

While the comprehensiveness of the study is its strength the authors do not present any novel markers, stainings, or imaging protocols. There is no fundamentally new mechanistic insight derived from this study although some earlier findings are consolidated by the higher spatial resolution.

In the following I want to comment on some major points.

Most importantly, in order to justify the authors claim to provide an "Atlas", I want to strongly suggest they share their raw 3D-imaging data (at least of the main figures) in a data repository. This would allow the readers to browse their structure of interest in 3D and significantly improve the impact of their study in the malaria cell biology field.

The organization of the manuscript can be improved. Aside some obvious modifications as citing the figures in the correct order (see also further comments and recommendations), I would maybe suggest one subsection and one figure per analyzed cellular structure/organelle (i.e. 13 sections). This would in my opinion improve readability and facilitate "browsing the atlas".

Considering the importance of reliability of the U-ExM protocol for this study the authors should provide some validation for the isotropic expansion of the sample e.g. by measuring one well defined cellular structure.

In the absence of time-resolved data and more in-depth mechanistic analysis the authors must down tone some of their conclusions specifically around mitochondrial membrane potential, supellicular microtubule depolymerization, and kinetics of the basal complex. More detailed suggestions for improvement are provided as further comments.

In conclusion the authors provide an exciting cell biological reference framework and new working hypotheses about the function of some subcellular structures, which are still largely enigmatic in the malaria parasite, and can be investigated in the future.

Reviewer #3 (Public Review):

The manuscript by Yang et al. investigated in mice how hypobaric hypoxia can modify the RBC clearance function of the spleen, a concept that is of interest. Via interpretation of their data, the authors proposed a model that hypoxia causes an increase in cellular iron levels, possibly in RPMs, leading to ferroptosis, and downregulates their erythrophagocytic capacity. However, most of the data is generated on total splenocytes/total spleen, and the conclusions are not always supported by the presented data. The model of the authors could be questioned by the paper by Youssef et al. (which the authors cite, but in an unclear context) that the ferroptosis in RPMs could be mediated by augmented erythrophagocytosis. As such, the loss of RPMs in vivo which is indeed clear in the histological section shown (and is a strong and interesting finding) can be not directly caused by hypoxia, but by enhanced RBC clearance. Such a possibility should be taken into account.

Major points:

1) The authors present data from total splenocytes and then relate the obtained data to RPMs, which are quantitatively a minor population in the spleen. Eg, labile iron is increased in the splenocytes upon HH, but the manuscript does not show that this occurs in the red pulp or RPMs. They also measure gene/protein expression changes in the total spleen and connect them to changes in macrophages, as indicated in the model Figure (Fig. 7). HO-1 and levels of Ferritin (L and H) can be attributed to the drop in RPMs in the spleen. Are any of these changes preserved cell-intrinsically in cultured macrophages? This should be shown to support the model (relates also to lines 487-88, where the authors again speculate that hypoxia decreases HO-1 which was not demonstrated). In the current stage, for example, we do not know if the labile iron increase in cultured cells and in the spleen in vivo upon hypoxia is the same phenomenon, and why labile iron is increased. To improve the manuscript, the authors should study specifically RPMs.

2) The paper uses flow cytometry, but how this method was applied is suboptimal: there are no gating strategies, no indication if single events were determined, and how cell viability was assessed, which are the parent populations when % of cells is shown on the graphs. How RBCs in the spleen could be analyzed without dedicated cell surface markers? A drop in splenic RPMs is presented as the key finding of the manuscript but Fig. 3M shows gating (suboptimal) for monocytes, not RPMs. RPMs are typically F4/80-high, CD11-low (again no gating strategy is shown for RPMs). Also, the authors used single-cell RNAseq to detect a drop in splenic macrophages upon HH, but they do not indicate in Fig. A-C which cluster of cells relates to macrophages. Cell clusters are not identified in these panels, hence the data is not interpretable).

3) The authors draw conclusions that are not supported by the data, some examples:

a) they cannot exclude eg the compensatory involvement of the liver in the RBCs clearance (the differences between HH sham and HH splenectomy is mild in Fig. 2 E, F and G)

b) splenomegaly is typically caused by increased extramedullary erythropoiesis, not RBC retention. Why do the authors support the second possibility? Related to this, why do the authors conclude that data in Fig. 4 G,H support the model of RBC retention? A significant drop in splenic RBCs (poorly gated) was observed at 7 days, between NN and HH groups, which could actually indicate increased RBC clearance capacity = less retention.

c) lines 452-54: there is no data for decreased phagocytosis in vivo, especially in the context of erythrophagocytosis. This should be done with stressed RBCs transfusion assays, very good examples, like from Youssef et al. or Threul et al. are available in the literature.

d) Line 475 - ferritinophagy was not shown in response to hypoxia by the manuscript, especially that NCOA4 is decreased, at least in the total spleen.

4) In a few cases, the authors show only representative dot plots or histograms, without quantification for n>1. In Fig. 4B the authors write about a significant decrease (although with n=1 no statistics could be applied here; of note, it is not clear what kind of samples were analyzed here). Another example is Fig. 6I. In this case, it is even more important as the data are conflicting the cited article and the new one: PMCID: PMC9908853 which shows that hypoxia stimulates efferocytosis. Sometimes the manuscript claim that some changes are observed, although they are not visible in representative figures (eg for M1 and M2 macrophages in Fig. 3M)

5) There are several unclear issues in methodology:

- what is the purity of primary RPMs in the culture? RPMs are quantitatively poorly represented in splenocyte single-cell suspensions. This reviewer is quite skeptical that the processing of splenocytes from approx 1 mm3 of tissue was sufficient to establish primary RPM cultures. The authors should prove that the cultured cells were indeed RPMs, not monocyte-derived macrophages or other splenic macrophage subtypes.<br /> - (around line 183) In the description of flow cytometry, there are several missing issues. In 1) it is unclear which type of samples were analyzed. In 2) it is not clear how splenocyte cell suspension was prepared.<br /> - In line 192: what does it mean: 'This step can be omitted from cell samples'?<br /> - 'TO method' is not commonly used anymore and hence it was unclear to this Reviewer. Reticulocytes should be analyzed with proper gating, using cell surface markers.<br /> - The description of 'phagocytosis of E. coli and RBCs' in the Methods section is unclear and incomplete. The Results section suggests that for the biotinylated RBCs, phagocytosis? or retention? Of RBCs was quantified in vivo, upon transfusion. However, the Methods section suggests either in vitro/ex vivo approach. It is vague what was indeed performed and how in detail. If RBC transfusion was done, this should be properly described. Of note, biotinylation of RBCs is typically done in vivo only, being a first step in RBC lifespan assay. The such assay is missing in the manuscript. Also, it is not clear if the detection of biotinylated RBCs was performed in permeablized cells (this would be required).

Reviewer #3 (Public Review):

The mechanistically diverse SLC26 transporters play a variety of physiological roles. The current manuscript establishes the SLC26A6 subtype as electroneutral chloride/bicarbonate exchanges and reports its high-resolution structure with chloride bound.

The claims in this manuscript are all well-supported by the data. Strengths include the comprehensive functional analysis of SLC26A6 in reconstituted liposome vesicles. The authors employ an array of assays, including chloride sensors, a newly developed fluorescent probe for bicarbonate, and assays to detect the electrogenicity of anion exchange. With this assortment of assays, the authors are able to establish the anion selectivity and stoichiometry of SLC26A6. Another strength of the manuscript is the functional comparison with SLC26A9, which permits fast, passive chloride transport, in order to benchmark the SLC26A6 activity. The structural analysis, including the assignment of the chloride binding site, is also convincing. The structural details and the chloride binding site are well-conserved among SLC26s. Finally, the authors present an interesting discussion comparing the structures of SLC26A5, SLC26A6, and SLC26A9, and how the details of the chloride binding site might influence the mechanistic distinctions between these similar transporters.

Reviewer #3 (Public Review):

Dux (or DUX4 in human) is a master transcription factor regulating early embryonic gene activation and has garnered much attention also for its involvement in reprogramming pluripotent embryonic stem cells to totipotent "2C-like" cells. The presented work starts with the recognition that DUX contains five conserved c. 100-amino acid carboxy-terminal repeats (called C1-C5) in the murine protein but not in that of other mammals (e.g. human DUX4). Using state-of-the-art techniques and cell models (BioID, Cut&Tag; rescue experiments and functional reporter assays in ESCs), the authors dissect the activity of each repeat, concluding that repeats C3 and C5 possess the strongest transactivation potential in synergy with a short C-terminal 14 AA acidic motif. In agreement with these findings, the authors find that full-length and active (C3) repeat containing Dux leads to increased chromatin accessibility and active histone mark (H3K9Ac) signals at genomic Dux binding sites. A further significant conclusion of this mutational analysis is the proposal that the weakly activating repeats C2 and C4 may function as attenuators of C3+C5-driven activity.

By next pulling down and identifying proteins bound to Dux (or its repeat-deleted derivatives) using BioID-LC/MS/MS, the authors find a significant number of interactors, notably chromatin remodellers (SMARCC1), a histone chaperone (CHAF1A/p150) and transcription factors previously (ZSCAN4D) implicated in embryonic gene activation.

The experiments are of high quality, with appropriate controls, thus providing a rich compendium of Dux interactors for future study. Indeed, a number of these (SMARCC1, SMCHD1, ZSCAN4) make biological sense, both for embryonic genome activation and for FSHD (SMCHD1).

A critical question raised by this study, however, concerns the function of the Dux repeats, apparently unique to mice. While it is possible, as the authors propose, that the weak activating C1, C2 C4 repeats may exert an attenuating function on activation (and thus may have been selected for under an "adaptationist" paradigm), it is also possible that they are simply the result of Jacobian evolutionary bricolage (tinkering) that happens to work in mice. The finding that Dux itself is not essential, in fact appears to be redundant (or cooperates with) the OBOX4 factor, in addition to the absence of these repeats in the DUX protein of all other mammals (as pointed out by the authors), might indeed argue for the second, perhaps less attractive possibility.

In summary, while the present work provides a valuable resource for future study of Dux and its interactors, it fails, however, to tell a compelling story that could link the obtained data together.

Reviewer #3 (Public Review):

Summary. This study sought to clarify the connection between inositol pyrophosphates (PP-IPs) and their regulation of phosphate homeostasis in the yeast Saccharomyces cerevisiae to answer the question of whether any of the PP-IPs (1-IP7, 5-IP7, and IP8) or only particular PP-IPs are involved in regulation. PP-IPs bind to SPX domains in proteins to affect their activity, and there are several key proteins in the PHO pathway that have an SPX domain, including Pho81. The authors use the latest methodology, capillary electrophoresis and mass spectrometry (CE-MS), to examine the cytosolic concentrations of PP-IPs in wild-type and strains carrying mutations in the enzymes that metabolize these compounds in rich medium and during a phosphate starvation time-course for the wild-type.

Major strengths and weaknesses. The authors have strong premises for performing these experiments: clarifying the regulatory molecule(s) in yeast and providing a unifying mechanism across eukaryotes. They use the latest methodologies and a variety of approaches including genetics, biochemistry, cell biology and protein structure to examine phosphate regulation. Their experiments are rigorous and well controlled, and the story is clearly told. The consideration of physiological levels of PP-IPs throughout the study was critical to the interpretation of the data and the strength of the manuscript.

There were a few places in which a deeper discussion of the data was warranted: not discussed was an explanation for the decrease in the levels of all of the PP-IPs upon phosphate starvation, nor of the phosphate regulation of two target genes of Pho4 when Pho4 is constitutively nuclear.

Appraisal. The authors achieved their goal of determining the mechanistic details for phosphate regulation, revising the prior model with new insights. Additionally, they provided strong support for the idea that IP8 regulates phosphate metabolism across eukaryotes - including animals and plants in addition to fungi.

Impact. This study is likely to have a broad impact because it addresses prior findings that are inconsistent with current understanding, and they provide good reasoning as to how older methods were inadequate.

Reviewer #3 (Public Review):

The study by Thommen et al. sought to identify the native role of the Plasmodium falciparum FKBP35 protein, which has been identified as a potential drug target due to the antiplasmodial activity of the immunosuppressant FK506. This compound has multiple binding proteins in many organisms; however, only one FKBP exists in P. falciparum (FKBP35). Using genetically-modified parasites and mass spectrometry-based cellular thermal shift assays (CETSA), the authors suggest that this protein is in involved in ribosome homeostasis and that the antiplasmodial activity of FK506 is separate from its activity on the FKBP35 protein. The authors first created a conditional knockdown using the destruction domain/shield system, which demonstrated no change in asexual blood stage parasites. A conditional knockout was then generated using the DiCre system. FKBP35KO parasites survived the first generation but died in the second generation. The authors called this "a delayed death phenotype", although it was not secondary to drug treatment, so this may be a misnomer. This slow death was unrelated to apicoplast dysfunction, as demonstrated by lack of alterations in sensitivity to apicoplast inhibitors. Quantitative proteomics on the FKBP35KO vs FKBP35WT parasites demonstrated enrichment of proteins involved in pre-ribosome development and the nucleolus. Interestingly, the KO parasites were not more susceptible to cycloheximide, a translation inhibitor, in the first generation (G1), suggesting that mature ribosomes still exist at this point. The SunSET technique, which incorporates puromycin into nascent peptide chains, also showed that in G1 the FKBP35KO parasites were still able to synthesize proteins. But in the second generation (G2), there was a significant decrease in protein synthesis. Transcriptomics were also performed at multiple time points. The effects of knockout of FKBP35 were transcriptionally silent in G1, and the parasites then slowed their cell cycles as compared to the FKBP35WT parasites.

The authors next sought to evaluate whether killing by FK506 was dependent upon the inhibition of PfKBP35. Interestingly, both FKBP35KO and FKBP35WT parasites were equally susceptible to FK506. This suggested that the antiplasmodial activity of FK506 was related to activity targeting essential functions in the parasite separate from binding to FKBP35. To identify these potential targets, the authors used MS-CETSA on lysates to test for thermal stabilization of proteins after exposure to drug, which suggests drug-protein interactions. As expected, FK506 bound FKBP35 at low nM concentrations. However, given that the parasite IC50 of this compound is in the uM range, the authors searched for proteins stabilized at these concentrations as putative secondary targets. Using live cell MS-CETSA, FK506 bound FKBP35 at low nM concentrations; however, in these experiments over 50 ribosomal proteins were stabilized by the drug at higher concentrations. Of note, there was also an increase in soluble ribosomal factors in the absence of denaturing conditions. The authors suggested that the drug itself led to these smaller factors disengaging from a larger ribosomal complex, leading to an increase in soluble factors. Ultimately, the authors conclude that the native function of FKBP35 is involved in ribosome homeostasis and that the antiplasmodial activity of FK506 is not related to the binding of FKBP35, but instead results from inhibition of essential functions of secondary targets.

Strengths:

This study has many strengths. It addresses an important gap in parasite biology and drug development, by addressing the native role of the potential antiplasmodial drug target FKBP35 and whether the compound FK506 works through inhibition of that putative target. The knockout data provide compelling evidence that the KBP35 protein is essential for asexual parasite growth after one growth cycle. Analysis of the FKBP35KO line also provides evidence that the effects of FK506 are likely not solely due to inhibition of that protein, but instead must have secondary targets whose function is essential. These data are important in the field of drug development as they may guide development away from structure-based FK506 analogs that bind more specifically to the FKBP35 protein.

Weaknesses:

There are also a few notable weaknesses in the evidence that call into question the conclusion in the article title that FKBP35 is definitely involved in ribosomal homeostasis. While the proteomics supports alterations in ribosome biogenesis factors, it is unclear whether this is a direct role of the loss of the FKBP35 protein or is more related to non-specific downstream effects of knocking down the protein. The CETSA data clearly demonstrate that FK506 binds PfKB35 at low nM concentrations, which is different than the IC50 noted in the parasite; however, the evidence that the proteins stabilized by uM concentrations of drug are actual targets is not completely convincing. Especially, given the high uM amounts of drug required to stabilize these proteins. This section of the manuscript would benefit from validation of a least one or two of the putative candidates noted in the text. In the live cell CETSA, it is noted that >50 ribosomal components are stabilized in drug treated but not lysate controls. Similarly, the authors suggest that the -soluble fraction of ribosomal components increases in drug-exposed parasites even at 37{degree sign}C and suggests that this is likely from smaller ribosomal proteins disengaging from larger ribosomal complexes. While the evidence is convincing that this protein may play a role in ribosome homeostasis in some capacity, it is not sure that the title of the paper "FKBP secures ribosome homeostasis" holds true given the lack of mechanistic data. A minor weakness, but one that should nonetheless be addressed, is the use of the term "delayed death phenotype" with regards to the knockout parasite killing. This term is most frequently used in a very specific setting of apicoplast drugs that inhibit apicoplast ribosomes, so the term is misleading. It is also possible that the parasites are able to go through a normal cycle because of the kinetics of the knockout and that the time needed for protein clearance in the parasite to a level that is lethal.

Overall, the authors set out to identify the native role of FKB35 in the P. falciparum parasites and to identify whether this is, in fact, the target of FK506. The data clearly demonstrate that FKBP35 is essential for parasite growth and provide evidence that alterations in its levels have proteomic but not transcriptional changes. However, the conclusion that FKBP35 actually stabilizes ribosomal complexes remains intermediate. The data are also very compelling that FK506 has secondary targets in the parasite aside from FKBP35; however, the high uM concentrations of the drug needed to attain results and the lack of biological validation of the CETSA hits makes it difficult to know whether any of these are actually the target of the compound or instead are nonspecific downstream consequences of treatment.

Reviewer #3 (Public Review):

The authors investigated the mechanism of transport of the GLUT5 sugar porter using enhanced sampling molecular dynamics simulations and biochemical analysis.<br /> The results suggest a possible general mechanism by which binding to a transported substrate stabilizes an occluded intermediate conformation between outward and inward-facing states of the alternating access conformational change of the protein, thereby enabling transport.

The authors also identified key elements of this transition, associated with residues involved in sugar binding, and through elegant biochemical experiments demonstrated how mutations of the latter affect the protein function, including mutations of gating residues that can recover the function of inactive mutants.<br /> The general computational methodology used by authors is appropriate for addressing these questions and compared to other techniques has the advantage of bringing forth an unbiased molecular description of the transport process. The results are overall qualitatively in line with the proposed conclusions.

A major weakness of this work is that, in contrast to previous studies with the same type of methodology, the authors do not report error analysis or careful statistical assessment of the computational results. Therefore, it is not clear whether the latter is solid or if they support the proposed conclusions. The computational data might generally benefit from an improved methodological design, such as including more degrees of freedom (or collective variables) in the description of the minimum free energy pathway, e.g. the salt-bridges.

Another weakness is that some of the details of the computational analysis are not reported, therefore other investigators would not know how to reproduce the results.

Once these issues are addressed, this work could potentially provide important insights into the mechanism of transport of sugar porters, which as suggested by other recent studies might also apply to other types of membrane transporters.

Reviewer #3 (Public Review):

In this work, Elbahnsi and colleagues use enhanced sampling MD simulation, to recapitulate step by step, the electromechanical coupling between VSD and the pore in HCN1 channels. Building on the available cryoEM structures of HCN1 with the VSD in resting and active state, the authors characterize by MD a subset of interactions that seemingly stabilize the open channel. This subset is, in turn, used in enhanced-sampling simulations to guide channel opening.<br /> The main findings are that S4 movement induces a rearrangement of the hydrophobic interaction at the level of S1- S4- and S5 interfaces. Occupancy of lipids seems therefore state-dependent and highlights their regulatory role in HCN gating.

The approach is rather innovative, and it apparently allows the reconstruction of the whole mechanism of gating, pushing the predictive power of MD simulation well beyond its actual temporal limitations. At the same time, the initial choice of interactions is crucial for this approach, because the result cannot differ from the inputs. And reading the paper it does not emerge clearly how the correctness of the reconstructed gating pathway can be verified, if not by functional validation.

Here are my comments on the main interactions that were used to feed the final MD simulation:

1. W281-N300: this interaction, previously identified and studied in SpH channels (Ramentol et al, 2020; Wu et al, 2021), has been elegantly confirmed in this paper. Its inclusion in the initial subset seems appropriate.<br /> In the other two cases, the choice of interactions requires further explanations and experimental validation.

2. D290 and K412: the validation of this interaction shown in Figure 3 and suppl Figure 1 is missing a control, i.e., the effect of the addition of Cd++ on the wt channel. Please add.

3. Modelling the open state of HCN1 pore (page 18), is done on the structure of the distantly related hERG rather than on the available open pore structure of HCN4. This choice is justified as follows by the authors:

a) "Available structures in the CNBD channel family for which representative structures have been solved in closed and open states".<br /> b) "The structural mechanism of pore gating (i.e. the ⍺ to 𝜋 helix occurring at the glycine657 hinge in hERG) observed in rEAG/hERG may be a conserved gating transition in the CNBD family of channels"<br /> I encourage the authors to consider the following:

a) The structure of hERG channel is not available in the closed/open configuration, indeed the comparison must be done with the closed configuration of the related channel rEAG. On the contrary, HCN4 is available in the closed/open configurations. Moreover, one of the open pore structures shows S4-S5-S6 in a very similar conformation to the lock open mutant (F186C/S264C) of HCN1 (Saponaro et al, 2021). With an available HCN4 open structure, forcing HCN1 to the open pore structure of hERG channel (which opens in depolarization and is not regulated by cAMP) seems not necessary.

To my knowledge, hERG is the only channel of the CNBD family for which the transition ⍺ to 𝜋 helix reported by the Authors, occurs in S6. It is not reported for other CNBD family members, in particular for the CNG channels mentioned by the Authors (Zheng et al., 2020; Xue et al., 2021, 2022). Task 4 (Zheng et al) does not show it. Its pore opens by a right-handed twist of S6 at glycine 399, a conserved glycine in all CNG. Human CNGA1 too, opens the pore by a rotational movement of S6 hinged at the equivalent glycine (glycine 385) (Xue et al, 2021). And the same occurs in the non-symmetrical channel CNGA1/B1 (Xue te al, 2022). So, it seems that CNG channels do not show the ⍺ to 𝜋 helix transition in the open pore. Moreover, hERG excluded, all other members of the CNBD family, CNG, EAG, and HCN4 included, do not bend at the hinge glycine 657 of hERG, but at another glycine (gly 648 in hERG numbering) located upstream. Further, their opening is due to a rotation of S6 associated with an outward movement, rather than to the lifting of the lower part of S6, as in hERG.

4- V390-I302: this interaction is predicted to stabilize the open pore configuration and was included in the subset. The contact between V390 on S6 and I302 on S5 is observed in the homology model discussed above when the S6 is twisted at the glycine hinge, rotating the preceding residue (V390) out of its pore-lining position and is.<br /> Again, I can only disagree with this hypothesis because it has been experimentally demonstrated (Cheng et al, J Pharmacol Exp Ther. 2007 Sep;322(3):931-9) that the side chain of Valine390 is inside the cavity of the open pore of HCN1 channels as it controls the affinity for the pore blocker ZD7288.

In conclusion, modelling the open state pore of HCN1 on hERG rather than on that of HCN4 seems not justified based on accumulated evidence in the published literature. Therefore, the choice of the authors to use it as the open pore model of HCN1 channels needs to be experimentally validated. One possibility is to mutate the glycine hinge, gly391 in HCN1, into an Alanine in order to remove the flexible hinge. If this mutation alters pore gating, it will support the choice of the Authors.

Reviewer #3 (Public Review):

Parab et al. investigate the requirement of specific Vegf ligands during the embryonic development of new blood vessels in different brain regions. The authors implement their previously published experimental paradigm (Parab et al 2021 eLife) combined with new transgenic and mutant zebrafish lines to show that vegf ligands (vegfaa, vegfab, vegfc, and vegfd) are required in various combinations to drive angiogenesis in distinct brain regions. Specifically, they show that individual loss of different vegf ligands causes either undetectable or partial effects in angiogenesis, while combined loss of vegf ligands results in severe defects in brain region-specific angiogenesis. As different blood vessel types (i.e. arteries, veins, lymphatics) require specific angiogenic cues, this study provides interesting new data on how the combination of these signals drives brain region-specific vascular development.

While the conclusions of the paper are generally well supported by the data, the authors overstate some of their findings, particularly with respect to the development of fenestrated capillaries. In this study, the authors use the zebrafish transgenic reporter line, plvap:EGFP, as an indicator of fenestrations. However, the authors do not provide any evidence of fenestrations of the blood vessels of the choroid plexuses or the cranial vessels used for quantification (Figures 1, 3, and 4). While expression of Plvap protein is often used as a marker for non-blood brain barrier endothelial cells, as Plvap is the major component of the diaphragms of fenestrated capillaries, plvap:EGFP expression alone does not indicate fenestrations. This is an important point because previous work has demonstrated that targeted deletion of Plvap does not cause a loss of fenestrations, but instead a loss of the diaphragms associated with fenestrations (Stan et al 2012 Dev Cell; Gordon et al 2019 Development). Similarly, Plvap expression alone does not necessarily indicate fenestrations as an expression of Plvap is not sufficient for fenestration formation. In fact, Plvap has initially been expressed in brain endothelial cells during initial angiogenesis to the brain without evidence of fenestrations, and subsequently, Plvap expression disappears during the maturation of the BBB. Thus, to conclude that specific vegf ligands are required for the development of fenestrated capillaries, transmission electron microscopy (TEM) should be used on the capillaries examined in this study or the language describing the results should be modified accordingly. Conversely, the authors did show TEM for the choriocapillaris (Figure 5A-C) but did not show plvap:EGFP expression in these vessels.

Additionally, the authors' usage of the phrase "development of fenestrated vessels" suggests that the study was examining signals that regulate the formation of fenestrations and not angiogenesis of vessels that may become fenestrated as demonstrated here. Therefore, as Plvap expression does not necessarily equate fenestrations (and vice-versa), the title and some of the major claims of the study are somewhat overstated.

Reviewer #3 (Public Review):

The study presents a systematic analysis of how a range of dystroglycan mutations alter CCK/CB1 axonal targeting and inhibition in hippocampal CA1 and impact seizure susceptibility. The study follows up on prior literature identifying a role for dystroglycan in CCK/CB1 synapse formation. The careful assay includes comparison of 5 distinct dystroglycan mutation types known to be associated with varying degrees of muscular dystrophy phenotypes: a forebrain specific Dag1 knockout in excitatory neurons at 10.5, a forebrain specific knockout of the glycosyltransferase enzyme in excitatory neurons, mice with deletion of the intracellular domain of beta-Dag1 and 2 lines with missense mutations with milder phenotypes. They show that forebrain glutamatergic deletion of Dag1 or glycosyltransferase alters cortical lamination while lamination is preserved in mice with deletion of the intracellular domain or missense mutation. The study extends prior works by identifying that forebrain deletion of Dag1 or glycosyltransferase in excitatory neurons impairs CCK/CB1 and not PV axonal targeting and CB1 basket formation around CA1 pyramidal cells. Mice with deletion of the intracellular domain or missense mutation show limited reductions in CCK/CB1 fibers in CA1. Carbachol enhancement of CA1 IPSCs was reduced both in forebrain knockouts. Interestingly, carbachol enhancement of CA1 IPSCs was reduced when the intracellular domain of beta-Dag1was deleted, but not I the missense mutations, suggesting a role of the intracellular domain in synapse maintenance. All lines except the missense mutations , showed increased susceptibility to chemically induced behavioral seizures. Together, the study, is carefully designed, well controlled and systematic. The results advance prior findings of the role for dystroglycans in CCK/CB1 innervations of PCs by demonstrating effects of more selective cellular deletions and site specific mutations in extracellular and intracellular domains. The interesting finding that deletion of intracellular domain reduces both CB1 terminals in CA1 and carbachol modulation of IPSCs warrants further analysis. Lack of EEG evaluation of seizure latency is a limitation.

Specific comments<br /> 1. Whether CCK/CB1 cell numbers in the CA1 are differentially affected in the transgenic mice is not clarified.<br /> 2. Whether basal synaptic inhibition is altered by the changes in CCK innervation is not examined.

Reviewer #3 (Public Review):

With a soft-spoken, matter-of-fact attitude and almost unwittingly, this brilliant study chisels away one of the pillars of hippocampal neuroscience: the special role(s) ascribed to theta oscillations. These oscillations are salient during specific behaviors in rodents but are often taken to be part of the intimate endowment of the hippocampus across all mammalian species, and to be a fundamental ingredient of its computations. The gradual anticipation or precession of the spikes of a cell as it traverses its place field, relative to the theta phase, is seen as enabling the prediction of the future - the short-term future position of the animal at least, possibly the future in a wider cognitive sense as well, in particular with humans. The present study shows that, under suitable conditions, place cell population activity "sweeps" to encode future positions, and sometimes past ones as well, even in the absence of theta, as a result of the interplay between firing rate adaptation and precise place coding in the afferent inputs, which tracks the real position of the animal. The core strength of the paper is the clarity afforded by the simple, elegant model. It allows the derivation (in a certain limit) of an analytical formula for the frequency of the sweeps, as a function of the various model parameters, such as the time constants for neuronal integration and for firing rate adaptation. The sweep frequency turns out to be inversely proportional to their geometric average. The authors note that, if theta oscillations are added to the model, they can entrain the sweeps, which thus may superficially appear to have been generated by the oscillations.

The main weakness of the study is the other side of the simplicity coin. In its simple and neat formulation, the model envisages stereotyped single unit behavior regulated by a few parameters, like the two time constants above, or the "adaptation strength", the "width of the field" or the "input strength", which are all assumed to be constant across cells. In reality, not only assigning homogeneous values to those parameters seems implausible, but also describing e.g. adaptation with the simple equation included in the model may be an oversimplification. Therefore, it remains important to understand to what extent the mechanism envisaged in the model is robust to variability in the parameters or to eg less carefully tuned afferent inputs.

The weak adaptation regime, when firing rate adaptation effectively moves the position encoded by population activity slightly ahead of the animal, is not novel - I discussed it, among others, in trying to understand the significance of the CA3-CA1 differentiation (2004). What is novel here, as far as I know, is the strong adaptation regime, when the adaptation strength m is at least larger than the ratio of time constants. Then population activity literally runs away, ahead of the animal, and oscillations set in, independent of any oscillatory inputs. Can this really occur in physiological conditions? A careful comparison with available experimental measures would greatly strengthen the significance of this study.

Reviewer #3 (Public Review):

In this manuscript, D'Ambra and colleagues report the effects of stimulating the deep cerebellar nuclei (DCN) on neurons in the core and the medial shell of the nucleus accumbens (NAc). Electrical stimulation results in both excitation and inhibition, with excitation preceding the inhibition. In general, neurons that underwent excitation had lower baseline activity than neurons that underwent inhibition. They observed no relationship between the location of the stimulation site within the DCN, and the type of response observed in the NAc. In order to identify disynaptic connections between the two areas, the authors combined the injection of a retrograde tracer in the NAc with an anterograde tracer in the DCN. These experiments led them to describe co-localization of the anterograde and retrograde signals within two regions, the intralaminar thalamus (IL), and the ventral tegmental area (VTA). In order to confirm these results, they then used an anterograde transsynaptic viral tracing strategy to mark neurons in the IL and the VTA that project to the NAc. In addition, by injecting an excitatory opsin into the DCN, and stimulating these axons within the VTA and the IL, the authors were able to demonstrate increased activity in the NAc and describe the latency of these responses. Thus, using a series of rigorous and complementary experiments, the authors provide evidence for a disynaptic connection between the DCN and the NAc, via the VTA and the IL.

Novelty and relationship to previous studies: The presence of a disynaptic connection between the DCN and the NAc has previously been shown, as has the projection from the DCN to the parafascicular nucleus of the intralaminar thalamus (Fujita et al. 2020); however, the intermediary nodes of the disynaptic connection between the DCN and NAc have not previously been mapped. Some other pieces of these results have also been shown previously: DCN to VTA: Watabe-Uchida et al. 2012, DCN-VTA-NAc Beier et al. 2015, Xiao and Schieffele 2018) Interestingly, the Beier et al. paper suggests that the connection from DCN-VTA-NAc is an extremely small proportion of the total inputs to the NAc. In contrast to the Fujita et al. paper, here the authors also stimulate or trace projections from the two other deep cerebellar nuclei, the lateral and the interposed (this is relevant for a comment below). In addition, previous studies have shown a projection from the DCN to the IL and, separately, from the IL to the NAc. Thus, the existence of the pathways described here is in line with previous work. Moreover, this study expands on previous ones through its electrophysiological measurement and description of neural responses to stimulation of DCN and DCN projections.

Strengths: The strengths of this paper include the authors' use of multiple techniques to confirm the presence of the connections that they describe. Any one of the experiments using electrical stimulation, combining anterograde and retrograde tracing, transsynaptic tracing, or optical stimulation of DCN axons in the IL and VTA has its own caveats. However, the combination of these techniques nullifies many of these caveats.

Weaknesses: While this is an interesting and exciting paper, there are a few weaknesses, listed below:

- The novelty of this paper lies in the mapping of projections from the interposed and the lateral nuclei of the cerebellum, as the authors themselves mention. However, in some of the experiments the medial nucleus is also clearly injected (Fig. 4B and 6B). In those experiments, it is impossible to distinguish which nucleus these projections come from, and they could be the ones from the medial nucleus that were previously described (see above).<br /> - A strength of the paper is the use of both electrical and optogenetic stimulation. However, the responses to the two in the NAc are very different - electrical stimulation results in both excitation and inhibition, whereas opto stimulation mostly results in only excitation.<br /> - The stimulation frequency at which the electrical stimulation in Fig 1 is done to identify responses in the NAc is 200 Hz for 25 ms. Is this physiological? In addition, responses in the NAc are measured for 500 ms after, which is a very long response time.<br /> - Previous studies have described how different cell types within the DCN have different downstream projections (Fujita et al. 2020). However, the experiments here bundle together all this known heterogeneity.<br /> - Previous studies have also highlighted the importance of different cell types within the NAc and how input streams are differentially targeted to them. Here, that heterogeneity is also obscured.<br /> - In Fig. 4C, E and F, the experiments on overlap between anterograde and retrograde tracers are not particularly convincing - it's hard to see the overlap.

Reviewer #3 (Public Review):

This work proposes a novel computational methodology that, using available structures of homologous proteins in different structural states, evolutionary couplings and machine-learning protocols, allows to predict structural states of a membrane transporter during the transport cycle. The core of the methodology is to use convolutional neural networks to distinguish state specific evolutionary contacts and drive alphafold2 models into a specific state based on the predicted contacts (using rosettaMP and short MD relaxation). The authors then derived the free energy landscape of the alternating access transition of GLUT5 (in absence of substrate) from enhanced sampling simulations biased along variables based on the previously mentioned contacts. The variables are constructed using a machine learning approach that allows distinguishing different structural states.

The advantage of this approach is that it uses a combination of advanced modeling and innovative computational techniques that might help the structural characterization of the alternating access cycle of membrane transporters. An important innovation is the use of machine learning methods that, based on previous structural information, allow to construct collective variables for free energy calculations in an objective, data-driven manner.

The results of the modellng part of the work are encouraging but could benefit from using more specific descriptors that better distinguish structural differences between states.

An important weakness of this work is that there are critical flaws in the simulation analysis. Another weakness is that the different free energy landscapes calculated do not appear strongly consistent to each other, which suggests the presence of significant errors in the calculations that are not discussed. An additional important point is that a quantitative assessment of the quality of the models used in the simulations is currently lacking and this could affect the reliability of the simulation results. In this regard, previous systematic studies (Proteins 2012; 80:2071-2079) have shown that small imperfections in the predicted models (such as in backbone and side chains conformations) could lead to simulations that drift away from the initial structure in the multi microseconds time domain.

Reviewer #3 (Public Review):

There is a lack of consensus about the best way to isolate EVs from biofluids, mainly due to EVs being present at low levels in clinically relevant samples and difficult to quantify. As a following study of one previous eLife paper (https://elifesciences.org/articles/70725) from the same group, the authors have extended their Simoa assay to ApoB-100, the major protein component of several lipoproteins. Combining with previously developed Simoa assays, the authors developed a quick framework to quantify EVs, albumin, and lipoproteins on the same platform. Additionally, the authors developed a new EV isolation method that combines two additional resins (i.e., cation-exchange resin and Capto Core 700) as a bottom layer below the SEC layer. Although not greater than the density gradient centrifugation, EVs isolated using the newly developed method showed better purity than with SEC alone or dual-mode chromatography. A device automatically running columns in parallel for EV isolation was further developed to increase the throughput and reproducibility of column-based EV isolation. The development of Simoa assay to ApoB-100 and the Tri-Mode Chromatography would be of great relevance to EV studies.

Reviewer #3 (Public Review):

In this manuscript, Chao et al seek to understand the role of brummer, a triglyceride lipase, in the Drosophila testis. They show that Brummer regulates lipid droplet degradation during differentiation of germ and somatic cells, and that this process is essential for normal development to progress. These findings are interesting and novel, and contribute to a growing realisation that lipid biology is important for differentiation.

Major comments:

1) The data in Figs 1 and 2, while helpful in setting the scene, do not add much to what was previously shown by the same group, namely that lipid droplets are present in both early germ cells and early somatic cells in the testis, and that Bmm regulates their degradation (PMID: 31961851). Measuring the distance of lipid droplets from the hub, while helpful in quantifying what is apparent, that only stem and early differentiated stages have lipid droplets, is not as informative as the way data are presented later (Fig. 2I), where droplets in specific stages are measured. Much of this could be condensed without much overall loss to the manuscript.

2) It would be important to show images of the clones from which the data in Fig. 2I are generated. The main argument is that Bmm regulates lipid droplets in a cell autonomous manner; these data are the strongest argument in support of this and should be emphasised at the expense of full animal mutants (which could be moved to supplementary data). Similarly, the title of Fig. S2 ("brummer regulates lipid droplets in a cell autonomous manner") should be changed as the figure has no experiments with cell (or cell-type)-specific knockdowns/mutants. This figure does show changes in lipid droplets in both lineages in bmm mutants, so an appropriate title could be "brummer regulates lipid droplets in both germ and soma".

3) Interestingly, the clonal data show that bmm is dispensable in germ cells until spermatocyte stages, as no increase in lipid droplet number is seen until then. This should be more clearly stated, as it indicates that the important function of Bmm is to degrade lipid droplets at the transition from spermatogonial to spermatocyte stages. This is consistent with the phenotypes observed in which late stage germ cells are reduced or missing. However, the effect on niche retention of the mutant GSCs at the expense of neighbouring wildtype GSCs is hard to explain. Are lipid droplets in mutant GSCs larger than in control? Is there any discernible effect of bmm mutation on lipids in GSCs? Additionally, bam expression is delayed, suggesting that bmm may have roles on cell fate in earlier stages than its roles that can be detected on lipid droplets.

4) The bmm loss-of-function phenotype could be better described. Some of the data is glossed over with little description in the text (see for example the reference to Fig. 3A-C). For instance, in the discussion, the text states "loss of bmm delays germline differentiation leading to an accumulation of early-stage germ cells" (p13, l.259-60). However, this accumulation has not been clearly shown, or at least described in the manuscript. Most of the data show a reduction (or almost complete absence) of differentiated cell types. This could indeed be due to delayed differentiation, or alternatively to a block in differentiation or to death of the differentiated cells. The clonal data presented show a decrease in the number of cells recovered, but do not allow inferences as to the timing of differentiation, making it hard to distinguish between the various possibilities for the lack of differentiated spermatids. Apart from data showing that GSCs are more likely to remain at the niche, no further data are shown to support the fact that mutant germ cells accumulate in early stages. While additional experiments could help resolve some of these issues, much of this could also be resolved by tempering the conclusions drawn in the text.

5) In the discussion (p.14, l-273 onwards), the authors suggest that products of triglyceride breakdown are important for spermatogenesis. However, an alternative interpretation of the results presented here (especially those using the midway mutant) could be that triglycerides impede normal differentiation directly. Indeed, preventing the cells' ability to produce triglycerides in the first place can rescue many of the defects observed. A better discussion of these results with a model for the function of triglycerides and their by-products would be a great improvement to this manuscript.

Reviewer #3 (Public Review):

In this manuscript, Man et al. describe a new signaling pathway for regulation of the voltage-gated calcium channel Cav1.2 and show that it can modulate synaptic plasticity in the hippocampus. Studies with specific inhibitors, phosphopecific antibodies, and gene knockdown show that activation of alpha-1 adrenergic receptors induces downstream activation of the serine/threonine protein kinase PKC and the tyrosine protein kinases Pyk2 and Src, which bind to the Cav1.2 channel through its large intracellular segment connecting domains II and III. This signaling complex leads to tyrosine phosphorylation of Cav1.2 and increased channel activity. Block of this novel signaling pathway in hippocampal slices with specific inhibitors of Pyk2 and Src reduced a specific component of long-term potentiation whose induction requires Cav1.2 channel activity.

This work is an important advance, as it presents a novel signaling pathway through which the ubiquitous neurotransmitter norepinephine and the neurohormone epinephrine can regulate synaptic plasticity, attention, learning, and memory. The experiments are comprehensive, carefully done, and clearly presented. The authors should consider revisions and responses to the points below.

1. Figure 2B, D. Inhibitors reduce Ica below control. Is there endogenous stimulation of this regulatory pathway under control conditions?

2. As noted by the authors, it would be interesting to know if peptides from the linker between domains II and III block this signaling pathway. This would be an important result because, without this information, it is not clear if this is the correct functional site of interaction for this regulatory complex.

3. Figure 4B. The Brain IP for Src has a weak signal. The authors should replace this panel with a more convincing immunoblot.

4. Scatter plots are provided for the electrophysiological results but not immunoblots. For immunoblots that are quanitified, it would be valuable to add a scatter plot of the replicates.

Reviewer #3 (Public Review):

This manuscript by Modi et al represents a novel and significant advance in the neurobiology of memory retrieval. The authors employ a novel behavioral paradigm in order to investigate memory generalization and discrimination. They investigate the role of two different populations of dopamine neurons (DANs) targeting different compartments involved in aversion learning, i.e. α3 (MB630B) and γ2α'1 (MB296B).

The behavioral platform is clear and convincing but lacks natural reinforcement comparisons. The entire paper uses optogenetic reinforcement of said DAN populations.

The authors identify that the gamma DANs can enable both easy and hard odour discrimination, while the alphas DANs can only do easy.

The odours can be separated by calcium imaging analysis of Kenyon cells. Subsequent calcium imaging of the gamma DANs themselves showed that a single training event was insufficient to enable easy odor discrimination at the gamma DAN level, but strangely not for the hard discrimination that gamma DANs can mediate. Seemingly, this is due to the lack of the temporal contiguity of odors (present in behavioral experiments but not in the initial imaging experiments.

However, in gamma DANs, Odour transitions enabled discrimination of odours in hard discrimination, based on the depression of calcium activity in DANs after training that was odour-specific. The same was not true for alpha DANs, though the authors used natural electric shock pairings instead of optogenetic stimulation of DANs for the alpha experiment. However, statistical comparisons are done within group and need also be provided for between the groups for both pre and post-training. The authors persuasively show that hard discrimination can only happen in transitions. They also argue that the same engram can be read in two different ways. This is convincing overall, but they claim it is happening downstream of the Kenyon cells just because they do not see it in the Kenyon cells, and I cannot comment on the modelling in Figure 5 (expertise).

Experimental methods used are appropriate, as are data analysis strategies.

The manuscript itself is well written in parts, though at times paragraphs are quite patchy, especially in the discussion. There are also a visible number of typos. The figures are well constructed, and generally well organized. The overall document is concise and has sufficient detail.

Reviewer #3 (Public Review):

Clay and colleagues investigate the proteostasis and longevity benefits derived from translation inhibition in C. elegans by examining the impacts of chemical translation initiation inhibitors (IIs) and translation elongation inhibitors (EIs) on thermotolerance, protein folding stress, aggregation and longevity. They observe somewhat distinct impacts by the two chemical groups. IIs increased longevity in wild-type animals in an hsf-1 dependent manner, whereas, EIs only extended hsf-1 mutants' lifespan. Only EIs protected against proteasome dysfunction. Both protected against heat stress but with differing hsf-1 dependence. The authors utilize these observations to derive conclusions regarding two dominant points of view on the mechanism by which translation inhibition improves lifespan and proteostasis.<br /> The study is based on interesting observations and several promising avenues of further investigation can be identified. However, the manuscript appears somewhat preliminary in nature, with many of the observations, while interesting, only explored superficially for mechanistic insights. The rationale behind some of the interpretations was also difficult to interpret. For example, the authors make conclusions about 'selective translation' being adopted upon IIs treatment without directly testing this. Protein aggregation, while possibly predictive, is not a reliable readout for selective translation of some mRNAs. Similarly, the evidence for a reduction in 'newly-synthesized protein load' by EIs is thin based on one reporter. Previous studies from the Blackwell lab have identified differential impacts of SKN-1 on select cytoprotective genes' expression and proteasomal gene expression based on inhibition of translation initiation or elongation. So there is precedence for both the differential impact of initiation vs. elongation inhibition as well as genetic background. There are several other such studies that reduce the impact of the observations presented here. With limited novelty and mechanistic insight, the impact of the study on the field is likely to be moderate.

Seventh chord identification 3

Triad identification (root position) 3

Schubert Symphony No 3 II

J.S. Bach BWV1002 Violin Partita No. 1 Sarabande

Intervals exercises 3

Reviewer #3 (Public Review):

How chromatin state is defined is an important question in the epigenetics field. Here, Jamge et al. proposed that the dynamics of histone variant exchange control the organization of histone modifications into chromatin states. They found 1) there is a tight association between H2A variants and histone modifications; 2) H2A variants are major factors that differentiate euchromatin, facultative heterochromatin, and constitutive heterochromatin; 3) the mutation in DDM1, a remodeler of H2A variants, causes the mis-assembly of chromatin states in TE region. The topic of this paper is of general interest and results are novel.

Overall, the paper is well-written and results are clearly presented. The biochemical analysis part is solid.

Reviewer #3 (Public Review):

Neininger-Castro and colleagues developed software tools for the quantification of sarcomeres and sarcomere-precursor features in immunostained human induced pluripotent stem cell-derived cardiac myocytes (hiCMs). In the first part they used a deep-learning- based model called a U-Net to construct and train a network for binarization of immunostained cardiomyocyte images. They also wrote graphical user interface (GUI) software that will assist other labs in using this approach and made it publicly available. They did not compare their approach to existing ones, but an example from one image suggests their binarization tool outperforms Otsu thresholding binarization.

In the second part they developed a software tool called sarcApp that classifies sarcomere structures in the binarized image as a Z-Line or Z-Body and assigns each to either a myofibril or to stress fibers. The tools can then automatically count and measure multiple features (33 per cell and 24 per myofibril) and report them on a per-cell, per-myofibril, and per- stress fiber basis.

To test the tools they used Blebbistatin to inhibit sarcomere assembly and showed that the sarcApp tool could capture changes in multiple features such as fewer myofibrils, fewer Z-Lines, decreased myofibril persistence, decreased Z-Line length and altered myofibril orientation in the Blebbistatin treated cells. With some changes the tool was also shown to quantify sarcomeres in titin and myomesin stained cardiomyocytes.

Finally they used sarcApp to quantify the changes in sarcomere assembly after siRNA mediated knockout of MYH7, MYH7, or MYOM. The analysis indicates that neither MYH6 nor MYH7 knockdown perturbed the assembly of Z- or M-lines, and that knockdown of MYOM perturbed the A-band/M-Line but not the Z-Line assembly according to features captured by the sarcApp tool.

Overall the authors developed and made publicly available an excellent software tool that will be very useful for labs that are interested in studying sarcomere assembly. Multiple features that are difficult to measure or count manually can be automatically measured by the software quickly and accurately.

There are however some remaining questions about these tools:<br /> 1. The binarization tool which is tailored to sarcomere image binarization appears promising but was not systematically compared with existing approaches.<br /> 2. How robust is the tool? The tool was tested on images from one type of cardiomyocytes (hiCMs) taken from one lab using Nikon Spinning Disk confocal microscope equipped with Apo TIRF Oil 100X 1.49 NA objective or instant Structured Illumination Microscopy (iSIM), using deconvolution (Microvolution software) and in a specific magnification. It remains to be seen whether the tool would be equally effective with images taken with other microscopy systems, with other cardiomyocytes (chick or neonatal rat), with different magnifications, live imaging, etc.<br /> 3. The tool was developed for evaluation of sarcomere assembly. The authors show that for this application it can detect the perturbation by Blebbistatin, or knockdown of sarcomeric genes. It remains to be seen if this tool is also useful for assessment of sarcomere structure for other questions beside sarcomere assembly and in other sarcomere pathologies.

Reviewer 3 (Public Review):

The main question of this article is as follows: "To what extent does having information on brain-age improve our ability to capture declines in fluid cognition beyond knowing a person's chronological age?" While this question is worthwhile, considering that there is considerable confusion in the field about the nature of brain-age, the authors are currently missing an opportunity to convey the inevitability of their results, given how brain-age and the brain-age gap are calculated. They also argue that brain-cognition is somehow superior to brain-age, but insufficient evidence is provided in support of this claim.

Specific comments follow:

- "There are many adjustments proposed to correct for this estimation bias" (p3). Regression to the mean is not a sign of bias. Any decent loss function will result in over-predicting the age of younger individuals and under-predicting the age of older individuals. This is a direct result of minimizing an error term (e.g., mean squared error). Therefore, it is inappropriate to refer to regression to the mean as a sign of bias. This misconception has led to a great deal of inappropriate analyses, including "correcting" the brain age gap by regressing out age.

- "Corrected Brain Age Gap in particular is viewed as being able to control for both age dependency and estimation biases (Butler et al., 2021)" (p3). This summary is not accurate as Butler and colleagues did not use the words "corrected" and "biases" in this context. All that authors say in that paper is that regressing out age from the brain age gap - which is referred to as the modified brain age gap (MBAG) - makes it so that the modified brain age gap is not dependent on age, which is true. This metric is meaningless, though, because it is the variance left over after regressing out age from residuals from a model that was predicting age. If it were not for the fact that regression on residuals is not equivalent to multiple regression (and out of sample estimates), MBAG would be a vector of zeros. Upon reading the Methods, I noticed that the authors use a metric from Le et al. (2018) for the "Corrected Brain Age Gap". If they cite the Butler et al. (2021) paper, I highly recommend sticking with the same notation, metrics and terminology throughout. That would greatly help with the interpretability of the present manuscript, and cross-comparisons between the two.

- "However, the improvement in predicting chronological age may not necessarily make Brain Age to be better at capturing Cognitionfluid. If, for instance, the age-prediction model had the perfect performance, Brian Age Gap would be exactly zero and would have no utility in capturing Cognitionfluid beyond chronological age" (p3). I largely agree with this statement. I would be really careful to distinguish between brain-age and the brain-age gap here, as the former is a predicted value, and the latter is the residual times -1 (i.e., predicted age - age). Therefore, together they explain all of the variance in age. Changing the first sentence to refer to the brain-age gap would be more accurate in this context. The brain-age gap will never be exactly zero, though, even with perfect prediction on the training set, because subjects in the testing set are different from the subjects in the training set.

- "Can we further improve our ability to capture the decline in cognitionfluid by using, not only Brain Age and chronological age, but also another biomarker, Brain Cognition?". This question is fundamentally getting at whether a predicted value of cognition can predict cognition. Assuming the brain parameters can predict cognition decently, and the original cognitive measure that you were predicting is related to your measure of fluid cognition, the answer should be yes. Upon reading the Methods, it became clear that the cognitive variable in the model predicting cognition using brain features (to get predicted cognition, or as the authors refer to it, brain-cognition) is the same as the measure of fluid cognition that you are trying to assess how well brain-cognition can predict. Assuming the brain parameters can predict fluid cognition at all, it is then inevitable that brain-cognition will predict fluid cognition. Therefore, it is inappropriate to use predicted values of a variable to predict the same variable.

- "However, Brain Age Gap created from the lower-performing age-prediction models explained a higher amount of variation in Cognitionfluid. For instance, the top performing age-prediction model, "Stacked: All excluding Task Contrast", generated Brain Age and Corrected Brain Age that explained the highest amount of variation in Cognitionfluid, but, at the same time, produced Brian Age Gap that explained the least amount of variation in Cognitionfluid" (p7). This is an inevitable consequence of the following relationship between predicted values and residuals (or residuals times -1): y=(y-y ̂ )+y ̂. Let's say that age explains 60% of the variance in fluid cognition, and predicted age (y ̂) explains 40% of the variance in fluid cognition. Then the brain age gap (-(y-y ̂)) should explain 20% of the variance in fluid cognition. If by "Corrected Brain Age" you mean the modified predicted age from Butler et al (2021), the "Corrected Brain Age" result is inevitable because the modified predicted age is essentially just age with a tiny bit of noise added to it. From Figure 4, though, this does not seem to be the case, because the lower left quadrant in panel (a) should be flat and high (about as high as the predictive value of age for fluid cognition). So it is unclear how "Corrected Brain Age" is calculated. It looks like you might be regressing age out of brain-age, though from your description in the Methods section, it is not totally clear. Again, I highly recommend using the terminology and metrics of Butler et al (2021) throughout to reduce confusion. Please also clarify how you used the slope and intercept. In general, given how brain-age metrics tend to be calculated, the following conclusion is inevitable: "As before, the unique effects of Brain Age indices were all relatively small across the four Brain Age indices and across different prediction models" (p10).

"On the contrary, the unique effects of Brain Cognition appeared much larger" (p10). This is not a fair comparison if you do not look at the unique effects above and beyond the cognitive variable you predicted in your brain-cognition model. If your outcome measure had been another metric of cognition other than fluid cognition, you would see that brain-cognition does not explain any additional variance in this outcome when you include fluid cognition in the model, just as brain-age would not when including age in the model (minus small amounts due to penalization and out-of-sample estimates). This highlights the fact that using a predicted value to predict anything is worse than using the value itself.

"First, how much does Brain Age add to what is already captured by chronological age? The short answer is very little" (p12). This is a really important point, but the paper requires an in-depth discussion of the inevitability of this result, as discussed above.

"Third, do we have a solution that can improve our ability to capture Cognitionfluid from brain MRI? The answer is, fortunately, yes. Using Brain Cognition as a biomarker, along with chronological age, seemed to capture a higher amount of variation in Cognitionfluid than only using Brain Age" (p12). I suggest controlling for the cognitive measure you predicted in your brain-cognition model. This will show that brain-cognition is not useful above and beyond cognition, highlighting the fact that it is not a useful endeavor to be using predicted values.

"Accordingly, a race to improve the performance of age-prediction models (Baecker et al., 2021) does not necessarily enhance the utility of Brain Age indices as a biomarker for Cognitionfluid. This calls for a new paradigm. Future research should aim to build prediction models for Brian Age indices that are not necessarily good at predicting age, but at capturing phenotypes of interest, such as Cognitionfluid and beyond" (p13). I whole-heartedly agree with the first two sentences, but strongly disagree with the last. Certainly your results, and the underlying reason as to why you found these results, calls for a new paradigm (or, one might argue, a pre-brain-age paradigm). As of now, your results do not suggest that researchers should keep going down the brain-age path. While it is difficult to prove that there is no transformation of brain-age or the brain-age gap that will be useful, I am nearly sure this is true from the research I have done. If you would like to suggest that the field should continue down this path, I suggest presenting a very good case to support this view.

Reviewer #3 (Public Review):

The current study examined 13 monosomic yeast strains that lost different individual chromosomes. By comparing the fitness of monosomic strains and several heterozygous deletion strains, the authors observed strong positive epistasis for fitness. The transcriptomes of monosomic strains indicated that general gene-dose compensation is not the reason for fitness gains. On the other hand, gene expression of ribosomal proteins was up-regulated and proteasome subunit expression was down-regulated in all tested monosomic strains. The authors speculated that overexpression in combination with decreased degradation of the insufficient proteins might explain the positive epistasis observed in monosomic strains. This study investigates an important biological question and has some interesting results. However, I have some reservations about the data interpretations listed below.

1) In Figure 3b (and line 179), the authors stated that those haploinsufficient genes were not transcribed at elevated rates, but almost half of them are in reddish colors (indicating that the expression is higher than 1-fold). Obviously, many haploinsufficient genes are up-regulated in monosomic strains. What the data really show is that the level of overexpression is not correlated with the fitness effect of the deletion (since all the p values are not significant). The authors need to correct their conclusions.

2) Why are some monosomic strains removed from the transcriptomics analysis, especially when the chromosome IV and XV strains show very strong positive epistasis? The authors need to provide an explanation here.

3) The authors stated that diploidy observed in chromosome VII and XIII strains were due to endoreplication after losing the marked chromosomes (lines 97 and 117). Isn't chromosome missegregation an equally possible explanation? Since monosomic cells are generated by chromosome missegregation during mitosis, another chromosome missegregation event may occur to rescue the fitness (or viability) of monosomic cells in these strains.

Reviewer #3 (Public Review):

Understanding the changes in the brain during the progression of neurodegenerative diseases may provide a critical entry point towards medical treatments. Many genes have been directly or indirectly implemented in an array of neurodegenerative diseases, including the microtubule associated protein tau (MAPT). Various studies have shown that misexpression of tau can cause behavioral, genetic as well as molecular phenotypes that display properties of human neurodegenerative diseases connected to tauopathies. Here the authors use the fruit fly as model to assess phenotypic defects at single-cell resolution. Pan-neuronal misexpression of a mutant form of tau (R406W) and single-cell RNAseq at different time points provides the basis for the investigation.

The authors assess which cell-types are affected (by comparing it with previously described brain cell atlas identities) and find that certain cell types are missing (or less abundant) while other appear unaffected. They do this comparison in relative abundance; both neurons and glia cells are affected.

As next step they compare this with the cell-cluster changes during aging and compare both types of analysis; the investigation here includes the analysis of differentially expressed genes in defined cell clusters. One particularly affected pathway in response to tau is the NFκB signaling pathway. The authors investigate the gene expression changes of the NFκB signaling pathway in the current dataset in more detail. In the last section the authors compare single-cell transcriptomic analyses between fly and human postmortem tissue, showing that the NFκB signaling pathway might be a conserved aspect of neurodegeneration.

The manuscript is overall an elegant example of how single-cell RNAseq can be employed as tool to study the impact of genetic modulators of neurodegeneration (in this case tau) and that it allows direct comparison with human tissues. The results are clean, logically presented and accordingly discussed. It shows that such approaches are indeed powerful for genetic dissection of mechanisms at a descriptive level and opening doors for functional studies.

Reviewer #3 (Public Review):

Buruli ulcer is a severe skin infection in humans that is caused by a bacterium, Mycobacterium ulcerans. The main clinical sign is a massive tissue necrosis subsequent to an edema stage. The main virulence factor called mycolactone is a polyketide with a lactone core and a long alkyl chain that is released within vesicles by the bacterium. Mycolactone was already shown to account for several disease phenotypes characteristic of Buruli ulcer, for instance tissue necrosis, host immune response modulation and local analgesia. A large number of cellular pathways in various cell types was reported to be impacted by mycolactone. Among those, the Sec61 translocon involved in the transport of certain proteins to the endoplasmic reticulum was first identified by the authors of the study and is currently the most consensual target. Mycolactone disruption of Sec61 function was then shown to directly impact on cell apoptosis in macrophages, limited immune responses by T-cells and increased autophagy in dermal endothelial cells and fibroblasts. In their manuscript, Tzung-Harn Hsieh and their collaborators investigated the Sec61- dependent role of mycolactone on morphology, adhesion and migration of primary human dermal microvascular endothelial cells (HDMEC). They used a combination of sugar and proteomic studies on a live image-based phenotypic assay on HDMEC to characterize the effect of mycolactone. First, they showed that upon incubation of monolayer of HDMEC with mycolactone at low dose (10 ng/mL) for 24h, the cells become elongated before rounding and eventually detached from the culture dish at 48h. Next, mycolactone was probed on a scratch assay and migration of the cells ceased upon a 24h incubation. The same effect as mycolactone on these two assays was observed for two other Sec61 inhibitors Ipomoeassin F and ZIF-80. Then, the authors resorted to the widely established mouse footpad model of M. ulcerans infection to evidence fibrinogen accumulation outside the blood vessel within the endothelium at 28 days post-infection, correlating with severe endothelial cell morphology changes.

To dissect the molecular pathways involved in these phenotypes, the authors performed an HDMEC membrane protein analysis and showed a decrease in the numbers of proteins involved in glycosylation and adhesion. As protein glycosylation mainly occurs in the Golgi apparatus, a deeper analysis revealed that enzymes involved in glycosaminoglycan (GAG) synthesis were lost in mycolactone treated HDMEC. A combination of immunofluorescence and flow cytometry approaches confirmed the impact of mycolactone on the ability of endothelial cells to synthesize GAG chains. The mycolactone effect on cell elongation was phenocopied by knock-down of galactosyltransferase II (B3Galt6) involved in GAG biosynthesis. A second extensive analysis of the endothelial basement membrane component and their ligands identified multiple laminins affected by mycolactone. Using similar functional studies as for GAG, the impact of mycolactone on cell rounding and migration could be reversed by the addition of laminin α5.

The major strengths of the study relies on a combination of cleverly designed phenotypic assays and in-depth cleverly designed membrane proteomic studies and follow-up analysis.<br /> The results really support the conclusions. Congratulations!<br /> The discussion takes into account the current state of the art, which has mostly been established by the authors of the present manuscript.

Reviewer #3 (Public Review):

I found this paper fascinating. It is a study that needed to be done in the field of behavioral endocrinology, as it addresses our understanding of exactly how steroid hormone action might regulate behavioral output like few other published studies. For decades, researchers have been implanting animals with steroids and observing corresponding changes in behavior, noting that some behavioral traits are immediately expressed, while others take time to be expressed. Why would this be? The answer lies in the temporal dynamics of steroid action, but few have ever addressed this. Having said this, I do have several issues with the manuscript that I think need to be addressed.

1) My biggest concern is the sample size. Most of the time points only have 5 or 6 individuals represented, and I question whether these numbers provide sufficient statistical power to uncover the effects the authors are trying to explore. This is a particular problem when it comes to evaluating the supposed "transient" of testosterone on gene expression. There is currently little basis for distinguishing such effects from noise that accrues because of low power. This can be a major problem with studies of gene expression in non-model species, like canaries, where among-individual variability in transcript abundance is quite high. Thus, it is possible that one or two outliers at a given time point cause the effect testosterone at this time point to become indistinguishable from the controls; if so, then a gene may get put into the transient category, when in fact its regulation was not likely transient.

2) More on the transient categorization. Would a gene whose expression is not immediately upregulated (within 1 hour), but is upregulated later on (say in the 14d group) be considered transient? If so, this seems problematic. Aren't the authors setting the null expectation of "non-transient" as a gene that does not increase immediately after 1 hour of treatment? The authors even recognize that it is quite surprising that gene expression changes after an hour. It may be that some genes whose regulation is classified as transient are simply slower to upregulate; but, really, would we say their expression in transient per se? Maybe I'm misunderstanding the categorizations?

3) The authors don't fully explain the logic for using females in this study to measure a "male-typical" behavior (singing). My understanding is that females have underlying circuitry to sign, and T administration triggers it; thus, this situation that creates a natural experiment in which we can explore T's on brain and behavior, unlike in males which have fluctuating T. First, it might be good to clarify this logic for readers, unless perhaps I'm misunderstanding something. Second, I found myself questioning this logic a little. Our understanding of basic sex differences and the role that steroid hormones play in generating them has changed over the last few decades. There are, for example, a variety of genetic factors that underlie the development of sex differences in the brain (I'm especially thinking about the incredible work from Art Arnold and many others that harness the experimental power of the four core genotype mice). Might some of these factors influence female development, such that T's effects on the female brain and subsequent ability to increase HVC size and sing is not the same as males.

4) I was surprised by the authors assertion that testosterone would only influence several tens or hundreds of genes. My read of the literature says that this is low, and I would have expected 100s, if not 1,000s, of genes to be influenced. I think that the total number of genes influenced by T is therefore quite consistent with the literature.

5) I found the GO analyses presented herein uncompelling. As the authors likely know, not all GO terms are created equally. Some GO terms are enriched by hundreds of genes and thus reflect broad functional categories, whereas other GO terms are much more specific and thus are enriched by only a few genes. The authors report broad GO terms that don't tell us much about what is happening in the HVC functionally. This is particularly the case when a good 50% of the genome is being differentially regulated.

6) The Genomatix analyses are similarly uncompelling. This approach to finding putative response elements can uncover many false positives, and these should always be validated thoroughly. Don't get me wrong-I appreciate that these validations are not trivial, and I value the authors response element analysis.

7) I'm sceptical about the section of the paper that speculates about modification of steroid sensitivity in the HVC. These conclusions are based on analyses of mRNA expression of AKR1D1, SRD5A2, and the like. However, this does not reflect a different in the capacity to metabolize steroids, or at least there is little evidence to suggest this. Note that many of these transcripts have different isoforms, which could also influence steroidal metabolism.

Reviewer #3 (Public Review):

Overall, the data quality and analyses are solid. The authors have extracted a lot of detailed information about gene expression in specific cell types of the sea star embryo, and this descriptive narrative forms much of the Results section. However, the most interesting analyses will be the between-species comparisons. The authors identify several striking differences in the apparent presence or absence of specific cell types between seastar and sea urchins. Some confirm well-known differences, such as the absence of pigmented and skeletogenic mesenchyme cells in seastar embryos based on morphological comparisons. Other findings are novel, such as transcriptionally distinct left and right coelomic pouches as early as late gastrula and the apparent absence of germ cells in seastar embryos. These findings are based on solid evidence, highly informative regarding molecular details, and will no doubt inspire many future studies, both into developmental mechanisms per se and into the evolution of development. While the descriptive part of this study is solid and highly informative, the evolutionary interpretations are more problematic. The Abstract and Introduction emphasize the promise of sc/snRNAseq to shed light on the evolution of cell types and novelty, but the data themselves tell a less clear-cut story. Indeed, for me, the biggest takeaway from reading this manuscript is that it is quite difficult to identify when a novel cell type has evolved based solely on analysis of embryonic stages. The last stage examined is late gastrula, which means that some cell types may appear to be missing simply because they have not yet begun to differentiate transcriptionally. An example would be germ cells since adults make gametes. Another limitation is that just two species are compared. This means that for any given difference in cell type composition, it is not possible to distinguish whether this represents a novel cell type in one species or the loss (or delay in differentiation) of a cell type in the other species. The authors are generally careful to identify these limitations when presenting results, but it does lead me to wonder why they did not choose to examine later stages of development when more cells are clearly differentiated.

Reviewer #3 (Public Review):

The goal of this work was to better understand how cell fate decisions at the neural plate border (NPB) occur. There are two prevailing models in the field for how neural, neural crest and placode fates emerge: (i) binary competence which suggests initial segregation of ectoderm into neural/neural crest versus placode/epidermis; (ii) neural plate border, where cells have mixed identity and retain the ability to generate all the ectodermal derivatives until after neurulation begins.

The authors use single-cell sequencing to define the development of the NPB at a transcriptional level and suggest that their cell classification identified increased ectodermal cell diversity over time and that as cells age their fate probabilities become transcriptionally similar to their terminal state. The observation of a placode module emerging before the neural and neural crest modules is somewhat consistent with the binary competence model but the observation of cells with potentially mixed identity at earlier stages is consistent with the neural plate border model.

Differences in the timing of analyses and techniques used can account for the generation of these two original models, and in essence, the authors have found some evidence for both models, possibly due to the period over which they performed their studies. However, the authors propose recognizing the neural plate border as an anatomical structure, containing transcriptionally unstable progenitors and that a gradient border model defines cell fate choice in concert with spatiotemporal positioning.

The idea that the neural plate border is an anatomical structure is not new to most embryologists as this has been well-recognized in lineage tracing and transplantation assays in many different species over many decades. The authors don't provide molecular evidence for transcriptional instability in any cells. It's a molecular term and phenomenon inaccurately applied to these cells that are simply bipotential progenitors. Lastly, there's no evidence of a gradient that fits the proper biochemical or molecular definition. Graded or sequential are more appropriate terms that reflect the lineage determination or segregation events the authors characterize, but there's no data provided to support a true role for a gradient such as that achieved by a concentration or time-dependent morphogen.

A limitation of the study is that much of it reads like a proof-of-principle because validation comes primarily from known genes, their expression patterns in vivo, and their subsequent in vivo functions. Thus, the authors need to qualify their interpretations and conclusions and provide caveats throughout the manuscript to reflect the fact that no functional testing was performed on any novel genes in the emerging modules classified as placode versus neural or neural crest.

Lastly, a limitation of gene expression studies is that it provides snapshots of cells in time, and while implying they have broad potential or are lineage fated, do not actually test and confirm their ultimate fate. Therefore, in parallel with their studies, the authors really need to consider, the wealth of lineage tracing data, especially single-cell lineage tracing, which has been performed using the embryos of the same stage as that sequenced in this study, and which has revealed critical data about the potential cells through when and where lineage segregation and cell fate determination occurs.

Reviewer #3 (Public Review):

This manuscript investigates how a seemingly random choice of odourant receptor (OR) gene expression is organised into sterotypic zones of OR expression along the olfactory epithelium. Using a varietty of functional genomics methods, the authors find that along the differentiation axis (progenitor to mature olfactory sensory neuron, OSN) multiple ORs are initally transcribed and from among these, only one OR is selected for expression. The rest are suppressed through chromatin silencing. In addition to this, the authors report a dorso-ventral gradient in OR expression at the immature stage - dorsally expressed ORs are also expressed ventrally and these then get silenced. The expression of the ventrally expressed ORs, on the other hand, are restricted to the ventral region. They suggest a role for the transcription factor NF1 in this dorsoventral process.

This is a valuable study. The data are compelling and generally well presented.

Reviewer #3 (Public Review):

In this article by Bastidas et al. the authors examine the functions of the Chlamydia deubiquitinating enzyme 1 (Cdu1) during infections of human cells. First, a mutant lacking Cdu1 but not Cdu2 was constructed using targetron and quantitative proteomics was used to identify differences in ubiquitinated proteins (both host and bacterial) during infection. While they found minimal changes in host protein ubiquitination, they identified three Chlamydia effector proteins, IpaM, InaC and CTL0480 were all ubiquitinated in the absence of Cdu1. Microscopy and immunoprecipitations found Cdu1 directly interacts with these Chlamydia effectors and confirmed that Cdu1 mediates the stabilization of these effectors at the inclusion membrane during late infection time points. Surprisingly rather than deubiquitination driving this stabilization, the acetylation function of Cdu1 was required, and acetylation on lysine residues prevented degradative ubiquitination of Cdu1, IpaM, InaC and CTL0480. In line with this observation the authors show that loss of Cdu1 phenocopies the loss of single effector mutants of InaC, IpaM and CTL0480, including golgi stack formation and the recruitment of MYPT1 to the inclusion. The aggregation of changes to the Chlamydia inclusion does not alter growth but controls extrusion of chlamydia from cells with reduced extrusion in Cdu1 mutant Chlamydia infections. The strengths of the manuscript are the range of assays used to convincingly examine the biochemical and cellular biology underlying Cdu1 functions. The finding that acetylation of lysine residues is a mechanisms for bacterial effectors to block degradative ubiqutination is impactful and will open new investigations into this mechanism for many intracellular pathogens. There are a few weaknesses that temper enthusiasm for the manuscript in its current form. These include caveats related to the timing of proteomics, the lack of an effect of Cdu1 directly on bacterial growth, and discussion of previous studies. Altogether this is an important series of findings that help to understand the mechanisms underpinning Chlamydia pathogenesis using orthologous methods with a few caveats that lower the overall impact.

Reviewer #3 (Public Review):

Lu et al. describe Vangl2 as a negative regulator of inflammation in myeloid cells. The primary mechanism appears to be through binding p65 and promoting its degradation, albeit in an unusual autolysosome/autophagy dependent manner. Overall, the findings are novel and the crosstalk of PCP pathway protein Vangl2 with NF-kappaB is of interest. Whether PCP is anyway relevant or if this is a PCP-independent function of Vangl2 is not directly explored (the later appears more likely from the manuscript/discussion). PCP pathways intersect often with developmentally important pathways such as WNT, HH/GLI, Fat-Dachsous and even mechanical tension. It might be of importance to investigate whether Vangl2-dependent NF-kappaB is influenced by developmental pathways. Are Vangl2 phosphorylations (S5, S82 and S84) in anyway necessary for the observed effects on NF-kappaB or would a phospho-mutant (alanine substitution mutant) Vangl2 phenocopy WT Vangl2 for regulation of NF-kappaB? Another area to strengthen might be with regards to specificity of cell types where this phenomenon may be observed. LPS treatment in mice resulted in Vangl2 upregulation in spleen and lymph nodes, but not in lung and liver. What explains the specificity of organ/cell-type Vangl2 upregulation and its consequences observed here? Why is NF-kappaB signaling not more broadly or even ubiquitously affected in all cell types in a Vangl2-dependent manner, rather than being restricted to macrophages, neutrophils and peritoneal macrophages, or, for that matter, in spleen and LN and not liver and lung? After all, one may think that the PCP proteins, as well as NF-kappaB, are ubiquitous. Regardless, Vangl2 as a negative regulator of NF-kappaB is an important finding. There are, however, some concerns about methodology and statistics that need to be addressed.

Reviewer #3 (Public Review):

The authors describe a method to tether proteins via DNA linkers in magnetic tweezers and apply it to a model membrane protein. The main novelty appears to be the use of DBCO click chemistry to covalently couple to the magnetic bead, which creates stable tethers for which the authors report up to >1000 force-extension cycles. Novel and stable attachment strategies are indeed important for force spectroscopy measurements, in particular for membrane proteins that are harder and therefore less studied in this regard than soluble proteins, and recording >1000 stretch and release cycles is an impressive achievement. Unfortunately, I feel that the current work falls short in some regards to exploring the full potential of the method, or at least does not provide sufficient information to fully assess the performance of the new method. Specific questions and points of attention are included below.

- The main improvement appears to be the more stable and robust tethering approach, compared to previous methods. However, the stability is hard to evaluate from the data provided. The much more common way to test stability in the tweezers is to report lifetimes at constant force(s). Also, there are actually previous methods that report on covalent attachment, even working using DBCO. These papers should be compared.

- The authors use the attachment to the surface via two biotin-traptavidin linkages. How does the stability of this (double) bond compare to using a single biotin? Engineered streptavidin versions have been studied previously in the magnetic tweezers, again reporting lifetimes under constant force, which appears to be a relevant point of comparison.

- Very long measurements of protein unfolding and refolding have been reported previously. Here, too, a comparison would be relevant. In light of this previous work, the statement in the abstract "However, the weak molecular tethers used in the tweezers limit a long time, repetitive mechanical manipulation because of their force-induced bond breakage" seems a little dubious. I do not doubt that there is a need for new and better attachment chemistries, but I think it is important to be clear about what has been done already.

- Page 5, line 99: If the PEG layer prevents any sticking of beads, how do the authors attach reference beads, which are typically used in magnetic tweezers to subtract drift?

- Figure 3 left me somewhat puzzled. It appears to suggest that the "no detergent/lipid" condition actually works best, since it provides functional "single-molecule conjugation" for two different DBCO concentrations and two different DNA handles, unlike any other condition. But how can you have a membrane protein without any detergent or lipid? This seems hard to believe.<br /> Figure 3 also seems to imply that the bicelle conditions never work. The schematic in Figure 1 is then fairly misleading since it implies that bicelles also work.

- When it comes to investigating the unfolding and refolding of scTMHC2, it would be nice to see some traces also at a constant force. As the authors state themselves: magnetic tweezers have the advantage that they "enable constant low-force measurements" (page 8, line 189). Why not use this advantage?<br /> In particular, I would be curious to see constant force traces in the "helix coil transition zone". Can steps in the unfolding landscape be identified? Are there intermediates?

- Speaking of loading rates and forces: How were the forces calibrated? This seems to not be discussed. And how were constant loading rates achieved? In Figure 4 it is stated that experiments are performed at "different pulling speeds". How is this possible? In AFM (and OT) one controls position and measures force. In MT, however, you set the force and the bead position is not directly controlled, so how is a given pulling speed ensured?<br /> It appears to me that the numbers indicated in Figures 4A and B are actually the speeds at which the magnets are moved. This is not "pulling speed" as it is usually defined in the AFM and OT literature. Even more confusing, moving the magnets at a constant speed, would NOT correspond to a constant loading rate (which seems to be suggested in Figure 4A), given that the relationship between magnet positions and force is non-linear (in fact, it is approximately exponential in the configuration shown schematically in Figure 1).

- Finally, when it comes to the analysis of errors, I am again puzzled. For the M270 beads used in this work, the bead-to-bead variation in force is about 10%. However, it will be constant for a given bead throughout the experiment. I would expect the apparent unfolding force to exhibit fluctuations from cycle to cycle for a given bead (due to its intrinsically stochastic nature), but also some systematic trends in a bead-to-bead comparison since the actual force will be different (by 10% standard deviation) for different beads. Unfortunately, the authors average this effect away, by averaging over beads for each cycle (Figure 4). To me, it makes much more sense to average over the 1000 cycles for each bead and then compare. Not surprisingly, they find a larger error "with bead size error" than without it (Figure 5A). However, this information could likely be used (and the error corrected), if they would only first analyze the beads separately.<br /> What is the physical explanation of the first fast and then slow decay of the error (Figure 5B)? I would have expected the error for a given bead after N pulling cycles to decrease as 1/sqrt(N) since each cycle gives an independent measurement. Has this been tested?

Reviewer #3 (Public Review):

Fulton et al. look to apply approaches for tackling the readout of gene regulatory networks (GRNs) to a system where cell position itself is continually changing. The objective is highly laudable. GRN analysis has proven to be a powerful approach for understanding how cell fates are determined by morphogenetic inputs, but it has thus far been applied in a limited number of systems. Here, the authors look to substantially extend the application of GRNs to more dynamic systems. The theoretical and experimental approaches are integrated to achieve the analysis of the GRN. In principle, this has wide potential impact and applicability to other systems.

Unfortunately, in its current form, the manuscript does not do justice to the central aims of the authors. The manuscript is unclear in nearly all sections, and figures and analysis can be substantially improved. The quantifications are not shown in a fitting manner. The modelling itself stands as the strongest part of the manuscript, but improvements are needed. Currently, the main claims of the authors cannot be evaluated based on the quality of the presented data.

Reviewer #3 (Public Review):

This study by Wang et al. investigates the role of the focal adhesion protein vinculin in osteocytes and its effect on bone mass. First, they showed decreased levels of vinculin in osteocytes in trabecular bone from aged individuals compared to young, suggesting a potential role for vinculin in regulating bone mass with aging. Next, they deleted vinculin in late osteoblasts and osteocytes in young and older mice and found decreased bone mineral density and trabecular bone mass. This was due to impaired bone formation, which the authors attributed to increased sclerostin levels. Further in vitro experiments showed that vinculin regulates sclerostin via the transcription factor Mefc2. Conditional knockout of vinculin in late osteoblasts and osteocytes had no effect on the bone of mice lacking Sost, further implicating an essential role for sclerostin in mediating the effects of vinculin in osteocytes. Interestingly, the vinculin conditional knockout mice had an impaired response to mechanical loading, suggesting an important role for vinculin in the osteocyte mechanoresponse. Finally, the authors showed that while ovariectomy increased osteoclast formation and bone resorption in control mice, it had no effect on the bone of the vinculin conditional knockout mice.

Overall, the authors show convincing data for the important role of vinculin in osteocytes in regulating the anabolic effects of bone formation under physiological conditions. They also show that osteocyte vinculin may be a regulator of bone resorption under conditions mimicking postmenopausal osteoporosis. However, not all of the conclusions are fully supported by the data.

Strengths:

The use of both in vivo and in vitro approaches to determine the role of vinculin in osteocytes provides compelling evidence for its importance under basal conditions and in regulating the anabolic effects of mechanical loading. The in vitro assays nicely demonstrate a potential mechanism through Mef2c/ECR5.

The creation of the vinculin and Sost double conditional knockout mouse model provides further convincing evidence for the causative role of sclerostin in the effects of vinculin knockout in osteocytes.

The use of both young and older male mice links nicely with the human samples where vinculin expression appears to be reduced in osteocytes with aging. The authors need to be careful in describing 14-month-old mice as aged though, as these mice would not be typically thought of as old.

Weaknesses:

The methods section is lacking in basic details (e.g., there is no information on the CRISPR deletion of Vcl in the MLO-Y4 cells). While referencing their previous papers is fine, a brief description of the methods should be included in this paper.

While much of the data linking vinculin to sclerostin is convincing, it is surprising that the authors show decreased trabecular bone volume in the vinculin cKO mice, yet show increased sclerostin levels in the cortical bone. If increased sclerostin is responsible for impaired bone formation in the vinculin cKO mice, why is there no cortical bone phenotype? It would be important for the authors to also show the sclerostin immunostaining in the trabecular bone of these animals.

The authors do not provide any potential explanation for the effects of vinculin cKO in the ovariectomized mice. Under physiological conditions, osteocyte vinculin has no effect on osteoclast number or bone resorption. How is osteocyte vinculin affecting osteoclasts after ovariectomy? Are there differences in the osteocyte expression of Rankl or Opg in response to the loss of estrogen in the vinculin cKO and control mice?

From their in vitro experiments, the authors deduce that loss of vinculin affects osteocyte attachment. However, their images would suggest that it is the formation of dendrites that is strongly inhibited in the cells lacking vinculin. It is surprising that no investigation of osteocyte dendrite number or connectivity was performed in the vinculin cKO mice. This is particularly important as a decrease in osteocyte dendrites and connectivity has been observed in the bones of aged mice (see Tiede-Lewis et al., Aging. 2017) and osteocyte dendrites are important for mechanosensation.

Reviewer #3 (Public Review):

The authors propose a mechanistic model of how the interplay between activity-independent growth and an activity-dependent synaptic strengthening/weaken model influences the dendrite shape, complexity and distribution of synapses. The authors focus on a model for stellate cells, which have multiple dendrites emerging from a soma. The activity independent component is provided by a random pool of presynaptic sites that represent potential synapses and that release a diffusible signal that promotes dendritic growth. Then a spontaneous activity pattern with some correlation structure is imposed at those presynaptic sites. The strength of these synapses follow a learning rule previously proposed by the lab: synapses strengthen when there is correlated firing across multiple sites, and synapses weaken if there is uncorrelated firing with the relative strength of these processes controlled by available levels of BDNF/proBDNF. Once a synapse is weakened below a threshold, the dendrite branch at that site retracts and loses its sensitivity to the growth signal

The authors run the simulation and map out how dendrites and synapses evolve and stabilize. They show that dendritic trees growing rapidly and then stabilize by balancing growth and retraction (Figure 2). They also that there is an initial bout of synaptogenesis followed by loss of synapses, reflecting the longer amount of time it takes to weaken a synapse (Figure 3). They analyze how this evolution of dendrites and synapses depends on the correlated firing of synapses (i.e. defined as being in the same "activity group"). They show that in the stabilized phase, synapses that remain connected to a given dendritic branch are likely to be from same activity group (Figure 4). The authors systemically alter the learning rule by changing the available concentration of BDNF, which alters the relative amount of synaptic strengthening, which in turn affects stabilization, density of synapses and interestingly how selective for an activity group one dendrite is (Figure 5). In addition the authors look at how altering the activity-independent factors influences outgrowth (Figure 6). Finally, one of the interesting outcomes is that the resulting dendritic trees represent "optimal wiring" solutions in the sense that dendrites use the shortest distance given the distribution of synapses. They compare this distribute to one published data to see how the model compared to what has been observed experimentally.

There are many strengths to this study. The consequence of adding the activity-dependent contribution to models of synapto- and dendritogenesis is novel. There is some exploration of parameters space with the motivation of keeping the parameters as well as the generated outcomes close to anatomical data of real dendrites. The paper is also scholarly in its comparison of this approach to previous generative models. This work represented an important advance to our understanding of how learning rules can contribute to dendrite morphogenesis

Reviewer #3 (Public Review):

In this study, the authors sought to test the hypothesis that blocking triglyceride storage in adipose tissue by knockout of DGAT1 and DGAT2 in adipocytes would lead to ectopic lipid deposition, lipodystrophy, and impaired glucose homeostasis. Surprisingly, the authors found the opposite result, with DGAT1/2 DKO in adipocytes leading to increased energy expenditure, minimal ectopic lipid deposition, and improved glucose homeostasis with HFD feeding. These metabolic improvements were largely attributed to increased beiging of the white fat and increased brown adipose tissue activity. This study provides an interesting new paradigm whereby impairing fat storage, the major function of adipose tissue, does not lead to severe metabolic disease, but rather improves it. The authors provide a comprehensive assessment of the metabolism of these DKO mice under chow and HFD conditions, which support their claims. The study lacks in mechanistic insight, which would strengthen the study, but does not detract from the authors' major conclusions.

The conclusions of this paper are mostly well-supported, but some aspects should be clarified and extended.

1. The authors claim the beiging of WAT of ADGAT DKO mice is partially through the SNS; however, housing these mice at thermoneutrality did not block the beiging, which seems to negate that claim. Is there evidence of increased cAMP/PKA activation in the adipose tissues of ADGAT DKO to support the premise that the beiging is activated by the SNS, even at thermoneutrality? Alternatively, if the authors block beta-adrenergic receptors with antagonists, such as propranolol, does this block the beiging?

2. It's been shown that autocrine FGF21 signaling is sufficient to promote beiging of iWAT (PMID 34192547). The authors show Fgf21 mRNA is increased in iWAT of chow-fed ADGAT DKO mice. Is Fgf21 also increased in iWAT of HFD-fed mice? This and measurement of local FGF21 secretion by adipocytes would strengthen this study.

3. The primary adipocytes in Figure S6A do not appear to have any depletion in TG stores, suggesting this may not be an appropriate model to study the cell autonomous effects of ADGAT DKO on beiging. The authors should use DGAT inhibitors instead to corroborate or investigate this question.

4. Multiple studies have shown the importance of lipolysis for the activation of brown and beige thermogenic programs (PMID 35803907, 34048700) and can be potentiated by HFD feeding (PMID 34048700). In the absence of DGAT activity in ADGAT DKO mice, it seems plausible that free fatty acids could be elevated, especially in the context of HFD. Are free fatty acids elevated in the adipose tissues, which could promote thermogenic gene expression?

5. The lack of ectopic lipid deposition in the ADGAT DKO mice is striking, especially under HFD conditions. Can the increased energy expenditure fully account for the difference in whole body fat accumulation between Control and DKO mice or have the mice activated other energy disposal mechanisms? Please discuss or include measurement of fat excretion in the feces to strengthen this study.

Reviewer #3 (Public Review):

This study examines how the correlation structure of a perceptual decision making task influences history biases in responding. By manipulating whether stimuli were more likely to be repetitive or alternating, they found evidence from both behavior and a neural signal of decision formation that history biases are flexibly adapted to the environment. On the whole, these findings are supported across an impressive range of detailed behavioral and neural analyses. The methods and data from this study will likely be of interest to cognitive neuroscience and psychology researchers. The results provide new insights into the mechanisms of perceptual decision making.

The behavioral analyses are thorough and convincing, supported by a large number of experimental trials (~600 in each of 3 environmental contexts) in 38 participants. The psychometric curves provide clear evidence of adaptive history biases. The paper then goes on to model the effect of history biases at the single trial level, using an elegant cross-validation approach to perform model selection and fitting. The results support the idea that, with trial-by-trial accuracy feedback, the participants adjusted their history biases due to the previous stimulus category, depending on the task structure in a way that contributed to performance.

The paper then examines MEG signatures of decision formation, to try to identify neural signatures of these adaptive biases. Looking specifically at motor beta lateralization, they found no evidence that starting-level bias due to the previous trial differed depending on the task context. This suggests that the adaptive bias unfolds in the dynamic part of the decision process, rather than reflecting a starting level bias. The paper goes on to look at lateralization relative to the chosen hand as a proxy for a decision variable (DV), whose slope is shown to be influenced by these adaptive biases.

This analysis of the buildup of action-selective motor cortical activity would be easier to interpret if its connection with the DV was more explicitly stated. The motor beta is lateralized relative to the chosen hand, as opposed to the correct response which might often be the case. It is therefore not obvious how the DV behaves in correct and error trials, which are combined together here for many of the analyses.

Reviewer #3 (Public Review):

In this manuscript, Yang et al. showed that two nuclear receptor genes, COUP-TFI and -TFII, displayed distinct expression patterns and functions during the development of the dorsal and ventral hippocampus. The phenotypes in the presented single and double conditional knockout mice are striking and intriguing, which expands our knowledge of hippocampus development, especially the ventral part. Nevertheless, the manuscript is a bit descriptive without in-depth molecular mechanisms.

My major concerns as follows:

1. Quantification and statistical analysis to support their conclusions are almost absent throughout the whole manuscript, especially in relation to the numbers of DG, CA1, and CA3 neurons.<br /> 2. Only TFI conditional knockout mice, not TFII knockout mice, were used to test for behavioral abnormalities. It is important to determine whether the abnormal ventral hippocampus in TFII loss leads to any psychiatric illness.<br /> 3. Behavior defects were only tested on TFI conditional knockout mice but not on TFII knockout mice. TFII loss predominantly affects the ventral hippocampus which is involved in psychiatric disorders, and this should be tested.

Reviewer #3 (Public Review):

This manuscript describes interesting experiments on how information from the two eyes is combined in cortical areas, sub-cortical areas, and perception. The experimental techniques are strong and the results are potentially quite interesting. But the manuscript is poorly written and tries to do too much in too little space. I had a lot of difficulty understanding the various experimental conditions, the complicated results, and the interpretations of those results. I think this is an interesting and useful project so I hope the authors will put in the time to revise the manuscript so that regular readers like myself can better understand what it all means.

Now for my concerns and suggestions:

The experimental conditions are novel and complicated, so readers will not readily grasp what the various conditions are and why they were chosen. For example, in one condition different flicker frequencies were presented to the two eyes (2Hz to one and 1.6Hz to the other) with the flicker amplitude fixed in the eye presented to the lower frequency and the flicker amplitude varied in the eye presented to the higher frequency. This is just one of several conditions that the reader has to understand in order to follow the experimental design. I have a few suggestions to make it easier to follow. First, create a figure showing graphically the various conditions. Second, come up with better names for the various conditions and use those names in clear labels in the data figures and in the appropriate captions. Third, combine the specific methods and results sections for each experiment so that one will have just gone through the relevant methods before moving forward into the results. The authors can keep a general methods section separate, but only for the methods that are general to the whole set of experiments.

I wondered why the authors chose the temporal frequencies they did. Barrionuevo et al (2014) showed that the human pupil response is greatest at 1Hz and is nearly a log unit lower at 2Hz (i.e., the change in diameter is nearly a log unit lower; the change in area is nearly 2 log units lower). So why did the authors choose 2Hz for their primary frequency? And why did the authors choose 1.6Hz which is quite close to 2Hz for their off frequency? The rationale behind these important decisions should be made explicit.

By the way, I wondered if we know what happens when you present the same flicker frequencies to the two eyes but in counter-phase. The average luminance seen binocularly would always be the same, so if the pupil system is linear, there should be no pupil response to this stimulus. An experiment like this has been done by Flitcroft et al (1992) on accommodation where the two eyes are presented stimuli moving oppositely in optical distance and indeed there was no accommodative response, which strongly suggests linearity.

Figures 1 and 2 are important figures because they show the pupil and EEG results, respectively. But it's really hard to get your head around what's being shown in the lower row of each figure. The labeling for the conditions is one problem. You have to remember how "binocular" in panel c differs from "binocular cross" in panel d. And how "monocular" in panel d is different than "monocular 1.6Hz" in panel e. Additionally, the colors of the data symbols are not very distinct so it makes it hard to determine which one is which condition. These results are interesting. But they are difficult to digest.

The authors make a strong claim that they have found substantial differences in binocular interaction between cortical and sub-cortical circuits. But when I look at Figures 1 and 2, which are meant to convey this conclusion, I'm struck by how similar the results are. If the authors want to continue to make their claim, they need to spend more time making the case.

Figure 5 is thankfully easy to understand and shows a very clear result. These perceptual results deviate dramatically from the essentially winner-take-all results for spatial sinewaves shown by Legge & Rubin (1981); whom they should cite by the way. Thus, very interestingly the binocular combination of temporal variation is quite different than the binocular combination of spatial variation. Can the pupil and EEG results also be plotted in the fashion of Figure 5? You'd pick a criterion pupil (or EEG) change and use it to make such plots.

My main suggestion is that the authors need to devote more space to explaining what they've done, what they've found, and how they interpret the data. I suggest therefore that they drop the computational model altogether so that they can concentrate on the experiments. The model could be presented in a future paper.

Reviewer #3 (Public Review):

Yang and colleagues investigated whether information on two task-irrelevant features that induce response conflict is represented in a common cognitive space. To test this, the authors used a task that combines the spatial Stroop conflict and the Simon effect. This task reliably produces a beautiful graded congruency sequence effect (CSE), where the cost of congruency is reduced after incongruent trials. The authors measured fMRI to identify brain regions that represent the graded similarity of conflict types, the congruency of responses, and the visual features that induce conflicts.

Using several theory-driven exclusion criteria, the authors identified the right dlPFC (right 8C), which shows 1) stronger encoding of graded similarity of conflicts in incongruent trials and 2) a positive correlation between the strength of conflict similarity type and the CSE on behavior. The dlPFC has been shown to be important for cognitive control tasks. As the dlPFC did not show a univariate parametric modulation based on the higher or lower component of one type of conflict (e.g., having more spatial Stroop conflict or less Simon conflict), it implies that dissimilarity of conflicts is represented by a linear increase or decrease of neural responses. Therefore, the similarity of conflict is represented in multivariate neural responses that combine two sources of conflict.

The strength of the current approach lies in the clear effect of parametric modulation of conflict similarity across different conflict types. The authors employed a clever cross-subject RSA that counterbalanced and isolated the targeted effect of conflict similarity, decorrelating orientation similarity of stimulus positions that would otherwise be correlated with conflict similarity. A pattern of neural response seems to exist that maps different types of conflict, where each type is defined by the parametric gradation of the yoked spatial Stroop conflict and the Simon conflict on a similarity scale. The similarity of patterns increases in incongruent trials and is correlated with CSE modulation of behavior. However, several potential caveats need to be considered.

One caveat to consider is that the main claim of recruitment of an organized "cognitive space" for conflict representation is solely supported by the exclusion criteria mentioned earlier. To further support the involvement of organized space in conflict representation, other pieces of evidence need to be considered. One approach could be to test the accuracy of out-of-sample predictions to examine the continuity of the space, as commonly done in studies on representational spaces of sensory information. Another possible approach could involve rigorously testing the geometric properties of space, rather than fitting RSM to all conflict types. For instance, in Fig 6, both the organized and domain-specific cognitive maps would similarly represent the similarity of conflict types expressed in Fig1c (as evident from the preserved order of conflict types). The RSM suggests a low-dimensional embedding of conflict similarity, but the underlying dimension remains unclear.

Another important factor to consider is how learning within the confined task space, which always negatively correlates the two types of conflicts within each subject, may have influenced the current results. Is statistical dependence of conflict information necessary to use the organized cognitive space to represent conflicts from multiple sources? Answering this question would require a paradigm that can adjust multiple sources of conflicts parametrically and independently. Investigating such dependencies is crucial in order to better understand the adaptive utility of the observed cognitive space of conflict similarity.

Taken together, this study presents an exciting possibility that information requiring high levels of cognitive control could be flexibly mapped into cognitive map-like representations that both benefit and bias our behavior. Further characterization of the representational geometry and generalization of the current results look promising ways to understand representations for cognitive control.

Reviewer #3 (Public Review):

The manuscript by Lin et al. reveals a novel positive regulatory loop between ZEB2 and ACSL4, which promotes lipid droplets storage to meet the energy needs of breast cancer metastasis. It is of interest, however, some concerns should be addressed to strengthen the finding.

Major concerns:

1. The effect of ZEB2 overexpression is not fully demonstrated in the whole study. This point should be addressed.

2. Does the addition of oleate restore the ability of migration or invasion in ACSL4 knockdown cells?

3. Which cellular compartment does ACSL4 localize in and interact with ZEB2 to stabilize ZEB2?

4. The ubiquitination assay and Co-IP assay are just performed in HEK293T cells. This result should be confirmed in MDA-MB-231 cells or Taxol-resistant MCF-7 cells.

5. How does ACSL4 regulate ZEB2 at the mRNA level？Please verify.

6. In Fig. 2F, the silencing efficiency for ACSL4 and ZEB2 should be shown. In addition, the protein level of ZEB2 or ACSL4 in shZEB2 and shZEB2+ACSL4 groups should also be addressed.

7. What is the survival status of patients with both high expression of ACSL4 and ZEB2 in TCGA. In addition, more survival data from databases especially patients with both high expression of ACSL4 and ZEB2 are needed to analyze to support the finding.

Reviewer #3 (Public Review):

The study examines how different cell types in various regions of the mouse dorsal cortex respond to visuomotor integration and how antipsychotic drugs impacts these responses. Specifically, in contrast to most cell types, the authors found that activity in Layer 5 intratelencephalic neurons (Tlx3+) and Layer 6 neurons (Ntsr1+) differentiated between open loop and closed loop visuomotor conditions. Focussing on Layer 5 neurons, they found that the activity of these neurons also differentiated between negative and positive prediction errors during visuomotor integration. The authors further demonstrated that the antipsychotic drugs reduced the correlation of Layer 5 neuronal activity across regions of the cortex, and impaired the propagation of visuomotor mismatch responses (specifically, negative prediction errors) across Layer 5 neurons of the cortex, suggesting a decoupling of long-range cortical interactions.

The data when taken as a whole demonstrate that visuomotor integration in deeper cortical layers is different than in superficial layers and is more susceptible to disruption by antipsychotics. Whilst it is already known that deep layers integrate information differently from superficial layers, this study provides more specific insight into these differences. Moreover, this study provides a first step into understanding the potential mechanism by which antipsychotics may exert their effect.

Whilst the paper has several strengths, the robustness of its conclusions is limited by its questionable statistical analyses. A summary of the paper's strengths and weaknesses follow.

Strengths:

The authors perform an extensive investigation of how different cortical cell types (including Layer 2/3, 4 , 5, and 6 excitatory neurons, as well as PV, VIP, and SST inhibitory interneurons) in different cortical areas (including primary and secondary visual areas as well as motor and premotor areas), respond to visuomotor integration. This investigation provides strong support to the idea that deep layer neurons are indeed unique in their computational properties. This large data set will be of considerable interest to neuroscientists interested in cortical processing.

The authors also provide several lines of evidence that visuomotor information is differentially integrated in deep vs. superficial layers. They show that this is true across experimental paradigms of visuomotor processing (open loop, closed loop, mismatch, drifting grating conditions) and experimental manipulations, with the demonstration that Layer 5 visuomotor integration is more sensitive to disruption by the antipsychotic drug clozapine, compared with cortex as a whole.

The study further uses multiple drugs (clozapine, aripiprazole and haloperidol) to bolster its conclusion that antipsychotic drugs disrupt correlated cortical activity in Layer 5 neurons, and further demonstrates that this disruption is specific to antipsychotics, as the psychostimulant amphetamine shows no such effect.

In widefield calcium imaging experiments, the authors effectively control for the impact of hemodynamic occlusions in their results, and try to minimize this impact using a crystal skull preparation, which performs better than traditional glass windows. Moreover, they examine key findings in widefield calcium imaging experiments with two-photon imaging.

Weaknesses:

A critical weakness of the paper is its statistical analysis. The study does not use mice as its independent unit for statistical comparisons but rather relies on other definitions, without appropriate justification, which results in an inflation of sample sizes. For example, in Figure 1, independent samples are defined as locomotion onsets, leading to sample sizes of approx. 400-2000 despite only using 6 mice for the experiment. This is only justified if the data from locomotion onsets within a mouse is actually statistically independent, which the authors do not test for, and which seems unlikely. With such inflated sample sizes, it becomes more likely to find spurious differences between groups as significant. It also remains unclear how many locomotion onsets come from each mouse; the results could be dominated by a small subset of mice with the most locomotion onsets. The more disciplined approach to statistical analysis of the dataset is to average the data associated with locomotion onsets within a mouse, and then use the mouse as an independent unit for statistical comparison. A second example, for instance, is in Figure 2L, where the independent statistical unit is defined as cortical regions instead of mice, with the left and right hemispheres counting as independent samples; again this is not justified. Is the activity of cortical regions within a mouse and across cortical hemispheres really statistically independent? The problem is apparent throughout the manuscript and for each data set collected.

An additional statistical issue is that it is unclear if the authors are correcting for the use of multiple statistical tests (as in for example Figure 1L and Figure 2B,D). In general, the use of statistics by the authors is not justified in the text.

Finally, it is important to note that whilst the study demonstrates that antipsychotics may selectively impact visuomotor integration in L5 neurons, it does not show that this effect is necessary or sufficient for the action of antipsychotics; though this is likely beyond the scope of the study it is something for readers to keep in mind.

Reviewer #3 (Public Review):

This paper reveals interesting physical connections between Elg1 and CST proteins that suggest a model where Elg1-mediated PCNA unloading is linked to regulation of telomere length extension via Stn1, Cdc13, and presumably Ten1 proteins. Some of these interactions appear to be modulated by sumolyation and connected with Elg1's PCNA unloading activity. The strength of the paper is in the observations of new interactions between CST, Elg1, and PCNA. These interactions should be of interest to a broad audience interested in telomeres and DNA replication.

What is not well demonstrated from the paper is the functional significance of the interactions described. The model presented by the authors is one interpretation of the data shown, and proposes that the role of sumolyation is temporally regulate the Elg1, PCNA and CST interactions at telomeres. This model makes some assumptions that are not demonstrated by this work (such as Stn1 sumolyation, as noted) and are left for future testing. Alternative models that envision sumolyation as a key in promoting spatial localization could also be proposed based on the data here (as mentioned in the discussion), in addition to or instead of a role for sumolyation in enforcing a series of switches governing a tightly sequenced series of interactions and events at telomeres. Critically, the telomere length data from the paper indicates that the proposed model depicts interactions that are not necessary for telomerase activation or inhibition, as telomeres in pol30-RR strains are normal length and telomeres in elg1∆ strains are not nearly as elongated as in stn1 strains. One possibility mentioned in the paper is the PCNAS and Elg1 interactions are contributing to the negative regulation of telomerase under certain conditions that are not defined in this work. Could it also be possible that the role of these interactions is not primarily directed toward modulating telomerase activity? It will be of interest to learn more about how these interactions and regulation by Sumo function intersect with regulation of telomere extension.

Reviewer #3 (Public Review):

Most farming is done by subtracting or adding what people want based in nature. However, in nature, crops interact with various objects, and mostly we are unaware of their effects. In order to increase agricultural productivity, finding useful objects is very important. However, in an uncontrolled environment, it coexists with so many biological objects that it is very inefficient to verify them all experimentally. It is therefore necessary to develop an effective screening method to identify external environmental factors that can increase crop productivity. This study identified factors presumed to be important to crop growth based on metabarcoding analysis, field sampling, and non-linear analysis/information theory, and conducted a mesocosm experiment to verify them experimentally. In conclusion, the object proposed by the author did not increase rice yield, but rather rice growth rate.

Strength<br /> In actual field data, since many variables are involved in a specific phenomenon, it is necessary to effectively eliminate false positives. Based on the metabarcoding technique, various variables that may affect rice growth were quantitatively measured, although not perfectly, and the causal relationship between these variables and rice growth was analyzed by using information transfer analysis. Using this method, two new players capable of manipulating rice growth were verified, despite their unknown functions until now. I found this process to be very logical, and I think it will be valuable in subsequent ecological studies.

Weaknesses<br /> CK treatment's effectiveness remains questionable. Rice's growth was clearly altered by CK treatment. The validation of the CK treatment itself is not clear compared to the GN treatment, and the transcriptome data analysis results do not show that DEG is not present. The possibility of a side effect caused by a variable that the author cannot control remains a possibility in this case. Even though this part is mentioned in Discussion, it is necessary to discuss various possibilities in more detail.

Reviewer #3 (Public Review):

This important work provides convincing evidence that artificial recurrent neural networks can be used to model neural activity during remapping events while an animal is moving along a one-dimensional circular track. This will be of interest to neuroscientists studying the neural dynamics of navigation and memory, as well as the community of researchers seeking to make links between artificial neural networks and the brain.

Low et al. trained artificial recurrent neural networks (RNNs) to keep track of their location during a navigation task and then compared the activity of these model neurons to the firing rates of real neurons recorded while mice performed a similar task. This study shows that a simple set of ingredients, namely, keeping track of spatial location along a one-dimensional circular track, along with storing the memory of a binary variable (representing which of the two spatial maps are currently being used), are enough to obtain model firing rates that reproduce features of real neural recordings during remapping events. This offers both a normative explanation for these neural activity patterns as well as a potential biological implementation.

One advantage of this modeling approach using RNNs is that this gives the authors a complete set of firing rates that can be used to solve the task. This makes analyzing these RNNs easier, and opens the door for analyses that are not always practical with limited neural data. The authors leverage this to study the stable and unstable fixed points of the model. However, in this paper there appear to be a few places where analyses that were performed on the RNNs were not performed on the neural data, missing out on an opportunity to appreciate the similarity, or identify differences and pose challenges for future modeling efforts. For example, in the neural data, what is the distribution of the differences between the true remapping vectors for all position bins and the average remapping vector? What is the dimensionality of the remapping vectors? Do the remapping vectors vary smoothly over position? Do the results based on neural data look similar to the results shown for the RNN models (Figures 2C-E)?

I enjoyed that the authors leveraged the RNNs to model remapping in a 2D navigation task that is harder to understand from data alone, at least with current experimental capabilities. I would recommend clarifying that you're studying a 2D environment that consists of two circular variables. Currently, this is not clear from the text, and it is more natural to interpret the task schematic in Figure 4A as depicting an arena without periodic boundary conditions. Figure 4F depicts neural activity for this task as a standard torus, however, I suspect the neural activity might actually lie along the surface of a Clifford torus as Cueva, Ardalan et al. 2021 found when they trained a RNN to store two circular variables. As a disclaimer, I am one of the authors of that study.

There are many choices that must be made when simulating RNNs and there is a growing awareness that these choices can influence the kinds of solutions RNNs develop. For example, how are the parameters of the RNN initialized? How long is the RNN trained on the task? Are the firing rates encouraged to be small or smoothly varying during training? For the most part these choices are not explored in this paper so I would interpret the authors' results as highlighting a single slice of the solution space while keeping in mind that other potential RNN solutions may exist. For example, the authors note that the RNN and biological data do not appear to solve the 1D navigation and remapping task with the simplest 3-dimensional solution. However, it seems likely that an RNN could also be trained such that it only encodes the task relevant dynamics of this 3-dimensional solution, by training longer or with some regularization on the firing rates. Similarly, a higher-dimensional RNN solution may also be possible and this would likely be necessary to explain the more variable manifold misalignment reported in the experimental data of Low et al. 2021 as opposed to the more tightly aligned distribution for the RNNs in this paper. However, thanks to the modeling work done in this paper, the door has now been opened to these and many other interesting research directions.

Reviewer #3 (Public Review):

The manuscript presents novel findings regarding the metacognitive judgment of difficulty of perceptual decisions. In the main task, subjects accumulated evidence over time about two patches of random dot motion, and were asked to report for which patch it would be easier to make a decision about its dominant color, while not explicitly making such decision(s). Using 4 models of difficulty decisions, the authors demonstrate that the reaction time of these decisions are not solely governed by the difference in difficulties between patches (i.e., difference in stimulus strength), but (also) by the difference in absolute accumulated evidence for color judgment of the two stimuli. In an additional experiment, the authors eliminated part of the uncertainty by informing participants about the dominant color of the two stimuli. In this case, reaction times were faster compared to the original task, and only depended on the difference between stimulus strength.

Overall, the paper is very well written, figures and illustrations clearly and adequately accompanied the text, and the method and modeling are rigor.

The weakness of the paper is that it does not provide sufficient evidence to rule out the possibility that judging the difficulty of a decision may actually be comparing between levels of confidence about the dominant color of each stimulus. One may claim that an observer makes an implicit color decision about each stimulus, and then compares the confidence levels about the correctness of the decisions. This concern is reflected in the paper in several ways:

1. It is not clear what were the actual instructors to the participants, as two different phrasings appear in the methods: one instructs participants to indicate which stimulus is the easier one and the other instructs them to indicate the patch with the stronger color dominance. If both instructions are the same, it can be assumed that knowing the dominant color of each patch is in fact solving the task, and no judgment of difficulty needs to be made (perhaps a confidence estimation). Since this is not a classical perceptual task where subjects need to address a certain feature of the stimuli, but rather to judge their difficulties, it is important to make it clear.

2. Two step model: two issues are a bit puzzling in this model. First, if an observer reaches a decision about the dominant color of each patch, does it mean one has made a color decision about the patches? If so, why should more evidence be accumulated? This may also support the possibility that this is a "post decision" confidence judgment rather than a "pre decision" difficulty judgment. Second, the authors assume the time it takes to reach a decision about the dominant color for both patches are equal, i.e., the boundaries for the "mini decision" are symmetrical. However, it would make sense to assume that patches with lower strength would require a longer time to reach the boundaries.

3. Experiment 2: the modification of the Difference model to fit the known condition (Figure 5b), can also be conceptualized as the two-step model, excluding the "mini" color decision time. These two models (Difference model with known color; two-step model) only differ from each other in a way that in the former the color is known in advance, and in the second, the subject has to infer it. One may wonder if the difference in patterns between the two (Figure 3C vs. Figure 6B) is only due to the inaccuracies of inferring the dominant color in the two-step model.

An additional concern is about the controlled duration task: Why were these specific durations chosen (0.1-1.65 sec; only a single duration was larger than 1sec), given the much longer reaction times in the main task (Experiment 1), which were all larger on average than 1sec? This seems a bit like an odd choice. Additionally, difficulty decision accuracies in this version of the task differ between known and unknown conditions (Figure 7), while in the reaction time version of the same task there were no detectable differences in performance between known and unknown conditions (Figure 6C), just in the reaction times. This discrepancy is not sufficiently explained in the manuscript. Could this be explained by the short trial durations?

Reviewer #3 (Public Review):

Wu and colleagues are characterising the function of Styxl2 during muscle development, a pseudo-phosphatase that was already described to have some function in sarcomere morphogenesis or maintenance (Fero et al. 2014). The authors verify a role for Styxl2 in sarcomere assembly/maintenance using zebrafish embryonic muscles by morpholino knock-down and by a conditional Styxl2 allele in mice (knocked-out in satellite cells with Pax7 Cre).

Experiments using a tamoxifen inducible Cre suggest that Styxl2 is dispensable for sarcomere maintenance and only needed for sarcomere assembly.

BioID experiments with Styxl2 in C2C 12 myoblasts suggest binding of nonmuscle myosins (NMs) to Styxl2. Interestingly, both NMs are downregulated when muscles differentiate after birth or during regeneration in mice. This down-regulation is reduced in the Styxl2 mutant mice, suggesting that Styxl2 is required for the degradation of these NMs.

Impressively, reducing one NM (zMyh10) by double morpholino injection in a Styxl2 morphant zebrafish, does improve zebrafish mobility and sarcomere structure. Degradation of Mhy9 is also stimulated in cell culture if Styxl2 is co-expressed. Surprisingly, the phosphatase domain is not needed for these degradation and sarcomere structure rescue effects. Inhibitor experiments suggest that Styxl2 does promote the degradation of NMs by promoting the selective autophagy pathway.

Strengths:

A major strength of the paper is the combination of various systems, mouse and fish muscles in vivo to test Styxl2 function, and cell culture including a C2C12 muscle cell line to assay protein binding or protein degradation as well as inhibitor studies that can suggest biochemical pathways.

Weakness:

The weakness of this manuscript is that the sarcomere phenotypes and also the western blots are not quantified. Hence, we rely on judging the results from a single image or blot.<br /> Also, Styxl2 role in sarcomere biology was not entirely novel.

Few high resolution sarcomere images are shown, myosins have not been stained for.

Reviewer #3 (Public Review):

The authors claim that this dataset covers a timepoint of embryogenesis that is not well covered in the other published single cell datasets (Tintori et al 2016 and Packer et al 2019). The Tintori data indeed do not cover the 28-102-cell stages sufficiently, but it is unclear how the data presented here compare to the Packer et al data. It is true that the Packer et al data have fewer cells at earlier timepoints than at later ones, but given that they sequenced tens of thousands of cells, they report that they still have ~10,000 cells <210 min of embryogenesis. If the authors want to make any claims about how their data enables exploration of a stage that was previously not accessible, this would require a better comparison to the available data.

The authors provide thorough support for how they assigned cell identities in their data. It is surprising though that at the 102-cell stage they only identify 37 unique cell identities. They suggest that this is because there are many equivalence groups at this stage. However, I would strongly encourage the authors to perform a similar analysis or otherwise compare their obtained identities with the data from Packer et al. 2019. It seems possible that given the low number of cells in this dataset, the authors are missing certain identities and it would be important to know this.

The main analysis the authors perform is to look at expression patterns of various classes of TFs and ask whether they are enriched in particular lineages or at specific timepoints. This analysis is interesting but would be more informative if the authors provided in Figure 3d the numbers of each class of TFs. The authors then focus on the homeodomain class of TFs as they display interesting lineage-specific expression patterns, which when mapped on the embryo form stripes. The stripe pattern however is not that obvious, at least not as shown in Figure 4b (for example all three darker shades of blue looks indistinguishable). Perhaps separate embryo schematics showing the different TF expression patterns would show this more clearly. Moreover, given the relatively small number of cell identities found in this dataset (particularly at the 102-cell stage), a similar analysis using the Packer data would provide further support to these patterns. The localization of cells with shared expression patterns does show a stripe pattern at the 28-cell stage, but also not so clearly beyond this timepoint.

I am also unsure about the validity/value of the comparison of the stripes to Drosophila and the centrality of homeodomain TFs to anterior-posterior positional identity. First, it would be important to map other TFs, very likely there are several other TFs that correlate with positional identity. Also, even if the expression of the homeodomain TFs in C. elegans form stripes, there are still several cells within that stripe that do not express these TFs, it is thus unclear whether these TFs encode positional information or the identity of cells with different positions in the embryo.

Reviewer #3 (Public Review):

Elkind et al. have devised a strategy to detect cells in whole brain samples of the large, publicly accessible Allen Mouse Brain Connectivity database. They put together an analysis pipeline to quantify cell numbers and -density as well as volumes for all annotated brain areas in these samples. This allowed them to make several important discoveries such as (1) strain-, sex- and hemisphere-specific differences in cell densities, (2) a large interindividual variability in cell numbers, and (3) an absence of linear scaling of cell count with volume, among others. The key strength of this work lies in its comprehensive analysis, the large sample size that the authors have drawn from (making their conclusions particularly robust), and the fact that they have made their analysis tools accessible. A weakness of the current manuscript is the dense layout and overplotting of several of the figures, and the lack of necessary information to understand them more easily. Another, conceptual weakness of using the autofluorescence channel for cell detection is that the identity (neuronal vs non-neuronal) of the underlying cells remains unresolved. Overall, however, I believe that this study has the potential to serve as a valuable reference point, and I would expect this work to have a lasting impact on quantitative studies of mouse brain cytoarchitecture.

Reviewer #3 (Public Review):

Trigo & Kawaguchi study how small somatic subthreshold depolarizations that do not trigger full blown APs can propagate to presynaptic endings and modulate transmitter release. To this end they directly recorded from small cerebellar MLI boutons. In paired somatic and presynaptic recordings, they demonstrate that small synaptic potentials can travel within 2 to 3 ms to the bouton and arrive there with an amplitude attenuated by 20 to 70% with respect to the somatically recorded potential. As expected, this amplitude attenuation depends on axon length. In recordings of MLI-Purkinje cell pairs the authors further demonstrate that small somatic subthreshold depolarizations of about 20 mV size can enhance AP-triggered IPSCs recorded in the Purkinje cells and change synaptic plasticity during AP trains. In order to address mechanisms of such presynaptic modulation, the authors measure presynaptic AP waveforms via cell attached recordings and found these very stable. On the other hand, presynaptic ICa(V) directly recorded in voltage-clamped MLI boutons facilitated in response to small pre-depolarizations and such facilitated ICa(V) produced larger IPSCs in paired recordings of MLI boutons and coupled Purkinje cells. The authors propose that an accumulation of partially gated channels during small presynaptic depolarizations is able to produce more rapid gating of VGCCs during the AP waveform on arrival of an invading presynaptic AP.

Electrotonic coupling between soma and presynaptic endings to the extent that small subthreshold depolarizations such as synaptic potentials can travel to the bouton has been demonstrated before. However direct quantification of such coupling is difficult because of the small size of presynaptic compartments. Trigo & Kawaguchi have now pioneered such very challenging direct presynaptic recordings in the form of recordings of MLI soma and bouton pairs or paired pre- and postsynaptic recordings.

The data is convincing and I do not see a need for additional experiments. But the manuscript in its present form falls short with respect to the presentation and discussion of the data. The authors conclusion about the mechanism of presynaptic ICa(V) facilitation should be verified with proper kinetic simulations using a kinetic scheme such as that proposed by Li, Bischofberger & Jonas (2007) J.Neurosci. which should be adapted to the presynaptic ICa(V) in MLI boutons. This would strengthen the manuscript which otherwise, regarding mechanisms, remains somewhat speculative.

Reviewer #3 (Public Review):

The fungus Entomophthora muscae infects flies and in turn manipulates the flies to produce a summiting behavior that is believed to enhance spore dispersal that happens upon the eventual death of the fly. In this study, the authors undertake a Herculean effort to identify the neural pathways that are manipulated by the fungus to cause summiting. In a major advance, the authors develop techniques that allow them to track behaviors of infected flies over the course of several days. This allows them to investigate summiting behaviors that occur just prior to death with unprecedented detail. In their analysis, the authors find that summiting flies show a burst of increased locomotion just prior to death. Importantly, they show that this burst of locomotion is not seen in flies that are dying from other causes (starvation or desiccation). The burst of locomotion is also found to coincide with an increase in elevation that occurs with summiting, but other results indicate that a change in elevation may be an indirect consequence of increased locomotion. With this new knowledge in hand, the authors screen for genes and neuronal pathways that either disrupt or enhance the burst of locomotion that is characteristic of summiting. These experiments clearly indicate that neurons and genes controlling circadian rhythms play a major role in summiting behaviors. The authors focus their attention on a particular subset of clock neurons (DN1p) as potentially mediating summiting behavior. It is worth noting that DN1p neurons have been implicated in a variety (and in some cases contradictory) of circadian processes and that the interpretation of manipulations of these neurons may be an oversimplification. In particular, prior studies have implicated these cells in temperature entrainment/compensation so interpreting thermogenetic manipulations of these cells might be complicated. The authors also zoom in on a specific region of the brain containing neurons of the pars intercebralis, since they find infiltration by the fungus in this region and the effects of drivers targeting the PI. Converging and convincing lines of evidence to suggest that the PI neurons output to the corpora allata and effects of summing may be mediated by the CA. The already impressive series of experiments are further clinched by the development of a machine vision-based classifier that allows the authors to automatically identify summiting flies so that they may be collected for metabolomic analyses. The authors are automatically emailed and seemingly roused themselves in the middle of the night in order to obtain the precious flies they needed. They find a bunch of compounds that appear in summiting flies and even inject hemolymph from the infected animals into naive flies to find that circulating compounds can affect behaviors. Overall, this paper is a tour de force that addresses a system of long-standing interest and brings it into the modern age. Many new questions are now raised for the future by this fascinating study.

Reviewer #3 (Public Review):

In this paper, Gochman et al examine TRPV1-3 channel sensitization by CBD, specifically in the context of 2-APB activation. The authors primarily used classic electrophysiological techniques to address their questions about channel behavior but have also used structural biology in the form of cryo-EM to examine drug binding to TRPV2. The authors have carefully observed and quantified sensitization of the rat TRPV2 channel to 2-APB by CBD. While this sensitization has been reported previously (Pumroy et al, Nat Commun 2022), the authors have gone into much more detail here and carefully examined this process from several angles, including a comparison to some other known methods of sensitizing TRPV2. Additionally, the authors have also revealed that CBD sensitizes rat TRPV1 and mouse TRPV3 to 2-APB, which has not been reported previously. Up to this point, the work is well thought through and cohesive.

The major weakness of this paper is that the authors' efforts to track down the structural and molecular basis for CBD sensitization neither give insight into how sensitization occurs nor provide a solid footing for future work on the topic. The structural work presented in this paper lacks proper controls to interpret the observed states and the authors do nothing to follow up on a potentially interesting second binding site for CBD. Overall, the structural work feels detached from the rest of the paper. The mutations chosen to examine sensitization are based on setting up TRPV1 in opposition to TRPV2 and TRPV3, which makes little sense as all three channels show sensitization by CBD, even if to different extents. The authors chose their mutations based on the assumption that response to CBD is the key difference between the channels for sensitization, yet the overall state of each channel or the different modes of activation by 2-APB seem to be more likely candidates. As a result, it is not particularly surprising that none of the mutations the authors make reduce CBD sensitization in TRPV2 or increase CBD sensitization in TRPV1.

A difficulty in examining TRPV1-3 as a group is that while they are highly conserved in sequence and structure, there are key differences in drug responses. While it does seem likely that CBD would bind to the same location in TRPV1-3, there is extensive evidence that 2-APB binds at different sites in each channel, as the authors discuss in the paper. Without more basic information about where 2-APB binds to each channel and confirmation that CBD does indeed bind TRPV1-3 at the same site, it may not be possible to untangle this particular mode of channel sensitization.

Reviewer #3 (Public Review):

In this paper, the authors used a cohort study to link immune signatures in blood 30 days after COVID-19 infection as possible predictors of prolonged symptomatology. The paper partially achieves its aims. While the selected analyses are comprehensive, the cohort design is appropriate and the mechanistic ex vivo work is clever and convincing, the strength of conclusions is somewhat limited by the selection of imprecise clinical endpoints, and the lack of analyses examining T regulatory signatures.

Strengths of the paper are:<br /> • The paper includes a comprehensive and structured immune analysis.<br /> • The paper is extremely clearly written.<br /> • The use of manual gating and unsupervised analysis in Fig 1 is complementary and helpful.<br /> • Bystander T cell experiments with IL-15 are useful and attempt to explore mechanisms from human samples which are traditionally very challenging.<br /> • The experiments shown in Figure 4 documenting equal Cov2 T cell responses in all 3 cohorts are an extremely important result.

Major concerns are:<br /> • The significance of the study is somewhat limited by the small sample size.<br /> • The symptomatic outcome scale for PASC is blunt and poorly captures severity. More state-of-the-start scales of symptomatic severity and heterogeneity exist for PASC. I suggest this and other papers as an example: https://pubmed.ncbi.nlm.nih.gov/36454631/<br /> • The omission of analyses examining T regulatory functions is a missed opportunity and these may be impaired in this population.<br /> • This is a challenging question that can be applied to many exploratory studies of this nature: how can we rule out the possibility that statistically significant differences in Figs 1, 2 & 3 are statistically significant but biologically meaningless? All cellular and cytokine measures of immune responses shown in these figures are not routinely measured in the clinic. Are there studies that can be cited to show that these differences are sufficient to have a causal impact on prolonged symptoms and tissue damage rather than just correlations with these outcomes?

Reviewer #3 (Public Review):

The co-suppressive molecule CTLA-4 has a critical role in the maintenance of peripheral tolerance, primarily by Treg mediated control of the co-stimulatory molecules CD80 and CD86. As stated by the authors, previous studies have found a variety of effects of anti-CTLA-4 antibody treatment or genetic loss of CTLA-4 on B-cells. These include increased B-cell activation and antibody production, autoantibody production, impairment of B-cell production in the bone marrow and loss of peripheral B-cells. In this article Muthana et al use a CTLA-4 humanized mouse model and examine the effects of drug conjugated CTLA-4 on the immune system. They observe a transient loss of B-cells in the blood of the treated mice. They then use a range of immune interventions such as T-cell depletion and blocking antibodies to demonstrate that this effect is dependent on T-cell activation.

Since anti-CTLA-4 immunotherapy is in active clinical use exploration of its effects are welcome, this is helped by the use of a humanized CTLA-4 system which should be considered a strength of the paper. However, currently, the central premise of this paper, that B-cells are depleted, seems underexplored. Direct evidence of T-cell killing of B-cells is never presented, rather it is inferred from the reduced numbers of B-cells in the blood. The status of B-cells in sites that contain a large proportion of B-cells such as the spleen and lymph nodes is not examined. Additionally, no examination of B-cell antibody production is performed.

Reviewer #3 (Public Review):

This is a well-written and referenced paper from the laboratory of an outstanding senior investigator. Dr. Corn and colleagues demonstrate convincingly that correction of three transcription factor binding sites in the delta-globin gene promoter results in high levels of delta-globin expression in HUDEP-2 clonal cell populations (Fig. 2B and C) and in CD34+ HSPC (hematopoietic stem and progenitor cells) clonal cell expansions (Fig. 3C). Although correction of the mutant KLF1 binding site has previously been shown to upregulate delta-globin gene transgenes, this new data demonstrate that correction of multiple factor binding sites is required to achieve high-level expression of the delta-globin gene in the endogenous beta-globin gene locus. The results are important because high delta-globin protein levels inhibit the formation of sickle hemoglobin (HbS) polymers that cause sickle cell disease.

Unfortunately, high levels of delta-globin gene expression were not observed after editing of pooled (non-clonal) populations of HUDEP-2 cells (Fig. 1D) or CD34+ HSPC pooled cell populations (Fig. 3B). This result suggests that correction of all 3 promoter elements on individual alleles in CD34+ HSPC populations is far below the level required to be clinically relevant. Also, NHEJ is high in CD34+ HSPC (Fig. 3A); therefore, promoter deletions will inactivate many alleles, and total hemoglobin levels in erythrocytes derived from populations of edited CD34+ HSPC will be much less than normal (29 pg/cell). These cells would be extremely beta-thalassemic.

Reviewer #3 (Public Review):

In this study, Nasser et al. aim to understand how early-life experience affects 1) developmental behavior trajectory and 2) individuality. They use early life starvation and longitudinal recording of C. elegans locomotion across development as a model to address these questions. They focus on one specific behavioral response (roaming vs. dwelling) and demonstrate that early life (right after embryo hatching) starvation reduces roaming in the first larval (L1) and adult stages. However, roaming/dwelling behavior during mid-larval stages (L2 through L4) is buffered from early life starvation. Using dopamine and serotonin biosynthesis null mutant animals, they demonstrated that dopamine is important for the buffering/protection of behavioral responses to starvation in mid-larval stages, while in contrast, serotonin contributes to early-life starvation's effects on reduced roaming in the L1 and adult stages. While the technique and analysis approaches used are mostly solid and support many of the conclusions made in the manuscript for part 1), there are some technical limitations (e.g., whether the method has sufficient resolution to analyze the behaviors of younger animals) and confounding factors (e.g., size of the animal) that the authors do not yet sufficient address, and can affect interpretation of the results. Additionally, much of the study is descriptive and lacks deep mechanistic insight. Furthermore, the focus on a single behavioral parameter (dwelling vs. roaming) limits the broad applicability of the study's conclusions. Lastly, the manuscript does not provide clear presentation or analysis to address part 2), the question of how early life experience affect individuality.

Reviewer #3 (Public Review):

The work by Ho et al describes the identification of Mec1/Tel1 independent activation of Rad53 after MMS treatment, which could lead to changes of GFP fusion signals for several dozens of proteins and this was partly dependent on Rtg3. Starting from an unbiased, targeted screen, the authors identified proteins whose GFP fusion signals changed intensity in rad53∆ but not in mec1∆ cells using live cell imaging, including Rad54. Using Rad54 as a readout for the subsequent experiments, a second screen amongst kinases/phosphatases and their regulators found that rtg2-3 mutants reduced Rad54-GFP intensity. Mass spectrometry data identified Rad53 phosphorylate sites in mec1∆ tel1∆ cells, consistent with the cell biological data described above. Overall, the work was well done and supported the main conclusions. The concept of Mec1/Tel1-independent and Rtg3-dependent Rad53 activation connects checkpoint signaling with the retrograde pathway.

Reviewer #3 (Public Review):

ZMYM2 is a transcriptional repressor known to bind to the post-translational modification SUMO2/3. It has been implicated in the silencing of genes and transposons in a variety of contexts, but lacking sequence-specific DNA binding, little is known about how it is targeted to specific regions. At least two reports indicate association with TRIM28 targets (Tsusaka 2020 Epigenetics & Chromatin, Graham-Paquin 2022 bioRxiv) but no physical association with TRIM28 targets had been observed. Tsusaka 2020 theorizes an indirect, potentially SUMO-independent, interaction via ATF7IP and SETDB1.

Here, Owen and colleagues show that a subset of ZMYM2-binding sites in U2OS cells are clearly TRIM28 sites, and further find that hundreds of genes are silenced by both ZMYM2 and TRIM28. They next demonstrate that ZMYM2 homes to chromatin, and interacts with TRIM28, in a SUMOylation-dependent manner, suggesting that ZMYM2 is recognizing SUMOylation on TRIM28 itself. ZMYM2 separately homes to SINE elements bound by the ChAHP complex, in an apparently SUMOylation independent manner. Although this is not the first report to show physical interaction between ZMYM2 and ChAHP, it is the first to show that ZMYM2 homes to ChAHP-binding sites and functions as a corepressor at these sites.

The mode by which ZMYM2 and TRIM28 coregulate genic targets remains somewhat unclear. TRIM28/ZMYM2 bind to LTR elements, loss of these proteins results in upregulation of genes distal to (but in the same TAD as) these binding sites.

Overall, the manuscript is well-written, convincing, and fills a significant hole in our understanding of ZMYM2's mechanistic function.

Reviewer #3 (Public Review):

A detailed understanding of how membrane receptor guanylyl cyclases (mGC) are regulated has been hampered by the absence of structural information on the cytoplasmic regions of these signaling proteins. The study by Caveney et al. reports the 3.9Å cryo-EM structure of the human mGC cyclase, GC-C, bound to the Hsp90-Cdc37 chaperone complex. This structure represents a first view of the intracellular functional domains of any mGC and answers without doubt that Hsp90-Cdc37 recognizes mGCs via their pseudokinase (PK) domain. This is the primary breakthrough of this study. Additionally, the new structural data reveals that the manner in which Hsp90-Cdc37 recognizes the GC-C PK domain C-lobe is akin to how kinase domains of soluble kinases docks to the chaperone complex. This is the second major finding of this study, which provides a concrete framework to understand, more broadly, how Hsp90-Cdc37 recruits a large number of other diverse client proteins containing kinase or pseudokinase domains. Finally, the Hsp90-Cdc37-GC-C structure offer clues as to how GC-C may be regulated by phosphorylation and/or ubiquitinylation by serving as a platform for recruitment of PP5 and/or E3 ligases.

Comments:

1. The authors used an interesting approach to obtain the GC-C-Hsp90-Cdc37 complex. Flag-tagged human GC-C was overexpressed in CHO cells with the expectation of co-purifying endogenous hamster homologs of Hsp90 and Cdc37. There are several points worth noting:<br /> a. It is not clear from the data presented (Figure 1C, Suppl Fig 1A) or the Methods the percentage of particles in the cryo-EM specimen that represent the GC-C-Hsp90-Cdc37 complex. Presumably, some fraction of GC-C isolated will not be associated with Hsp90-Cdc37. If a very large portion of GC-C is associated with Hsp90-Cdc37, it would be good to explain why this is to be expected. Are 2D/3D classes corresponding to the activated GC-C dimer found? If not, why?<br /> b. Figure 1A suggests that GC-C is phosphorylated before recruitment of Hsp90-Cdc37. What is the phosphorylation status of the GC-C specimen that was imaged by cryo-EM?<br /> c. The resolution of the cryo-EM map (3.9 Å) is too low for unambiguous identification of proteins. Please provide more precise justification for the claim that the densities observed do in fact correspond to hamster Hsp90 and Cdc37.<br /> d. The authors state that human GC-C pulls down hamster Hsp90-cdc37 but soluble kinases cannot, despite the high sequence identity between human and hamster Hsp90-cdc37. Is this because GC-C recognition is more promiscuous? Can this difference be understood in light of the new structural information presented?

2. A large portion of the enforced GC-C dimer was not visible in the cryo-EM maps. It is not easy to learn from Figure 1 exactly which parts of the GC-C construct was sufficiently ordered and observed structurally. Please improve Figure 1.

3. On page 4, the authors claim that they are able to orient the GC-C-Hsp90-Cdc37 complex "as it would sit on a membrane" and referred to Figure 1B. It is not clear what is implied here. Does Hsp90-Cdc37 binding constrain the complex to face the inner leaflet of the membrane in a specific orientation as shown in Figure 1B? If true, this could potentially have important functional implications. Please illustrate how this was deduced based on the information available.

4. Also on page 4, it is stated that it is sterically unlikely an additional Hsp90-Cdc37 complex is associated with the other copy of GC-C in the leucine zippered dimer. It is not obvious to the reader how this may be the case. An additional figure could help make this more clear. Additional biochemical evidence will also help. The absence of GC-C-Hsp90-Cdc37 dimers in cryo-EM micrographs can also support the argument.

5. Some comments on Figure 2:<br /> a. NTD and CTD are mislabeled in Figure 2A.<br /> b. The authors should show cryo-EM density to support their modeling of GC-C in Figures 2B and C.

6. The authors claim that Hsp90-Cdc37 clients are more similar structurally near the cdc37 interface. Please illustrate this with additional figures. Suppl. Figure 2 is inadequate for this purpose. The authors can also consider adding a more detailed discussion comparing the interactions between the pseudokinase/kinase C-lobe and Cdc37 in known structures. Is shape/charge complementarity a universal feature of cdc37-dependent kinase/pseudokinase recruitment? It would be interesting to also consider if it would be possible to predict which of the ~60 human pseudokinases are possible Hsp90-Cdc37 clients. New structural findings from this study and publicly available AI-predicted protein structures could help.

Reviewer #3 (Public Review):

Summary:

This manuscript sheds light on the cell cycle-dependent post-transcriptional regulation of the oncogenic kinase AURKA. AURKA mRNA is subjected to alternative polyadenylation (APA), resulting in a short and a long 3'UTR isoform. While the ratio long/short isoform is important for AURKA expression and might impact cancer development, it is not unclear how this is regulated throughout the cell cycle. Translation and decay rate of the long isoform only are targeted by let-7a miRNA and in a cell-cycle dependent manner. In contrast, the short isoform is translated highly and constantly throughout interphase. Finally, depletion of the long isoform led to an increase in proliferation and migration rates of cells. In Triple Negative Breast Cancer, where AURKA is typically overexpressed, the short isoform is predominant and its expression correlates with faster relapse times of patients, suggesting that this mechanism might play an important role in this cancer.

Originality and novelty:

The originality of this work is to show the cooperation between APA and miRNA-targeting in controlling gene expression dynamics of AURKA during cell cycle. To investigate this mechanism, the authors have developed an interesting transient single-cell and biochemical assay to rapidly study mRNA-specific gene expression in a way that measures post-transcriptional events. This manuscript puts an emphasis on the cell cycle dependent expression control of AURKA at the translation level. However, the magnitude of the changes in mRNA levels throughout the cell cycle is even greater than that of the changes in translation. Therefore, it remains unclear whether translation really is that important in controlling AURKA expression during the cell cycle. Moreover, (i) AURKA regulation by miRNA is already known (Fadaka et al., Oncotarget 2020, Zhang et al., Arch Med Sci 2020, Yuan et al., Technol Cancer Res Treat 2019, Ma et al., Oncotarget 2015), (ii) the concept of cooperation between APA and translation already is not new (Sandberg et al., Science 2008, Mayr and Bartel, Cell 2009, Masamha et al. Nature 2015), and (iii) previous transcriptome-wide studies already suggested a cell-cycle dependent control of AURKA at the translation level (Tanenbaun et al., eLife 2015, translation efficiency ratio G2/G1 = 1.59) as well as the mRNA level (Krenning et al., eLife 2022). The impact of this manuscript could be increased by investigating (i) the mechanism of cell cycle-dependent regulation by let7a expression (i.e is there changes in let7a expression or activity during the cell cycle in this model) and (ii) the origin of AURKA APA dysregulation in cancer (could it be modulated by CFIm25? (Masamha et al. Nature 2015, Tamaddon et al. Sci Rep 2020)).

Reviewer #3 (Public Review):

Moutard, Laura, et al. investigated the gene expression and functional aspects of Leydig cells in a cryopreservation/long-term culture system. The authors found that critical genetic markers for Leydig cells were diminished when compared to the in-vivo testis. The testis also showed less androgen production and androgen responsiveness. Although they did not produce normal testosterone concentrations in basal media conditions, the cultured testis still remained highly responsive to gonadotrophin exposure, exhibiting a large increase in androgen production. Even after the hCG-dependent increase in testosterone, genetic markers of Leydig cells remained low, which means there is still a missing factor in the culture media that facilitates proper Leydig cell differentiation. Optimizing this testis culture protocol to help maintain proper Leydig cell differentiation could be useful for future human testis biopsy cultures, which will help preserve fertility and child cancer patients.

Methods: In line 226, there is mention that the central necrotic area was carefully removed before RNA extraction. This is particularly problematic for the inference of these results, especially for the RT-qPCR data. Was the central necrotic area consistent between all samples and variables (16 and 30FT)? How big was the area? This makes the in-vivo testis not a proper control for all comparisons. Leydig cells are not evenly distributed throughout the testis. A lot of Leydig cells can be found toward the center of the gonad, so the results might be driven by the loss of this region of the testis.

What did the morphology of the testis look like after culturing for 16 and 30 days? These images will help confirm that the culturing method is like the Nature paper Sato et al. 2011 and also give a sense of how big the necrotic region was and how it varied with culturing time.

There are multiple comparisons being made. Bonferroni corrections on p-value should be done.

Results: In the discussion, it is mentioned that IGF1 may be a missing factor in the media that could help Leydig cell differentiation. Have the authors tried this experiment? Improving this existing culturing method will be highly valuable.

Add p-values and SEM for qPCR data. This was done for hormones, should be the same way for other results.

Regarding all RT-qPCR data-There is a switch between 3bHSD and Actb/Gapdh as housekeeping genes. There does not seem to be as some have 3bHSD and others do not. Why do Igf1 and Dhh not use 3bHSD for housekeeping? If this is the method to be used, then 3bHSD should be used as housekeeping for the protein data, instead of ACTB. Also, based on Figure 1B and Figure 2A (Hsd3b1) there does not seem to be a strong correlation between Leydig cell # and the gene expression of Hsd3b1. If Hsd3b1 is to be used as a housekeeper and a proxy for Leydig cell number a correlation between these two measurements is necessary. If there is no correlation a housekeeping gene that is stable among all samples should be used. Sorting Leydig cells and then conducting qPCR would be optimal for these experiments.

Figure 2A (CYP17a1): It is surprising that the CYP17a1 gene and protein expression is very different between D30FT and 36.5dpp, however, the immunostaining looks identical between all groups. Why is this? A lower magnification image of the testis might make it easier to see the differences in Cyp17a1 expression. Leydig cells commonly have autofluorescence and need a background quencher (TrueBlack) to visualize the true signal in Leydig cells. This might reveal the true differences in Cyp17a1.

Figure 3D: there are large differences in estradiol concentration in the testis. Could it be that the testis is becoming more female-like? Leydig and Sertoli cells with more granulosa and theca cell features? Were any female markers investigated?

Figure 3D and Figure 5A: It is hard to imagine that intratesticular estradiol is maintained for 16-30 days without sufficient CYP19 activity or substrate (testosterone). 6.5 dpp was the last day with abundant CYP19 expression, so is most of the estrogen synthesized on this first day and it sticks around? Are there differences in estradiol metabolizing enzymes? Is there an alternative mechanism for E production?

Reviewer #3 (Public Review):

Signore et al. the synthesized and functionally characterized the recombinant adult hemoglobin (Hb) proteins of extant, extinct, and ancestral sirenians to explore the putative role of Hb in helping Steller's sea cows adapt to life in extremely cold waters. The functional comparisons show that the Hb of the subarctic Steller's sea cows differs in multiple biochemical properties relative to the Hbs of the two extant sirenians in the study, the Florida manatee, and the dugong and also from the Hb inferred for the common ancestor of Steller's sea cow and dugong. Specifically, the Steller's sea cow shows reduced oxygen binding affinity, reduced sensitivity to the allosteric co-factors DPG, Cl-, and H+, increased solubility, and reduced thermal sensitivity. DPG plays an important role in regulating Hb oxygen affinity in mammals, and the lack of sensitivity to it is unique to the Hb of Steller's sea cow. Sequence comparisons show that the Hb of the Steller's sea cow differs at 11 amino acids from that of its sister group, the dugong, one of which is intriguing because it occurs in a position that is invariable among mammals at a site that is critical for DPG binding, a change from Lys to Ans in position 82 of the mature β/δ globin chain. To test the significance of this change, the authors use site directed mutagenesis to insert back a Lys in the Steller's sea cow Hb background (β/δ82Asn→Lys) and test its biochemical properties. The functional assays with the β/δ82Asn→Lys mutant indicate that reverting this position to its ancestral state drastically altered the biochemical properties of the Steller's sea cow Hb, making it functionally similar to the Hbs of manatee, dugong, and the Hb inferred for the common ancestor of Steller's sea cow and dugong.

The study's strength lies in comparing the different recombinant Hbs in an explicit evolutionary framework. The conclusions are supported by the analyses, and the results are relevant in the fields of evolutionary biology, physiology, and biochemistry because they suggest that a single amino acid substitution in a protein can have profound biochemical consequences that impact whole organism physiology.

Reviewer #3 (Public Review):

In this article, Briggs et al. used scRNAseq to get high-resolution cell cycle-regulated transcriptomes of both replicative forms of Trypanosoma brucei (PCF and BSF) without prior synchronization. Briggs et al. also demonstrated that performing the scRNAseq library immediately after thawing cryopreserved samples did not show significant differences. The authors used computational reconstruction of the cell cycle to get the dynamic expression patterns of cycling genes in both life cycle forms. They identified a core cycling transcriptome highly conserved between forms. However, some slight differences were found between them, e.g.: a switch in gene expression associated with the S-G2 transition is much more discrete in PCFs than BSFs. Moreover, as proteomics data across the cell cycle is not available for BSFs, the authors tagged the top most significant genes with transcripts peaking in G1, S, and G2/M phases with a fluorescent epitope. After comparing the transcript expression patterns with protein abundance, the authors found that the majority of genes with periodic cycling transcript and protein levels exhibited a relative delay between peak transcript and protein expression, which was expected. In summary, this work provides a valuable public tool for further investigation into gene expression dynamics throughout the cell cycle in T. brucei.

Reviewer #3 (Public Review):

Nurr1 is an orphan nuclear receptor that may be a significant target for the treatment of neurodegenerative disorders. Targeting Nurr1 with small molecule ligands has been challenging, but there has been some progress in the identification of synthetic ligands that appear to increase Nurr1 activity. Nurr1 functions as a monomer, but may also heterodimerize with RXR. Heterodimerization appears to repress Nurr1 transcriptional activation via NBRE-driven reporters. Importantly, small molecule ligands that appear to selectively activate the Nurr1/RXR heterodimer complex (and not the Nurr1 or RXR homodimers, individually) have been identified. Exactly how these ligands function in this manner is unclear.

Here, the authors demonstrate that Nurr-1/RXR agonists actually function by perturbing the heterodimer formation providing Nurr1 monomers that are much more active in driving transcription. The authors demonstrate this with a range of biochemical, biophysical, and cell-based methodologies. Cotransfection assays examining the activity of Nurr1 on an NBRE reporter illustrate that RXRalpha is a repressor of Nurr1 transcriptional activity and that this is mediated by the RXR LBD. Using this experimental model as well as RXR coactivator interaction assays (biochemical) and RXR/DR1 reporter cotransfection assays, the authors examined multiple classes of RXR ligands (RXR agonists, modulators, antagonists, and Nurr1/RXRa selective agonists) to compare their activities. The Nurr1/RXR heterodimer agonists were quite effective at inducing transcription in the Nurr1/RXR assay but relatively ineffective in the RXR - coactivator binding assay or the RXR cotransfection assay. Using the array of ligands, the authors show that the Nurr1/RXR activity does not correlate to the ability of compounds to induce RXR to recruit a coactivator or activate RXR-mediated transcription. This suggests that Nurr1/RXR heterodimer agonists may not be mediating transcriptional activation via the "standard" mechanism. One weakness here is that some compounds used in the Nurr1/RXR transcription assay are not included in the other assays and may not have been included in the correlation studies. Assessment of RXR/Nurr1 dimerization in the presence of the ligands was assessed by ITC and demonstrated a correlation between the weakening of heterodimer formation and Nurr1/transcriptional activity suggesting that modulation of dimerization may be a mechanism by which the Nurr1/RXR heterodimer specific ligands function. NMR and analytical SEC data support this hypothesis as well. With regard to the physiological significance of these observations, no studies were completed on actual Nurr1 target genes addressing this type of mechanism offering limitations on the applicability of the hypothesis. However, the mechanism proposed is strongly supported by the data and offers a novel paradigm for the development of drugs targeting this receptor and possibly other nuclear receptors as well.

Reviewer #3 (Public Review):

The authors described the one family showing autoinflammatory phenotypes with L236P variant of TNFAIP3 gene. The variant has not been reported on and they evaluated the function of this variant using in vitro and in silico methods. I think this is well-written manuscript and I agree with their interpretation about the pathogenicity of this variant, but the new finding is poor. The variant information was only a new finding.

I recommend the revision of the following points.

In Table 2, T647P seemed to be pathogenic which was evaluated with in vitro assay by Kadowaki.

Two other missense variants, V377I (Niwano, Rheumatology 2022) and T602S (Jiang W, Cellular Immunol 2022) were recently reported. These should be included in the discussion.

Reviewer #3 (Public Review):

Genome-wide association studies on asthma have been challenging, due to the accuracy of phenotyping and potential gene-environment interactions. Thus, the authors aimed to identify genetic loci associated with subtypes of childhood wheezing. One main strength of this investigation is that the data availability of wheeze from birth to adolescence among multiple birth cohorts allowed the sub-phenotyping on a large scale and high statistical power. The study is properly designed and the conclusions are well-supported. Understanding the heterogeneity in subtypes of childhood wheezing is of great clinical interest and may help inform future directions in disease prediction and prevention.

Reviewer #3 (Public Review):

Numerous experimental models are phenotyped in this manuscript including mouse neurons, iPSC-derived human neurons, knock-in mice, and knock-in iPSCs. Expression of acetylation-mimic or acetylation-null TDP-43 protein is achieved either with overexpression or CRISPR-Cas9-based knock-in. A complex phenotype is observed including loss of TDP-43 function (reduced autoregulation, increased cryptic splicing) and a gain of TDP-43 (increased insoluble TDP-43 protein). These correlate with downstream neurobehavioral changes which are most consistent with a cortical/hippocampal phenotype without a motor phenotype. Post-translational modifications of disease-associated proteins are thought to contribute to neurodegenerative disease pathogenesis, and this study succeeds in demonstrating that TDP-43 acetylation results in downstream molecular and behavioral phenotypes.

There are numerous additional strengths. TDP-43 acetylation is a post-translational modification that is known to be associated with TDP-43 inclusions that are characteristic of human diseases. An important strength is the rigorous use of multiple different experimental models (rodent cells, iPSC-derived neurons, mice, overexpression, knock-in) with overall consistent results. Moreover, multiple orthogonal endpoints are presented including histology/cytology/immunostaining, biochemistry, molecular biology, and neurobehavioral assays. As TDP-43 acetylation is known to block RNA binding, these novel cellular and mouse models represent interesting albeit complex tools to study the functional consequences of a partial loss of function. As TDP-43 regulates its own expression (i.e. autoregulation), the complexity lies at least in part due to the loss of RNA binding leading to a functional loss of TDP-43 function which includes the increased expression of the TARDBP transcript and TDP-43 protein.

Conceptually, there is a disconnect in that the mouse model exhibits primarily a cortical/hippocampal phenotype more akin to frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), while TDP-43 acetylation is only seen in ALS tissues and not in FTLD-TDP tissues because most of the pathologic protein in the latter is N-terminally truncated (i.e. the acetylation site is not present). That being said, there is no mouse model which completely and faithfully recapitulates the human disease, and this mouse model avoids overt overexpression (increased TDP-43 protein expression stemming from altered autoregulation) and avoids the use of synthetic/artificial mutation (such as mutation of the TDP-43 nuclear localization signal).

In terms of the CNS phenotype, it is difficult to interpret the reduced density of NeuN-positive neurons in the mouse model as a neurodegenerative phenotype. The reduction in NeuN density but not overall cellular density is only suggestive of neurodegeneration (as opposed to, for example, a developmental phenotype) without more rigorous stereological approaches that take into account potential volumetric changes. Indeed, the absence of astrocytosis and microgliosis argues against neurodegeneration.

Some of the loss of function measures (CFTR construct splicing, shift in SORT1 protein) are subtle, although RNA sequencing clearly shows many splicing aberrations including cryptic splicing events which overall supports that a loss of TDP-43 function is observed.

Finally, there are multiple instances where multiple measurements are made on a few biological replicates. ANOVA or t-tests are not appropriate in these instances (lack of independence).

Reviewer #3 (Public Review):

The current manuscript describes the expression of multiple Natriuretic peptides and their receptor during the early embryo development in the amphibian Xenopus laevis. This signaling pathway is well known to control a broad range of physiological processes but its role in embryogenesis has not been studied before. Thus, the study presents some important novel findings. After defining the combination of ligands and receptors expressed during embryogenesis, they used loss of function experiment to test the requirement of this signaling pathway to the development of neural crest cells.

The loss of function experiments are well controlled as they use both chemical inhibitors and Morpholino that block the translation of the protein. They also rescue the phenotypes by using either mRNA from the human protein (receptor), or purified peptides (Ligands).

The results clearly show that loss of either Npr1 or Npr3 affects the development of both neural crest and placodes, while Npr2 had no visible effect. Similarly, they found that the loss of the ligands Nppa and Nppc affected neural crest and placode development while Nppb had no effect. Again, the loss of function was achieved with both Morpholino KD and inhibitors. In general, the loss of neural crest and placodal marker are associated with an expansion of neural marker (Sox2) and a corresponding decrease of epidermal marker (Keratin).

For Npr3 the author show that the loss of the protein is associated with an increase in cell death and decrease in cell proliferation which match some previous work on the role of the receptor in other cell type. It is unclear how much this can account for the striking difference in patterning observed and experiment to test this have not been performed.<br /> Overall, this work is important for the field as it shows novel genes that are critical for craniofacial and sensory development. It is likely that mutations in any genes involved in this pathway could result in birth defect which could be corrected pharmacologically.

Reviewer #3 (Public Review):

The function of a brain area is defined by its interaction with other regions. Accordingly, two areas communicate via axons and dendrites, but the language is plurimodal along the neurotransmitter-receptor dimension. Consequently, reading its neurochemical constituents is the most advanced way to characterize the brain into functional territories.

Along this theoretical line of research, Rapan et al. produced an exceptional report on the structure of the macaque frontal lobes based on cytoarchitectural division complemented with functional connectivity and neurochemical data. Results are lavishly illustrated. They report 35 cytoarchitectural areas in the prefrontal lobe with precise, different connectivity and neurotransmitter profiles together with practical anatomical landmarks. All data is openly available to the community and will constitute a cornerstone for future neuroscientific research in the macaque frontal lobes.

I congratulate the authors for this already extraordinary work.

Reviewer #3 (Public Review):

The manuscript concerns the cleavage of the Gag polyprotein lattice from the HIV virion membrane, a key stage in HIV lifecycle, and one that is required for HIV to become infectious. Since cleavage requires homodimerization between the small fraction (5%) of such Gag polyproteins that carry a protease domain, referred to as Gag-Pol, this raises questions regarding how such homodimerization can take place, and whether it can happen on the required timescales, given that Gag-Pol is typically embedded in a lattice that is observed to form one large connected component.

The authors address these questions in silico, using particle-based reaction diffusion simulations. Such simulations are rigid-body and "structure-resolved" meaning that they rigidly incorporate the geometry of the polyproteins, and their various binding interfaces, based on existing structural data. Other aspects of the simulations are also in-line with available data, including copy numbers, lattice curvature, and dissociation rates. This focused approach is a strength of the work and allows the authors to make credible claims that their simulations have relevance to HIV (as does their commitment to comparison with HALO-SNAP-based measurements of dimerization kinetics as well as iPALM experiments that characterize lattice dynamics).

A central part of the model is that it allows for the "possibility of imperfect alignment of molecules in the lattice", presumably due to the incompatibility of regular hexagonal tiling and surfaces with non-zero Gaussian curvature, such as a sphere. This is implemented via the ad-hoc imposition of a free-energy penalty when complete hexamers are formed, implying that hexamers are less stable than six ideal bonds. By varying this strain penalty, the authors can change the stability of the lattice independently of individual binding affinities, allowing its use as an effective fitting parameter when comparing to HALO-SNAP data. In the latter case, agreement between simulation and experiment can only be found at moderate levels of lattice stability.

However, such energetic penalties are present whenever the polyprotein structure must undergo deformations which, on surfaces with nonzero Gaussian curvature, should be the case for partial tilings as well as complete ones (where all six interfaces form bonds). This, therefore, appears to be a weakness of the work. An elastic implementation of polyprotein structure, for example, would permit strain to accumulate (and therefore stresses to propagate) throughout the lattice naturally, irrespective of whether complete hexamers were formed, and might reasonably be expected to impact the likelihood of different lattice structures. Whilst it is not clear how or whether this would lead to qualitatively or quantitatively different results, it is nevertheless worth remarking upon since the authors high-level claim is that lattice structure is an important determinant of the mean-first-passage times to dimerization.

Overall, I find this to be a valuable study, carried out in a solid and comprehensive manner. The primary impact of the work appears to be twofold: the unification of different experimental measurements under a single model, and the further identification of the salient parts of that model that most impact biological function. The results advance the understanding of one of the steps of the HIV lifecycle, via a better description of the mechanisms underpinning Gag-Pol dimerization. Notably, the authors stop short of drawing parallels to many related concepts and models in statistical physics, such as those concerning percolation and diffusion limited aggregation as well as the notions of dislocations and defects in crystalline matter on curved surfaces. These might reasonably have provided a basis for better understanding and quantification of the authors' simulations, as well as improving the scope for extensions and conceptual clarity.

Reviewer #3 (Public Review):

This report by Mohebi et al. provides new answers to old questions by showing that the activity of striatal cholinergic interneurons (CINs) escalates progressively during specific reward-related behaviors and that this correlates with previously observed ramps in dopamine (DA) release in the nucleus accumbens core. The report is strong and provides evidence for the authors' hypothesis that DA ramps are independent of DA neuron activity, but are instead the result of CIN activity and corresponding acetylcholine (ACh) release. The authors further demonstrate that the fidelity of CIN activation and consequent driving of DA release is even more robust in vivo than observed ex vivo slice preparations, which is fundamental for understanding the role of ACh-DA interactions in behavior. The findings complement the authors' previous evidence ventral tegmental area (VTA) DA neuron firing patterns do not show a ramping pattern; the previously reported VTA data are appropriately included here (in Fig. 3) to illustrate the absence of VTA firing during the time-locked increases in CIN activity and DA release. The present studies stop short of showing a direct link between CIN activity and DA release, however, which would require examining DA release during behavior in the presence of an antagonist of nicotinic ACh receptors. The authors also extend the understanding of the regulation of DA release by acetylcholine (ACh) by showing that optical activation of CINs in vivo promotes DA release responses that do not attenuate with repetitive stimulation. This contrasts with previous results in ex vivo striatal slices in which ACh-evoked DA release has been found to decline progressively from rundown and/or receptor desensitization. The authors propose that in vivo, AChE may be more effective in curtailing local ACh levels than in slices because of the slightly lower temperature typically used for slice studies, as well as the use of superfusion that might facilitate some AChE washout (AChE inhibitors are still effective in slices, of course). Overall, the report not only provides evidence for the cellular substrate for DA ramps but also shows the robustness of ACh-driven DA release in vivo. A few points to strengthen the report are listed below.

1) The authors give a few details about how CINs were activated at the beginning of the results, but say only that DA dynamics were monitored using fiber photometry. Given that the methods are at the end, a brief summary should be given here to indicate whether this means direct monitoring of DA or indirect via GCaMP, for example. It would be helpful to note the sensor used in the abstract, as well. In this light, as it were, RdLight1 should be described upon the first mention.

2) The authors show that infusion of DHbE in the NAc likelihood of decisions to approach the center port, as did antagonism of DA receptors. This supports the authors' argument that ramping of CIN activity and consequent ACh release underlies observed ramps in DA release. However, to show a causal interaction requires testing whether the observed DA ramps are absent after DHbE infusion in the NAc, under the same conditions that attenuated behavior.

3) In Fig. 3, the y-axis title for the upper panels should specify VTA, not simply "rate". This is stated in the legend, but should also be specified in the figure panel.

4) A recent preprint in BioRxiv by AC Krok, NX Tritsch et al. shows a related correlation between ACh and DA release in vivo in a reward task, as well as differences in other conditions. This report shows also that cortical input to CINs indeed plays a role, as suggested in the concluding sections of the present report. Consideration of the data in the preprint in the context of the present results could be valuable for the field.

Reviewer #3 (Public Review):

In this manuscript, the authors explore the mechanism of ATRIP recruitment to single-stranded DNA (ssDNA), which is important for ATR activation and the subsequent control of DNA repair and cell cycle progression. Using Xenopus egg extracts, in vitro interaction assays, and ssDNA constructs, the authors found that AP endonuclease 1 (APE1) plays a role in the recruitment of ATRIP to ssDNA independently of RPA. Moreover, APE1 domains are characterized for ssDNA, ATRIP, and RPA interaction, determining that the nuclease activities of APE1 are not required for this new mode of ATRIP recruitment. Overall, the work presented makes a compelling case for a novel role for APE1 in ATRIP recruitment that seems crucial for ATR activation (at least in the Xenopus system). The results are likely to have an important impact on our understanding of the determinants for activation of ATR signaling and cellular responses to DNA damage and replication stress. It remains unclear whether the findings will be extended to other organisms and be relevant for different types of DNA lesions. Also, there are several points of concern in the manuscript that require further clarification, especially regarding some of the quantitative analyses presented and the claimed importance of the RPA-independent mode of ATRIP recruitment for ATR activation.

Reviewer #3 (Public Review):

This work describes transcriptome profiling of dissected skin of zebrafish at post-embryonic stages, at a time when adult structures and patterns are forming. The authors have used the state-of-the-art combinatorial indexing RNA-seq approach to generate single cell (nucleus) resolution. The data appears robust and is coherent across the four different genotypes used by the authors.

The authors present the data in a logical and accessible manner, with appropriate reference to the anatomy. They include helpful images of the biology and schematics to illustrate their interpretations.

The datasets are then interrogated to define cell and signalling relationships between skin compartments in six diverse contexts. The hypotheses generated from the datasets are then tested experimentally. Overall, the experiments are appropriate and rigorously performed. They ask very interesting questions of interactions in the skin and identify novel and specific mechanisms. They validate these well.

The authors use their datasets to define lineage relationships in the dermal scales and also in the epidermis. They show that circumferential pre-scale forming cells are precursors of focal scale forming cells while there appeared a more discontinuous relationship between lineages in the epidermis.

The authors present transcriptome evidence for enamel deposition function in epidermal subdomains. This is convincingly confirmed with an ameloblastin in situ. They further demonstrate distinct expression of SCPP and collagen genes in the SFC regions.

The authors then demonstrate that Eda and TH signalling to the basal epidermal cells generates FGF and PDGF ligands to signal to surrounding mesenchyme, regulating SFC differentiation and dermal stratification respectively.

Finally they exploit RNA-seq data performed in parallel in the bnc2 mutants to identify the hypodermal cells as critical regulators of pigment patterning and define the signalling systems used.

Whilst these six interactions in the skin are disparate, the stories are unified by use of the sci-RNA-seq data to define interactions. Overall, it's an assembly of work which identifies novel and interesting cell interactions and cross-talk mechanisms. There are some aspects that require clarification:

With respect to the discontinuous relationship noted in Figure 2I in the epidermis, the authors did not make mention of the fact that there are in fact two independent sources of periderm in the zebrafish. The first periderm derives from the EVL, is segregated a gastrulation, and gradually replaced from the basal epidermis during post-embryonic stages. Could this residual EVL-derived periderm have reduced sensitivity of the trajectory mapping from basal to periderm? The authors should comment whether their transcriptome dataset likely had residual EVL-derived periderm and if this might have impacted their trajectory continuity interpretation.

The authors ask if dermal SFCs express proteins associated with cartilage formation and use Col10a1 orthologues as markers (Fig 3B, I). I wonder if these are the best transcripts to answer this question as this has also been described to label osteoblasts in certain contexts in the fish and the authors might want to refer to Li et al 2009 or Avaron et al 2005. Were other markers of cartilage formation present such as collagen2 genes? These may be more definitive. The authors might want to reinterrogate their datasets for true cartilage markers or reframe their question.

Finally, of interest, were there any clear clusters on the UMAP plots (Fig 1 Supp3A) of unassigned identity? Even comment on these and how they were dealt with would be of significant interest to the field, as it is highly unlikely all cell types in the skin have been defined. This dataset promises to be a critical reference for defining these in the future.

Minor clarification:

Fig 2E top. The authors interpret that fate-mapped SFCs at the posterior margin are progressively displaced towards the scale focus. This is confusing as the margin SFC in Fig 2E seems to show them staying largely at the margin. Please clarify if this is what you meant.

Reviewer #3 (Public Review):

This paper addresses an apparent contradiction related to the interaction between spontaneous neural activity and neural plasticity during circuit development. It is well established that spontaneous activity contains instructive information for developmental downstream circuit refinement, for instance for the formation of retinotopic projections in the visual system. These developmental cues are contained in activity correlations, and thus can be picked up by Hebbian mechanisms. However, previous work has shown that informative correlations in spontaneous activity can be found on slow time scales (100s of milliseconds). Here it is shown that in this case correlations on fast time scales arise simultaneously from the integration of unrefined inputs and that these likely interfere with developmental plasticity. The paper shows that such fast, "parasitic" correlations can appear during retinal wave activity, and provides evidence that NMDA receptors can suppress them to avoid their influence during developmental plasticity.

This work is based on detailed biophysical models of thalamic relay neurons, which were fit to data from in-vitro whole-cell recordings. A genetic algorithm was used to fit these models, which provides a family of suitable models instead of a point estimate of good parameters. This approach is a real strength as it shows that the effects generalise well to many plausible models, and do not depend on specific parameter choices. The model neurons are then placed in simulated networks (without or with anatomically informed recurrent connections) and driven by retinal wave activity recorded from the mouse. Together the simulation and analysis show under which conditions fast, undesirable correlations do and do not appear. Specifically, the key model ingredient this work identifies for the suppression of fast correlations is the presence of NMDA receptors on the recipient synapses of the relay neurons.

An open question is how the parasitic correlations are actually suppressed by NMDA receptors. Is it correct that a stronger NMDAR contribution to the transmitted activity simply low-pass filters the incoming spike trains, and that fast correlations are smoothed out as a result? So are developing circuits tuned to slower activity? This could also explain why AMPA receptors subunits with slower kinetics are expressed during development in many circuits.

Taken together, I think this paper presents a very interesting set of results. The issue with parasitic correlations is quite obvious in retrospect, and clearly, a problem developing circuits will generally face. Additionally, the presence of NMDA receptors is often linked to plasticity and is seen as less important in shaping postsynaptic integration. Although no developmental plasticity has been modelled, would it be possible to predict the possible effects of experimental manipulations?

Dual of the dual is just the original space.

It's an example of theorem 2.7-9. A linear operator has boundedness and continuity being an equivalent conditions.

Reviewer #3 (Public Review):

In their study: Comprehensive characterization of tumor microenvironment in colorectal cancer via histopathology-molecular analysis, Wu et al., aim to examine the contribution of the tumour microenvironment (TME) on biological and clinical heterogeneity in colorectal cancer (CRC).

To achieve this the authors use a vast array of publicly available datasets across a variety of biological modalities (transcriptomic, epigenetic, mutational). Using thoughtfully curated genesets the authors classify CRC into 4 holistic comprehensive characterised CRC (CCCRC) subtypes which comprise immune, stromal, and tumour features of CRC biology.

The authors investigate the association of their novel CCCRC subtypes with current "gold standard" classification schemes.

The authors' integration of deep learning methods for HE classification and subsequent association with "Tumor level" CCCRC subtypes is a refreshing addition to the study. Comment on the degree of heterogeneity observed in HE samples and correlation to the heterogeneity of CCCRC subtypes would be a welcomed addition. It is likely publicly available datasets from such platforms as 10X Genomic Visium would be available for this type of analysis.

Whilst one of the main outcomes of the study is the addition of another classification scheme to the field of colorectal cancer, the CCCRC scheme represents a holistic perspective on CRC classification.

The authors provide a welcomed graphical overview of the complex narrative of the study in Figure 7.

The authors focus on the classification of inter-patient heterogeneity and its associated predictive and prognostic utility. There appears to be a significant degree of overlap between immunosuppressive and immune excluded, and proliferative and immuno-modulatory signatures in Figure 1A. One of the major limitations of patient response to treatment is intra-patient heterogeneity, it would be nice for the authors to comment briefly on the degree of intra-patient heterogeneity of the CCCRC subtypes.

Overall the authors succeed in providing a holistic deep characterisation of CRC from the perspective of a variety of biological modalities. The authors provide a novel classification scheme for the field of CRC which demonstrates prognostic and predictive utility, which would benefit from further validation from external datasets. The authors demonstrate a pathway for integration and interpretation of complex high-dimensional data into clinically translatable currency such as the H&E.

Reviewer #3 (Public Review):

The authors report a study where, using multiple datasets with [18F]FDG PET bolus + continuous infusion ("functional PET") and BOLD fMRI data, they re-evaluate the metabolic and hemodynamic properties of the default mode network (DMN) in a task-evoked context, with a focus on posteromedial DMN due to its relevance for across-network integration.<br /> They show how posterior DMN is differently engaged depending on the chosen task: while visual and motor tasks lead to BOLD deactivations and glucose metabolic decrease, specifically in the dorsal posterior cingulate cortex (PCC) area, working memory tasks produce BOLD deactivations but metabolic increases, specifically in ventral PCC, as shown in their previous paper (Stiernman et al. 2021, https://doi.org/10.1073/pnas.2021913118). This aims to solve the controversies elicited by findings of both increased and decreased glucose consumption in the presence of BOLD deactivation in the DMN.

Additionally, they show how task-evoked glucose metabolism in posterior DMN seems to be shaped by that of the corresponding task-positive networks, with a positive link with dorsal attention and a negative link with frontoparietal network metabolism. This is explored using a type of directional connectivity analysis called "metabolic connectivity mapping", drawn from their previous work (Riedl et al. 2016, https://doi.org/10.1073/pnas.1513752113; Hahn et al. 2020, https://doi.org/10.7554/eLife.52443). They go on to speculate that concomitant BOLD deactivation and reductions in glucose expense might relate to decreased glutamatergic signaling, while BOLD deactivations accompanied by increased glucose consumption might depend on increased GABAergic neuronal activity.

This is a relevant topic because it not only shows how the DMN is flexibly engaged in different tasks but also allows us to better understand the complex relationships between BOLD fMRI and [18F]FDG PET signals, which are still not fully characterized to this day. Of course, while in resting state the situation is further complicated by the more uncertain physiological meaning of the resting BOLD signal, task-evoked states are expected to provide a more interpretable intermodal link between metabolism and hemodynamics, due to the known major changes in blood flow, blood volume, and glucose metabolism - which underlie BOLD and [18F]FDG signal changes - in response to neural activation. However, even in task states, there is not always a strong association between the two responses, as previously shown by the authors themselves (Rischka et al. 2018, https://doi.org/10.1016/j.neuroimage.2018.06.079). This is something I think the authors should stress out a little more, as they have previously done (Rischka et al. 2018, https://doi.org/10.1016/j.neuroimage.2018.06.079), both in the introduction and in reference to Figure 1, which shows clear differences between BOLD and [18F]FDG activations/deactivations (e.g., widespread negative responses in the cerebellum for [18F]FDG).

Overall, the analyses reported in the manuscript are simple and seem mostly sound, drawing from well-established methods in PET and fMRI activation studies, with additional approaches previously developed by some of the authors themselves (e.g., "metabolic connectivity mapping", Riedl et al. 2016, https://doi.org/10.1073/pnas.1513752113). Moreover, a clear strength of the paper is the high number of subjects, at least from a PET perspective, i.e., n = 50 for the Tetris task, plus group averages of previously published data for working memory (Stiernman et al. 2021, https://doi.org/10.1073/pnas.2021913118) and motor tasks (Hahn et al. 2018, https://doi.org/10.1007/s00429-017-1558-0).

The conclusions are in line with the results, and, though a little speculative, are potentially relevant for further exploration aimed at characterizing the neurotransmitter pathways underlying positive and negative BOLD and [18F]FDG responses. Moreover, the language is sufficiently clear to allow a proper understanding of the aims and the results, as well as the details of the analyses. As a side note, the title should probably be adjusted to "Task-evoked metabolic demands of the posteromedial default mode network are shaped by dorsal attention and frontoparietal control networks", to emphasize that the findings do not necessarily generalize to the resting state.

In conclusion, I am overall quite positive about this manuscript, which seems to nicely position itself within the existing literature, making some additional contributions.

Reviewer #3 (Public Review):

In this paper, the authors explore the effects of the environment, specifically temperature, on male harm to females. Male harm is the phenomenon where males reduce female fitness in polyandrous systems, where a single female may mate with multiple males. The selection of males to increase their reproductive success in male-male competition can lead to genetic conflict that increases male fitness at the expense of female fitness. Typically, male harm has been studied in single environments under optimal conditions. However, there is an increasing focus on the effect of the environment on fitness costs of male harm to females, as a way to better understand the effect of male harm on population fitness in more realistic ecological contexts. In this paper, the authors add to these studies by exploring the effect of temperature on male harm and female fitness, using the fruit fly Drosophila melanogaster, as a model system. They find that temperature affects the impact of male harm on female fitness, with male harm having the greatest effect at 24˚C relative to 20˚C and 28˚C. The authors then go on to disentangle how temperature affects the various components of male harm that impact female fitness (e.g. harassment, ejaculate toxicity). The paper demonstrates that male harm depends on ecological context, which has implications for understanding its impact on population fitness under realistic ecological scenarios, particularly with respect to climate change.

The strength of the paper is that it demonstrates that male harm (presented as differences in female life reproductive success between monogamous and polyandrous matings) changes with temperature. The authors dissect this general observation by showing that different aspects of pre-copulatory reproductive behavior, for example, male-male aggression, copulation rate, and female rejection rate, also change with temperature. Further, they demonstrate that correlates for male ejaculate quality also change with temperature, suggesting that temperature also affects post-copulatory mechanisms of male harm.

The weakness of the paper is that the method and results section are difficult to follow, which negatively impacts the interpretation of the data. The experiments are complex and need to be for what the authors are studying. Nevertheless, the paper is written in a way that makes it challenging for the reader to fully understand how precisely the experiments were conducted. Further, the authors do not explain clearly how some of the experiments relate to the phenomenon ostensibly being assayed. For example, a more detailed explanation of why mating duration and remating latency are assays for ejaculate quality in the context of sperm competition would be very helpful in interpreting the data. Further, a clearer explanation of the statistical analyses conducted

Reviewer #3 (Public Review):

Henthorn and coworkers obtain a single cell atlas of the parasite nematode Brugia malayi to search for excretory secretory products. These are involved in therapeutic responses but it is unknown what are the cell types that express them. In fact this seems as an ideal question to be addressed with single cell transcriptomics. The authors analyse their dataset, coming to the conclusion that many of these ES products are expressed broadly throughout the parasite, including secretory and non secretory types. This would be a nice conclusion if supported by the data. Then they go on to compare responses to exposure of ivermectin at the single cell level.

I must praise the attempt of using single cell transcriptomics to examine this question. These relatively novel methods have been so far used to collect information of cell types, but have immense potential for the investigation of important questions in neglected diseases like this. Fundamental knowledge about the biology of Brugia malayi and the tissue and cell types present are key to understanding their pathogenesis and advancing new therapeutic options. The authors start this research project with the right model and right technique.

My major concern is the quality of their single cell data. The authors perform no FACS or other methods to clear their suspension from cellular debris. This arises from all cell types, and then gets encapsulated with single cells in the droplet-based single cell transcriptomic process. Then, all cell barcodes receive genes from a single cell but also from a collection of cellular debris particles (arising from all other cell types). This, and only this, can in principle explain one of the major findings in the abstract: secreted antigens are expressed broadly in many cell types. This same caveat might explain their finding of pan neuronal markers broadly expressed - conceptually very similar to what the authors hold for secreted antigens, but that the authors only mention briefly and do not explain.

Reviewer #3 (Public Review):

In this manuscript, the authors (Salas et al.) have investigated the communication strategies among various strains or species of Trichomonas vaginalis. T. vaginalis is a parasite that is responsible for non-viral sexually transmitted infections, worldwide. The authors have demonstrated that highly adherent parasite uses cytoneme-like membrane structures and extracellular vesicles (EVs) to communicate with poorly adherent isolates and mount a stronger response to hosts.

The major strength of this work is the use of state-of-the microscopic techniques to analyze cytonemes and EVs. However, the weakness is the experiments shown in the manuscript are more descriptive than mechanistic. The significance of this work is high because it demonstrates the presence of a unique communication strategy in Trichomonas vaginalis. Trichomonas uses cytoneme-like elongated membrane structures and extracellular vesicles to interact with each other and induce a robust pathogenic response in host cells.

The authors have used state-of-the-art cell biology techniques to conduct the study and the data analysis is solid.

Overall, the experiments are solid and the authors were able to accomplish their objectives of demonstrating parasite communication in T. vaginalis.

Reviewer #3 (Public Review):

A current topic in the translational control field revolves around the idea that "the ribosome" is not a singular monolith machine, but rather that there are a variety of ribosomes, some with specialized functions. The presence of evolutionarily conserved ribosomal protein gene paralogs provides a platform for testing this idea. Presumably, if a paralog is required to translate a specific mRNA or class of mRNAs in a cell or organ type specific manner, it's loss should generate an observable phenotype. In this study, Xu and colleagues exploit the evolutionarily conserved eS27 and eS27L proteins to probe this hypothesis. Technically, the work is on the cutting edge of the field. Advanced genetic engineering techniques were used to generate mice lacking either paralogous gene, to create reciprocal swaps of each coding sequence into the other locus, and even to create genetically homogenous mice. The authors also use state of the art molecular biology methods, e.g. paralog-specific ribosome profiling, to search for differences in the mRNAs translated by ribosomes containing either of the two homologs.

Some phylogenetic evidence was presented suggesting that the paralogs first appeared during a gene duplication event in vertebrates: however, only and bird and one amphibian are represented. It is recommended that this analysis go deeper, parsing the amphibians and fish more finely. Although not identifying evidence for specialized ribosomes, they did find that it is essential that at least two copies of eS27 or eS27L are retained. Interestingly, the embryonic lethality of truncation alleles of either of the two paralogs result manifested at different stages of development, pointing to some kind of functional differences during development. The finding that eS27L containing ribosomes are more prevalent in lactating mammary gland and liver is an interesting observation, and that such ribosomes are preferentially associated with mRNAs involved in the cell cycle. From this the authors conclude that the data support subfunctionalization model of eukaryotic ribosomal protein S27 evolution rather than a specialized ribosome model. I also note that this is the most comprehensive and technologically advanced study of its kind in the translational control field and that it represents a significant contribution to the field of evolutionary biology.

Reviewer #3 (Public Review):

Mating changes an animal's behavior. In Drosophila, mated females have higher energy needs, suggesting that their consumption of caloric foods may be altered. While previous studies have examined post-mating changes in the consumption of specific nutrients such as salt and protein, it was not known whether the intake of sugar, their primary energy source, is also changed. This study describes a post-mating increase in sugar intake and identifies the neural circuit that mediates this change. By using precise genetic manipulations, behavioral assays, and new connectome datasets, the authors provide high-quality data to support their claims.

This study reveals several new insights into the regulation of behavior after mating: 1) Female flies increase sugar intake after mating, and this is an "anticipatory" change rather than a homeostatic change resulting from energy depletion. 2) The post-mating change in sugar intake is mediated by the sex peptide circuit, SPSNs-SAG-pC1, which is known to regulate other post-mating changes. 3) The authors identify a new downstream target of pC1, the pCd-2 neurons, which regulate feeding. pCd-2 neurons do not affect egg-laying, and neurons downstream of pC1 that regulate egg-laying or receptivity after mating do not affect sugar intake. Thus, the SPSN-SAG-pC1 circuit that regulates post-mating behaviors diverges downstream of pC1 into multiple branches regulating different behaviors. 4) The authors identify cells downstream of pCd-2, median bundle cells expressing Lgr3, the receptor for Dilp8. These cells are inhibited by pCd-2, suggesting that they are active in mated females, and promote sugar consumption. Because previous studies showed that Dilp8 and Lgr3 are expressed more highly in fed flies and suppress feeding, the present study suggests that Lgr3+ cells integrate hunger and mating signals to regulate feeding. This is an interesting circuit motif that could extend to mammals. In future studies, it will be interesting to test how hunger and mating signals are integrated within these cells (e.g. do they function redundantly, additively, etc).

Reviewer #3 (Public Review):

Seelbinder et al present local heating of the cell nucleus in live cells as a perturbation of the nucleus, which they use to interrogate mechanical properties of the nucleus. The authors use their recently developed technique of generating local heat gradients (Mittasch et al, 2018) and apply it to the cell nucleus, where they then measure the displacements/strains of chromatin as a function of distance from the heat source. They show that during the heat perturbation the nuclear area and shape remain unchanged. They measure spatially resolved strains across the nucleus and find that different parts of the nucleus exhibit different mechanical behavior. Their analysis reveals that chromatin shows both elastic and viscous properties at the timescales of seconds, with heterochromatin showing solid-like properties. In addition, they find that the nucleolus shows high resistance to the heat-induced deformation at the seconds' timescale.

Conceptually, this is an interesting and thought-provoking work allowing for new ways to perturb the cell nucleus and study its internal mechanics.

Reviewer #3 (Public Review):

In this manuscript, Li et al. describe the contribution of the ATM-E6AP-MASTL pathway in recovery from DNA damage. Different types of DNA damage trigger an increase in protein levels of mitotic kinase MASTL, also called Greatwall, caused by increased protein stability. The authors identify E3 ligase E6AP to regulate MASTL protein levels. Depletion or knockout of E6AP increases MASTL protein levels, whereas overexpression of E6AP leads to lower MASTL levels. E6AP and MASTL were suggested to interact in conditions without damage and this interaction is abrogated after DNA damage. E6AP was shown to be phosphorylated upon DNA damage on Ser218 and a phosphomimicking mutant does not interact with MASTL. Stabilization of MASTL was hypothesized to be important for recovery of the cell cycle/mitosis after DNA damage.

The identification of this novel pathway involving ATM and E6AP in MASTL regulation in the DNA damage response is interesting. However, is surprising that authors state that not a lot is known about DNA damage recovery while Greatwall and MASTL have been described to be involved in DNA damage (checkpoint) recovery. In addition, PP2A, a phosphatase downstream of MASTL is a known mediator of checkpoint recovery, in addition to other proteins like Plk1 and Claspin. Although some of the publications regarding these known mediators of DNA damage recovery are mentioned, the discussion regarding the relationship to the data in this manuscript are very limited.

The regulation of MASTL stability by E6AP is novel, although the data regarding this regulation and the interaction are not entirely convincing. In addition, several experiments presented in this paper suggest that E6AP is (additionally) involved in checkpoint signalling/activation, whereas the activation of the G2 DNA damage checkpoint was described to be independent of MASTL. Has E6AP multiple functions in the DNA damage response or is ATM-E6AP-MASTL regulation not as straightforward as presented here?

Altogether, in my opinion, not all conclusions of the manuscript are fully supported by the data.

Reviewer #3 (Public Review):

Martino et al. demonstrated that BRCA2-deficient cells (but not BRCA1-deficient cells) bear additional vulnerabilities (i.e., cytokinesis failure) outside S phase that could represent new synthetic lethality targets. Strengths of the study include the ability of the authors to recapitulate the cell death by ROCK inhibition by inhibiting another key cytokinesis enzyme, CITK. The claims are well supported by the data, and because the study indicates HR failure/replication stress is not the only possible way to achieve synthetic lethality in BRACA mutant cancers the study will be of broad interest to potential readers.

Reviewer #3 (Public Review):

In the work by Zhou, et.al., the authors pursue a mechanistic understanding of chromosome size scaling in development, a problem first noted in 1912 by Conklin and largely unstudied until very recently. Using the tools available in the Xenopus genus developmental biology system (cell extracts, related species of differing size, etc.), they nicely show that condensin I levels directly correlates with chromosome size. Further, importin levels decrease leading to axial shortening of chromosomes during development. The combined physical outcome is that Mitotic Chromatin looping changes, resulting in axial compression of chromosomes. This work represents a major step in the molecular understanding of how the genome is regulated through development and changing cell size, which also occurs in many other adult tissues and cancers. Further work to understand other contributing factors and understanding how loop structure changes the polymer dynamics of mitotic chromatin will be exciting in the future.

Reviewer #3 (Public Review):

International researchers from the International Agency for Research on Cancer and cancer screening program experts from six countries in Latina America (Argentina, Colombia), Asia (Sri Lanka, Bangladesh, Thailand), and Northern Africa (Morocco), provide detailed information on the impact of the COVID Pandemic on cancer screening and diagnostic services.

The authors examine countries that have had screening programs and a surveillance system/registry to see how the volume of screening, diagnostic procedures, and detection of precancers or cancers are impacted. The data are presented as case studies with an explanation of the program, the technologies, and then the impact. They describe no matter how low or high income the country, there was a considerable impact on the volume of screening.

Usually, the impact of the pandemic on cancer screening has been limited to Europe or North America and is usually not quantified. This information will be helpful for these countries to examine the impact on stage distribution and eventually mortality impact through modeling studies. The authors also comment on some interesting hypotheses such that the impact on recovery based on if one is detecting precancers (e.g. colon cancer/cervical cancer) vs. invasive cancers (breast cancer). Strategies that require less frequent screening,self-collection, where screening and treatment can be combined in fewer visits, or where some visits can occur via telehealth are valuable strategies or lessons learned that will allow for quicker recovery time after a pandemic.

The authors acknowledge the limitations and strengths of these case-based studies well.

It is beautiful storytelling with both qualitative and quantitative data.

Reviewer #3 (Public Review):

Ketamine has been shown to be effective at producing a rapid-antidepressant effect at low doses, but the underlying molecular mechanism of this effect is still not clear. Previous studies have suggested that the effect of low-dose ketamine may occur by promoting neuronal plasticity in the hippocampus. However, this goes against the findings that ketamine acts as a noncompetitive NMDA receptor antagonist, which should prevent NMDAR-dependent plasticity. Furthermore, a therapeutic dose of ketamine has been shown to increase neuronal Ca2+ signaling, which again does not conform to its antagonistic action on NMDA receptors. In this paper, the authors provide evidence that therapeutic low-dose ketamine increases the expression of Ca2+-permeable AMPA receptors (CP-AMPARs) by increasing phosphorylation of GluA1 subunit of AMPARs and surface expression of GluA1-containing CP-AMPARs. They further provide evidence that this is likely mediated by a decrease in calcineurin activity and that blocking CP-AMPARs prevent the antidepressant effect of ketamine in mice. One interesting finding of this study is that the authors see heightened sensitivity of ketamine in female mice, both at the level of behavioral readout and for molecular correlates. This finding is interesting in light of the different pharmacokinetics of ketamine reported in females and that ketamine metabolites can bind estrogen receptors.

Based on their data and previous findings, the authors outline a plausible molecular signaling mechanism for the antidepressant effect of ketamine. Specifically, the authors propose that reduced neuronal activity, which could be triggered by ketamine-induced NMDAR antagonism, causes homeostatic plasticity to upregulate GluA1-containing CP-AMPARs. Their data would support this idea, as phosphorylation of GluA1 as well as increased surface expression and functional incorporation of CP-AMPARs at synapses have been shown before in models of homeostatic plasticity.

Overall, the study is well-done and the data presented support the main conclusions. One main question is whether the current finding provides a conceptual advancement in our understanding of the molecular signaling involved in ketamine's antidepressant effects. There are previous studies that showed an increase in CP-AMPARs in the nucleus accumbens and an increase in the expression of GluA1 in the hippocampus with low-dose ketamine. In addition, ketamine's antidepressant effect has been shown to require GluA1 phosphorylation. The main contribution of this paper might be that it provides the potential molecular signaling within the same preparation (i.e. hippocampal neurons) and provides a causal link of CP-AMPARs in mediating the behaviorally measured antidepressant effect of ketamine.

Another question is whether the behavioral effect of ketamine is due to molecular changes in the hippocampus as outlined in this paper. A more targeted inhibition of CP-AMPAR function could resolve this issue. With the systemic application of CP-AMPAR antagonist as done in this study, it would be hard to know the role of CP-AMPAR upregulation in the hippocampus in mediating ketamine's effect. Especially, considering that low-dose ketamine has been shown to upregulate CP-AMPARs in the nucleus accumbens. While it would have been nice to know the site of action, this does not alter the conclusion that CP-AMPARs are involved in mediating the antidepressant effect of ketamine on behavioral readouts.

Reviewer #3 (Public Review):

This paper describes fundamental work which attempts to understand how universal stress proteins Rv1636/Msmeg3811 function as a sink allowing mycobacteria to use intra-bacterial cAMP. Because cAMP is a major second messenger, Rv1636 remains essential to mycobacteria. A compound that inhibits cAMP binding of Rv1636 also can effectively inhibit mycobacterial growth. The major strength of the manuscript is that the authors probed their hypothesis by different approaches. In general, the conclusions from the results are largely justified. However, I find the manuscript quite difficult to follow. Also, the results and functional analyses are inadequate as they rely on a limited set of experiments, thereby making the evidences less than compelling.