Reviewer #2 (Public Review):

In their paper entitled "Molecular, Cellular, and Developmental Organization of the Mouse Vomeronasal Organ at Single Cell Resolution" Hills Jr. et al. perform single-cell transcriptomic profiling and analyze tissue distribution of a large number of transcripts in the mouse vomeronasal organ (VNO). The use of these complementary tools provides a robust approach to investigating many aspects of vomeronasal sensory neuron (VSN) biology based on transcriptomics. Harnessing the power of these techniques, the authors present the discovery of previously unidentified sensory neuron types in the mouse VNO. Furthermore, they report co-expression of chemosensory receptors from different clades on individual neurons, including the co-expression of VR and OR. Finally, they evaluated the correlation between transcription factor expression and putative surface axon guidance molecules during the development of different neuronal lineages. Based on such correlation analysis, authors further propose a putative cascade of events that could give rise to different neuronal lineages and morphological organization.

Taken together, Hills Jr. et al. present findings on (a) cell types in the VNO, (b) novel classes of sensory neurons, (c) developmental trajectories of the neuronal linage, (d) receptor expression in VSNs, (e) co-expression of chemosensory receptors, (f) a surface molecule code for individual receptor types, and (g) transcriptional regulation of receptor and axon guidance cues. Before outlining the major strengths and weaknesses of the manuscript, we need to disclose that, while we are comfortable reviewing aspects (a) to (e) of their work, we lack the expertise to provide constructive criticism on the two last points (f) and (g). Thus, we will not comment on these.

In general, interpretations/claims put forward by Hills Jr. et al. appear striking at first glance. Upon careful review of the manuscript, however, it becomes apparent that many of the groundbreaking discoveries lack compelling support. Several (not all) of the results presented in this work lack novelty, accurate interpretability, and corroboration. A recurrent theme throughout the manuscript is an incomplete, and somewhat misleading account of the current knowledge in the field. This is perhaps most apparent in the introductory paragraphs, where the authors present a biased report of previously published work, largely including only those results that do not overlap with their own findings, but ignoring results that would question the novelty of the data presented here. For example: "...In contrast, transcriptomic information of the VNO is rather limited (Ref 24,25)...". Indeed, transcriptomic information of the mouse VNO is limited. Here, however, the authors ignore recent reports of robust single-cell transcriptomic analysis from adult and juvenile mice. These papers are, in part, cited later in this manuscript (ref 88, 89, 90, 91), or are completely missing (doi.org/10.7554/eLife.77259). Regardless, previously published results on the same topics have to be included in the Introduction to put the background and novelty of the findings into perspective.

General comments on (a) cell types in the VNO

The authors performed single-cell transcriptomic analysis of a large number of cells from both adult and juvenile VNO, creating the largest dataset of its kind to date. This dataset contains a wealth of information and, once made public, could be a valuable resource to the community. However, the analysis implemented in this paper raises several questions:

Did the authors perform any cell selectivity, or any directed dissection, to obtain mainly neuronal cells? Previous studies reported a greater proportion of non-neuronal cells. For example, while Katreddi and co-workers (ref 89) found that the most populated clusters are identified as basal cells, macrophages, pericytes, and vascular smooth muscle, Hills Jr. et al. in this work did not report such types of cells. Did the authors check for the expression of marker genes listed in Ref 89 for such cell types?

The authors should report the marker genes used for cell annotation. This is important for data validation, comparison with other publicly available datasets, as well as future use of this dataset.<br /> The authors reported no differences between juvenile and adult samples, and between male and female samples. It is not clear how they evaluate statistically significant differences, which statistical test was used, or what parameters were evaluated.

"Based on our transcriptomic analysis, we conclude that neurogenic activity is restricted to the marginal zone." This conclusion is quite a strong statement, given that this study was not directed to carefully study neurogenesis distribution, and when neurogenesis in the basal zone has been proposed by other works, as stated by the authors.

General comments on (b) novel classes of sensory neurons

The authors report at least two new types of sensory neurons in the mouse VNO, a finding of huge importance that could have a substantial impact on the field of sensory physiology. However, the evidence for such new cell types is based solely on this transcriptomic dataset and, as such, is quite weak, since many crucial morphological and physiological aspects would be missing to clearly identify them as novel cell types. As stated before, many control and confirmatory experiments, and a careful evaluation of the results presented in this work must be performed to confirm such a novel and interesting discovery. The reported "novel classes of sensory neurons" in this work could represent previously undescribed types of sensory neurons, but also previously reported cells (see below) or simply possible single-cell sequencing artefacts.

The authors report the co-expression of V2R and Gnai2 transcripts based on sequencing data. That could dramatically change classical classifications of basal and apical VSNs. However, did the authors find support for this co-expression in spatial molecular imaging experiments?

Canonical OSNs: The authors report a cluster of cells expressing neuronal markers and ORs and call them canonical OSN. However, VSNs expressing ORs have already been reported in a detailed study showing their morphology and location inside the sensory epithelium (References 82, 83). Such cells are not canonical OSNs since they do not show ciliary processes, they express TRPC2 channels and do not express Golf. Are the "canonical OSNs" reported in this study and the OR-expressing VSNs (ref 82, 83) different? Which parameters, other than Gnal and Cnga2 expression, support the authors' bold claim that these are "canonical OSNs"? What is the morphology of these neurons? In addition, the mapping of these "canonical OSNs" shown in Figure 2D paints a picture of the negligible expression/role of these cells (see their prediction confidence).

Secretory VSN: The authors report another novel type of sensory neurons in the VNO and call them "secretory VSNs". Here, the authors performed an analysis of differentially expressed genes for neuronal cells (dataset 2) and found several differentially expressed genes in the sVSN cluster. However, it would be interesting to perform a gene expression analysis using the whole dataset including neuronal and non-neuronal cells. Could the authors find any marker gene that unequivocally identifies this new cell type?

When the authors evaluated the distribution of sVSN using the Molecular Cartography technique, they found expression of sVSN in both sensory and non-sensory epithelia. How do the authors explain such unexpected expression of sensory neurons in the non-sensory epithelium?

The low total genes count and low total reads count, combined with an "expression of marker genes for several cell types" could indicate low-quality beads (contamination) that were not excluded with the initial parameter setting. It looks like cells in this cluster express a bit of everything V1R, V2R, OR, secretory proteins...

General comments on (c) developmental trajectories of the neuronal linage

The authors evaluated a possible cascade of events leading to the development of different lineages of mature sensory neurons using GBCs as a starting point. They found the differential expression of several transcription factors at different stages of development. This analysis was performed correctly, and its interpretation is coherent. However, it is mysterious why the authors included only classical V1R and V2R-expressing neurons, while the novel sensory neurons, cOSN and sVSN, were not included. Furthermore, it is important to notice again the misreport of previously published works.

The authors wrote "...the transcriptomic landscape that specifies the lineages is not known...". This statement is not completely true, or at least misleading. There are still many undiscovered aspects of the transcriptomics landscape and lineage determination in VSNs. However, authors cannot ignore previously reported data showing the landscape of neuronal lineages in VSNs (Ref ref 88, 89, 90, 91 and doi.org/10.7554/eLife.77259). Expression of most of the transcription factors reported by this study (Ascl1, Sox2, Neurog1, Neurod1...) were already reported, and for some of them, their role was investigated, during early developmental stages of VSNs (Ref ref 88, 89, 90, 91 and doi.org/10.7554/eLife.77259). In summary, the authors should fully include the findings from previous works (Ref ref 88, 89, 90, 91 and doi.org/10.7554/eLife.77259), clearly state what has been already reported, what is contradictory and what is new when compared with the results from this work.

General comments on (d) receptor expression in VSNs

The authors evaluated the expression of chemosensory receptors in the VNO and correlated receptor expression with the expression of transcription factors. The analysis of such correlation showed that, while the expression of V1Rs is mainly correlated with the expression of the transcription factor Meis2, the expression of V2Rs is correlated with the combination of many transcription factors. These results are interesting, however, the co-expression of specific V2Rs with specific transcription factors does not imply a direct implication in receptor selection. Directed experiments to evaluate the VR expression dependent on a specific transcription factor must be performed.

This study reports that transcription factors, such as Pou2f1, Atf5, Egr1, or c-Fos could be associated with receptor choice in VSNs. However, no further evidence is shown to support this interaction. Based on these purely correlative data, it is rather bold to propose cascade model(s) of lineage consolidation.

General comments on (e) co-expression of chemosensory receptors

The authors use spatial molecular imaging to evaluate the co-expression of many chemosensory receptors in single VNO cells. Molecular Cartography is a powerful tool and the reported data in this work is truly interesting. The authors show some clear confirmation of previously reported V2R co-expression (Figure 5H), and new co-expression of chemosensory receptors including V1R, V2R, and Fpr (Figure 5G-K).

However, it is difficult to evaluate and interpret the results due to the lack of cell borders in spatial molecular imaging. The inclusion of cell border delimitation in the reported images (membrane-stained or computer-based) could be tremendously beneficial for the interpretation of the results.

It is surprising that the authors reported a new cell type expressing OR, however, they did not report the expression of ORs in Molecular Cartography technique. Did the authors evaluate the expression of OR using the cartography technique?

Reviewer #2 (Public Review):

Summary:

The authors present a method that allows for the identification and localization of molecular machinery at chemical synapses in unstained, unfixed native brain tissue slices. They believe that this approach will provide a 3D structural basis for understanding different mechanisms of synaptic transmission, plasticity, and development. To achieve this, the group used genetically engineered mouse lines and generated thin brain slices that underwent high-pressure freezing (HPF) and focused ion beam (FIB) milling. Utilizing cryo-electron tomography (cryo-ET) and integrating it with cryo-fluorescence microscopy, they achieved micrometer resolution in identifying the glutamatergic synapses along with nanometer resolution to locate AMPA receptors GluA2-subunits using Fab-AuNP conjugates. The findings are summarized with detailed examples of successfully prepared substrates for cryo-ET, specific morphological identification and localization, and the detailed structural organization of excitatory synapses, including synaptic vesicle clusters close to the postsynaptic density and in the cleft.

Strengths:

The study advances previous work that used cultured neurons or synaptosomes. Combining cryo-electron tomography (cryo-ET) with fluorescence-guided targeting and labeling with Fab-AuNP conjugates enabled the study of synapses and molecular structures in their native environment without chemical fixation or staining. This preserves their near-native state, offering high specificity and resolution. The methods developed are generalizable, allowing adaptation for identifying and localizing other key molecules at glutamatergic synapses and potentially useful for studying a variety of synapses and cellular structures beyond the scope of this research.

Weaknesses

The preparation and imaging techniques are complex and require highly specialized equipment and expertise, potentially limiting their accessibility and widespread adoption.

Additionally, the methods might need further modifications/tweaks to study other types of synapses or molecular structures effectively.

The reliance on genetically engineered mouse lines may again impact the generalizability of the findings.

Similarly, the requirement of monoclonal, high-affinity antibodies/Fab fragments to specifically label receptors/proteins would limit the wider employment of these methods.

Reviewer #3 (Public Review):

Summary:

Cheng et al. studied if and how blood flow regulates differentiation of vascular smooth muscle cells (VSMC) in the Circle of Willis (CW) in zebrafish embryos. They show that CW vessels gradually acquire arterial identity. VSMCs also undergo gradual differentiation, which correlates with blood flow velocity. Using cell culture they show that pulsatile blood flow promotes pericyte differentiation into smooth muscle cells. They further identify transcription factor klf2a as differentially regulated by blood flow, and show that klf2a inhibition results in VSMC differentiation. The authors conclude that pulsatile flow promotes VSMC differentiation through klf2a activation.

Strengths:

Overall this is an important study, because VSMC differentiation in CW has not been previously studied, although analogous observations regarding the role of blood flow and klf2 involvement have been previously made in other systems and other vascular beds, for example, mouse klf2 mutants, which have deficient VSMC coverage of the dorsal aorta (Wu et al., 2008, JBC 283: 3942-50). The results convincingly show that VSMC differentiation in CW depends on the blood flow, and that klf2a flow dependent function regulates VSMC differentiation.

Weaknesses:

(1) The provided data do not support correlation between wall shear stress (WSS) and acta2+ cell number. The number of acta2+ cells in CaDI increases dramatically between 54 hpf and 3 dpf (Fig. 2F). However, the graph provided in the response to reviewers shows that WSS in CaDI is actually lower at 3 dpf compared to 54 hpf. Authors argue that Pearson correlation analysis shows that both variables increase together, but this is calculated over the stage between 54 hpf and 4 dpf. acta2+ cells appear by 3 dpf, and at this stage WSS in CaDI is not increased (or even lower), which argues agains WSS being the cause of acta2+ cell differentiation. Furthermore, data in Fig. 3I-K show that WSS actually decreases in BCA and PCS between 54 hpf and 4 dpf, while the number of acta2+ increases in BCA and PCS by 4 dpf. This also argues against the argument that WSS affects differentiation of acta2+ cells.<br /> (2) In multiple instances, results are based on a single independent experiment (Fig. 3, Fig. 4H, I, Fig. S2 and Fig. S3) with only a few embryos analyzed in many cases. This falls short of expected standards in the field, and it is unclear if these results are reproducible.

Reviewer #2 (Public Review):

Summary:

This manuscript explores the utility of AlphaFold2 (AF2) and the author's own AF2-RAVE method for drug discovery. As has been observed elsewhere, the predictive power of docking against AF2 structures is quite limited, particularly for proteins like kinases that have non-trivial conformational dynamics. However, using enhanced sampling methods like RAVE to explore beyond AF2 starting structures leads to a significant improvement.

Strengths:

This is a nice demonstration of the utility of the authors' previously published RAVE method.

Weaknesses:

My only concern is the authors' discussion of induced fit. I'm quite confident the structures discussed are present in the absence of ligand binding, consistent with conformational selection. It seems the author's own data also argues for an important role in conformational selection. It would be nice to acknowledge this instead of going along with the common practice in drug discovery of attributing any conformational changes to induced fit without thoughtful consideration of conformational selection.

Reviewer #2 (Public Review):

Summary:

This study by Sun et al. presents a role for the S. pombe MAP kinase Pmk1 in the activation of the Spindle Assembly Checkpoint (SAC) via controlling the protein levels of APC/C activator Cdc20 (Slp1 in S. pombe). The data presented in the manuscript is thorough and convincing. The authors have shown that Pmk1 binds and phosphorylates Slp1, promoting its ubiquitination and subsequent degradation. Since Cdc20 is an activator of APC/C, which promotes anaphase entry, constitutive Pmk1 activation leads to an increased percentage of metaphase-arrested cells. The authors have used genetic and environmental stress conditions to modulate MAP kinase signalling and demonstrate their effect on APC/C activation. This work provides evidence for the role of MAP kinases in cell cycle regulation in S. pombe and opens avenues for exploration of similar regulation in other eukaryotes.

Strengths:

The authors have done a very comprehensive experimental analysis to support their hypothesis. The data is well represented, and including a model in every figure summarizes the data well.

Weaknesses:

As mentioned in the comments, the manuscript does not establish that MAP kinase activity leads to genome stability when cells are subjected to genotoxic stressors. That would establish the importance of this pathway for checkpoint activation.

Reviewer #2 (Public Review):

Summary:

The study conducted by Noh et al. investigated the effects of parathyroid hormone (PTH) and a dimeric PTH peptide on bone formation and serum biochemistry in ovariectomized mice as a model for postmenopausal osteoporosis. The authors claimed that the dimeric PTH peptide has pharmacological benefits over PTH in promoting bone formation, despite both molecules having similar effects on bone formation and serum Ca2+. However, after careful evaluation, I am not convinced that this manuscript adds a significant contribution to the literature on bone and mineral research.

Strengths:

Experiments are well performed, but strengths are limited to the methodology used to evaluate bone formation and serum biochemical analysis.

Weaknesses:

(1) Limited significance of this study:<br /> • this study follows a previous study (not cited) reporting the effect of the dimeric R25CPTH(1-34) on bone regeneration in an osteoporotic dog (Beagle) model (Jeong-Oh Shin et al., eLife 13:RP93830, 2024). It's unclear why the authors tested the dimeric R25C-PTH peptide on a rodent animal model, which has limitations because the healing mechanism of human bone is more similar in dogs than in mice.<br /> • the authors should clarify why they tested the effects of dimeric R25CPTH(1-34) and not dimeric R25CPTH(1-84)?<br /> • The study is descriptive with no mechanism.

(2) Statistics are inadequately described or performed for the experimental design:<br /> • the statistical analysis in Figure 5 needs to be written in a way that makes it clearer how statistics were done; t-test or one-way ANOVA?<br /> • Statistics in Figures 6 and 7 should be performed by one-way ANOVA to compare the mean values of one variable among three or more groups, and not t-test.

(3) Misleading and confused discussion:<br /> • The first paragraph lacks clarity in the PTH nomenclature and the authors should provide a clear statement that the PTH mutant found in patients is likely a monomeric R25CPTH(1-84), considering that there has been no proof of a dimeric form.<br /> • Moreover, the authors should discuss the study by White et al. (PNAS 2019), which shows that there are defective PTH1R signaling responses to monomeric R25CPTH(1-34). This results in faster ligand dissociation, rapid receptor recycling, a short cAMP time course, and a loss of calcium ion allosteric effect.<br /> • The authors should also clarify what they mean by "the dimeric form of R25CPTH can serve as a new peptide ...(lines 328-329)" The dimeric R25CPTH(1-34) induces similar bone anabolic effects and calcemic responses to PTH(1-34), so it is unclear what the new benefit of the dimeric PTH is.

Please address these concerns.

Reviewer #2 (Public Review):

Here I submit my previous review and a great deal of additional information following on from the initial review and the response by the authors.

* Initial Review *

Assessment:

This manuscript is based upon the unprecedented identification of an apparently highly unusual trigeminal nuclear organization within the elephant brainstem, related to a large trigeminal nerve in these animals. The apparently highly specialized elephant trigeminal nuclear complex identified in the current study has been classified as the inferior olivary nuclear complex in four previous studies of the elephant brainstem. The entire study is predicated upon the correct identification of the trigeminal sensory nuclear complex and the inferior olivary nuclear complex in the elephant, and if this is incorrect, then the remainder of the manuscript is merely unsupported speculation. There are many reasons indicating that the trigeminal nuclear complex is misidentified in the current study, rendering the entire study, and associated speculation, inadequate at best, and damaging in terms of understanding elephant brains and behaviour at worst.

Original Public Review:

The authors describe what they assert to be a very unusual trigeminal nuclear complex in the brainstem of elephants, and based on this, follow with many speculations about how the trigeminal nuclear complex, as identified by them, might be organized in terms of the sensory capacity of the elephant trunk.<br /> The identification of the trigeminal nuclear complex/inferior olivary nuclear complex in the elephant brainstem is the central pillar of this manuscript from which everything else follows, and if this is incorrect, then the entire manuscript fails, and all the associated speculations become completely unsupported.

The authors note that what they identify as the trigeminal nuclear complex has been identified as the inferior olivary nuclear complex by other authors, citing Shoshani et al. (2006; 10.1016/j.brainresbull.2006.03.016) and Maseko et al (2013; 10.1159/000352004), but fail to cite either Verhaart and Kramer (1958; PMID 13841799) or Verhaart (1962; 10.1515/9783112519882-001). These four studies are in agreement, the current study differs.

Let's assume for the moment that the four previous studies are all incorrect and the current study is correct. This would mean that the entire architecture and organization of the elephant brainstem is significantly rearranged in comparison to ALL other mammals, including humans, previously studied (e.g. Kappers et al. 1965, The Comparative Anatomy of the Nervous System of Vertebrates, Including Man, Volume 1 pp. 668-695) and the closely related manatee (10.1002/ar.20573). This rearrangement necessitates that the trigeminal nuclei would have had to "migrate" and shorten rostrocaudally, specifically and only, from the lateral aspect of the brainstem where these nuclei extend from the pons through to the cervical spinal cord (e.g. the Paxinos and Watson rat brain atlases), the to the spatially restricted ventromedial region of specifically and only the rostral medulla oblongata. According to the current paper the inferior olivary complex of the elephant is very small and located lateral to their trigeminal nuclear complex, and the region from where the trigeminal nuclei are located by others, appears to be just "lateral nuclei" with no suggestion of what might be there instead.

Such an extraordinary rearrangement of brainstem nuclei would require a major transformation in the manner in which the mutations, patterning, and expression of genes and associated molecules during development occurs. Such a major change is likely to lead to lethal phenotypes, making such a transformation extremely unlikely. Variations in mammalian brainstem anatomy are most commonly associated with quantitative changes rather than qualitative changes (10.1016/B978-0-12-804042-3.00045-2).

The impetus for the identification of the unusual brainstem trigeminal nuclei in the current study rests upon a previous study from the same laboratory (10.1016/j.cub.2021.12.051) that estimated that the number of axons contained in the infraorbital branch of the trigeminal nerve that innervate the sensory surfaces of the trunk is approximately 400 000. Is this number unusual? In a much smaller mammal with a highly specialized trigeminal system, the platypus, the number of axons innervating the sensory surface of the platypus bill skin comes to 1 344 000 (10.1159/000113185). Yet, there is no complex rearrangement of the brainstem trigeminal nuclei in the brain of the developing or adult platypus (Ashwell, 2013, Neurobiology of Monotremes), despite the brainstem trigeminal nuclei being very large in the platypus (10.1159/000067195). Even in other large-brained mammals, such as large whales that do not have a trunk, the number of axons in the trigeminal nerve ranges between 400 000 and 500 000 (10.1007/978-3-319-47829-6_988-1). The lack of comparative support for the argument forwarded in the previous and current study from this laboratory, and that the comparative data indicates that the brainstem nuclei do not change in the manner suggested in the elephant, argues against the identification of the trigeminal nuclei as outlined in the current study. Moreover, the comparative studies undermine the prior claim of the authors, informing the current study, that "the elephant trigeminal ganglion ... point to a high degree of tactile specialization in elephants" (10.1016/j.cub.2021.12.051). While clearly the elephant has tactile sensitivity in the trunk, it is questionable as to whether what has been observed in elephants is indeed "truly extraordinary".

But let's look more specifically at the justification outlined in the current study to support their identification of the unusual located trigeminal sensory nuclei of the brainstem.

(1) Intense cytochrome oxidase reactivity<br /> (2) Large size of the putative trunk module<br /> (3) Elongation of the putative trunk module<br /> (4) Arrangement of these putative modules correspond to elephant head anatomy<br /> (5) Myelin stripes within the putative trunk module that apparently match trunk folds<br /> (6) Location apparently matches other mammals<br /> (7) Repetitive modular organization apparently similar to other mammals.<br /> (8) The inferior olive described by other authors lacks the lamellated appearance of this structure in other mammals

Let's examine these justifications more closely.

(1) Cytochrome oxidase histochemistry is typically used as an indicative marker of neuronal energy metabolism. The authors indicate, based on the "truly extraordinary" somatosensory capacities of the elephant trunk, that any nuclei processing this tactile information should be highly metabolically active, and thus should react intensely when stained for cytochrome oxidase. We are told in the methods section that the protocols used are described by Purkart et al (2022) and Kaufmann et al (2022). In neither of these cited papers is there any description, nor mention, of the cytochrome oxidase histochemistry methodology, thus we have no idea of how this histochemical staining was done. In order to obtain the best results for cytochrome oxidase histochemistry, the tissue is either processed very rapidly after buffer perfusion to remove blood or in recently perfusion-fixed tissue (e.g., 10.1016/0165-0270(93)90122-8). Given: (1) the presumably long post-mortem interval between death and fixation - "it often takes days to dissect elephants"; (2) subsequent fixation of the brains in 4% paraformaldehyde for "several weeks"; (3) The intense cytochrome oxidase reactivity in the inferior olivary complex of the laboratory rat (Gonzalez-Lima, 1998, Cytochrome oxidase in neuronal metabolism and Alzheimer's diseases); and (4) The lack of any comparative images from other stained portions of the elephant brainstem; it is difficult to support the justification as forwarded by the authors. It is likely that the histochemical staining observed is background reactivity from the use of diaminobenzidine in the staining protocol. Thus, this first justification is unsupported.<br /> Justifications (2), (3), and (4) are sequelae from justification (1). In this sense, they do not count as justifications, but rather unsupported extensions.

(4) and (5) These are interesting justifications, as the paper has clear internal contradictions, and (5) is a sequelae of (4). The reader is led to the concept that the myelin tracts divide the nuclei into sub-modules that match the folding of the skin on the elephant trunk. One would then readily presume that these myelin tracts are in the incoming sensory axons from the trigeminal nerve. However, the authors note that this is not the case: "Our observations on trunk module myelin stripes are at odds with this view of myelin. Specifically, myelin stripes show no tapering (which we would expect if axons divert off into the tissue). More than that, there is no correlation between myelin stripe thickness (which presumably correlates with axon numbers) and trigeminal module neuron numbers. Thus, there are numerous myelinated axons, where we observe few or no trigeminal neurons. These observations are incompatible with the idea that myelin stripes form an axonal 'supply' system or that their prime function is to connect neurons. What do myelin stripe axons do, if they do not connect neurons? We suggest that myelin stripes serve to separate rather than connect neurons." So, we are left with the observation that the myelin stripes do not pass afferent trigeminal sensory information from the "truly extraordinary" trunk skin somatic sensory system, and rather function as units that separate neurons - but to what end? It appears that the myelin stripes are more likely to be efferent axonal bundles leaving the nuclei (to form the olivocerebellar tract). This justification is unsupported.

(6) The authors indicate that the location of these nuclei matches that of the trigeminal nuclei in other mammals. This is not supported in any way. In ALL other mammals in which the trigeminal nuclei of the brainstem have been reported they are found in the lateral aspect of the brainstem, bordered laterally by the spinal trigeminal tract. This is most readily seen and accessible in the Paxinos and Watson rat brain atlases. The authors indicate that the trigeminal nuclei are medial to the facial nerve nucleus, but in every other species the trigeminal sensory nuclei are found lateral to the facial nerve nucleus. This is most salient when examining a close relative, the manatee (10.1002/ar.20573), where the location of the inferior olive and the trigeminal nuclei matches that described by Maseko et al (2013) for the African elephant. This justification is not supported.

(7) The dual to quadruple repetition of rostro-caudal modules within the putative trigeminal nucleus as identified by the authors relies on the fact that in the neurotypical mammal, there are several trigeminal sensory nuclei arranged in a column running from the pons to the cervical spinal cord, these include (nomenclature from Paxinos and Watson in roughly rostral to caudal order) the Pr5VL, Pr5DM, Sp5O, Sp5I, and Sp5C. But, these nuclei are all located far from the midline and lateral to the facial nerve nucleus, unlike what the authors describe in the elephants. These rostrocaudal modules are expanded upon in Figure 2, and it is apparent from what is shown is that the authors are attributing other brainstem nuclei to the putative trigeminal nuclei to confirm their conclusion. For example, what they identify as the inferior olive in figure 2D is likely the lateral reticular nucleus as identified by Maseko et al (2013). This justification is not supported.

(8) In primates and related species, there is a distinct banded appearance of the inferior olive, but what has been termed the inferior olive in the elephant by other authors does not have this appearance, rather, and specifically, the largest nuclear mass in the region (termed the principal nucleus of the inferior olive by Maseko et al, 2013, but Pr5, the principal trigeminal nucleus in the current paper) overshadows the partial banded appearance of the remaining nuclei in the region (but also drawn by the authors of the current paper). Thus, what is at debate here is whether the principal nucleus of the inferior olive can take on a nuclear shape rather than evince a banded appearance. The authors of this paper use this variance as justification that this cluster of nuclei could not possibly be the inferior olive. Such a "semi-nuclear/banded" arrangement of the inferior olive is seen in, for example, giraffe (10.1016/j.jchemneu.2007.05.003), domestic dog, polar bear, and most specifically the manatee (a close relative of the elephant) (brainmuseum.org; 10.1002/ar.20573). This justification is not supported.

Thus, all the justifications forwarded by the authors are unsupported. Based on methodological concerns, prior comparative mammalian neuroanatomy, and prior studies in the elephant and closely related species, the authors fail to support their notion that what was previously termed the inferior olive in the elephant is actually the trigeminal sensory nuclei. Given this failure, the justifications provided above that are sequelae also fail. In this sense, the entire manuscript and all the sequelae are not supported.

What the authors have not done is to trace the pathway of the large trigeminal nerve in the elephant brainstem, as was done by Maseko et al (2013), which clearly shows the internal pathways of this nerve, from the branch that leads to the fifth mesencephalic nucleus adjacent to the periventricular grey matter, through to the spinal trigeminal tract that extends from the pons to the spinal cord in a manner very similar to all other mammals. Nor have they shown how the supposed trigeminal information reaches the putative trigeminal nuclei in the ventromedial rostral medulla oblongata. These are but two examples of many specific lines of evidence that would be required to support their conclusions. Clearly tract tracing methods, such as cholera toxin tracing of peripheral nerves cannot be done in elephants, thus the neuroanatomy must be done properly and with attention to details to support the major changes indicated by the authors.

So what are these "bumps" in the elephant brainstem?

Four previous authors indicate that these bumps are the inferior olivary nuclear complex. Can this be supported?

The inferior olivary nuclear complex acts "as a relay station between the spinal cord (n.b. trigeminal input does reach the spinal cord via the spinal trigeminal tract) and the cerebellum, integrating motor and sensory information to provide feedback and training to cerebellar neurons" (https://www.ncbi.nlm.nih.gov/books/NBK542242/). The inferior olivary nuclear complex is located dorsal and medial to the pyramidal tracts (which were not labelled in the current study by the authors but are clearly present in Fig. 1C and 2A) in the ventromedial aspect of the rostral medulla oblongata. This is precisely where previous authors have identified the inferior olivary nuclear complex and what the current authors assign to their putative trigeminal nuclei. The neurons of the inferior olivary nuclei project, via the olivocerebellar tract to the cerebellum to terminate in the climbing fibres of the cerebellar cortex.

Elephants have the largest (relative and absolute) cerebellum of all mammals (10.1002/ar.22425), this cerebellum contains 257 x109 neurons (10.3389/fnana.2014.00046; three times more than the entire human brain, 10.3389/neuro.09.031.2009). Each of these neurons appears to be more structurally complex than the homologous neurons in other mammals (10.1159/000345565; 10.1007/s00429-010-0288-3). In the African elephant, the neurons of the inferior olivary nuclear complex are described by Maseko et al (2013) as being both calbindin and calretinin immunoreactive. Climbing fibres in the cerebellar cortex of the African elephant are clearly calretinin immunopositive and also are likely to contain calbindin (10.1159/000345565). Given this, would it be surprising that the inferior olivary nuclear complex of the elephant is enlarged enough to create a very distinct bump in exactly the same place where these nuclei are identified in other mammals?

What about the myelin stripes? These are most likely to be the origin of the olivocerebellar tract and probably only have a coincidental relationship to the trunk. Thus, given what we know, the inferior olivary nuclear complex as described in other studies, and the putative trigeminal nuclear complex as described in the current study, is the elephant inferior olivary nuclear complex. It is not what the authors believe it to be, and they do not provide any evidence that discounts the previous studies. The authors are quite simply put, wrong. All the speculations that flow from this major neuroanatomical error are therefore science fiction rather than useful additions to the scientific literature.

What do the authors actually have?<br /> The authors have interesting data, based on their Golgi staining and analysis, of the inferior olivary nuclear complex in the elephant.

* Review of Revised Manuscript *

Assessment:

There is a clear dichotomy between the authors and this reviewer regarding the identification of specific structures, namely the inferior olivary nuclear complex and the trigeminal nuclear complex, in the brainstem of the elephant. The authors maintain the position that in the elephant alone, irrespective of all the published data on other mammals and previously published data on the elephant brainstem, these two nuclear complexes are switched in location. The authors maintain that their interpretation is correct, this reviewer maintains that this interpretation is erroneous. The authors expressed concern that the remainder of the paper was not addressed by the reviewer, but the reviewer maintains that these sequelae to the misidentification of nuclear complexes in the elephant brainstem renders any of these speculations irrelevant as the critical structures are incorrectly identified. It is this reviewer's opinion that this paper is incorrect. I provide a lot of detail below in order to provide support to the opinion I express.

Public Review of Current Submission:

As indicated in my previous review of this manuscript (see above), it is my opinion that the authors have misidentified, and indeed switched, the inferior olivary nuclear complex (IO) and the trigeminal nuclear complex (Vsens). It is this specific point only that I will address in this second review, as this is the crucial aspect of this paper - if the identification of these nuclear complexes in the elephant brainstem by the authors is incorrect, the remainder of the paper does not have any scientific validity.

The authors, in their response to my initial review, claim that I "bend" the comparative evidence against them. They further claim that as all other mammalian species exhibit a "serrated" appearance of the inferior olive, and as the elephant does not exhibit this appearance, that what was previously identified as the inferior olive is actually the trigeminal nucleus and vice versa.

For convenience, I will refer to IOM and VsensM as the identification of these structures according to Maseko et al (2013) and other authors and will use IOR and VsensR to refer to the identification forwarded in the study under review.<br /> The IOM/VsensR certainly does not have a serrated appearance in elephants. Indeed, from the plates supplied by the authors in response (Referee Fig. 2), the cytochrome oxidase image supplied and the image from Maseko et al (2013) shows a very similar appearance. There is no doubt that the authors are identifying structures that closely correspond to those provided by Maseko et al (2013). It is solely a contrast in what these nuclear complexes are called and the functional sequelae of the identification of these complexes (are they related to the trunk sensation or movement controlled by the cerebellum?) that is under debate.

Elephants are part of the Afrotheria, thus the most relevant comparative data to resolve this issue will be the identification of these nuclei in other Afrotherian species. Below I provide images of these nuclear complexes, labelled in the standard nomenclature, across several Afrotherian species.

(A) Lesser hedgehog tenrec (Echinops telfairi)

Tenrecs brains are the most intensively studied of the Afrotherian brains, these extensive neuroanatomical studies undertaken primarily by Heinz Künzle. Below I append images (coronal sections stained with cresol violet) of the IO and Vsens (labelled in the standard mammalian manner) in the lesser hedgehog tenrec. It should be clear that the inferior olive is located in the ventral midline of the rostral medulla oblongata (just like the rat) and that this nucleus is not distinctly serrated. The Vsens is located in the lateral aspect of the medulla skirted laterally by the spinal trigeminal tract (Sp5). These images and the labels indicating structures correlate precisely with that provide by Künzle (1997, 10.1016/S0168- 0102(97)00034-5), see his Figure 1K,L. Thus, in the first case of a related species, there is no serrated appearance of the inferior olive, the location of the inferior olive is confirmed through connectivity with the superior colliculus (a standard connection in mammals) by Künzle (1997), and the location of Vsens is what is considered to be typical for mammals. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

Decision letter image 1.

(B) Giant otter shrew (Potomogale velox)

The otter shrews are close relatives of the Tenrecs. Below I append images of cresyl violet (left column) and myelin (right column) stained coronal sections through the brainstem with the IO, Vsens and Sp5 labelled as per standard mammalian anatomy. Here we see hints of the serration of the IO as defined by the authors, but we also see many myelin stripes across the IO. Vsens is located laterally and skirted by the Sp5. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

Decision letter image 2.

(C) Four-toed sengi (Petrodromus tetradactylus)

The sengis are close relatives of the Tenrecs and otter shrews, these three groups being part of the Afroinsectiphilia, a distinct branch of the Afrotheria. Below I append images of cresyl violet (left column) and myelin (right column) stained coronal sections through the brainstem with the IO, Vsens and Sp5 labelled as per standard mammalian anatomy. Here we see vague hints of the serration of the IO (as defined by the authors), and we also see many myelin stripes across the IO. Vsens is located laterally and skirted by the Sp5. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

Decision letter image 3.

(D) Rock hyrax (Procavia capensis)

The hyraxes, along with the sirens and elephants form the Paenungulata branch of the Afrotheria. Below I append images of cresyl violet (left column) and myelin (right column) stained coronal sections through the brainstem with the IO, Vsens and Sp5 labelled as per the standard mammalian anatomy. Here we see hints of the serration of the IO (as defined by the authors), but we also see evidence of a more "bulbous" appearance of subnuclei of the IO (particularly the principal nucleus), and we also see many myelin stripes across the IO. Vsens is located laterally and skirted by the Sp5. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

Decision letter image 4.

(E) West Indian manatee (Trichechus manatus)

The sirens are the closest extant relatives of the elephants in the Afrotheria. Below I append images of cresyl violet (top) and myelin (bottom) stained coronal sections (taken from the University of Wisconsin-Madison Brain Collection, https://brainmuseum.org, and while quite low in magnification they do reveal the structures under debate) through the brainstem with the IO, Vsens and Sp5 labelled as per standard mammalian anatomy. Here we see the serration of the IO (as defined by the authors). Vsens is located laterally and skirted by the Sp5. This is in agreement with the authors, as they propose that ONLY the elephants show the variations they report.

Decision letter image 5.

These comparisons and the structural identification, with which the authors agree as they only distinguish the elephants from the other Afrotheria, demonstrate that the appearance of the IO can be quite variable across mammalian species, including those with a close phylogenetic affinity to the elephants. Not all mammal species possess a "serrated" appearance of the IO. Thus, it is more than just theoretically possible that the IO of the elephant appears as described prior to this study.

So what about elephants? Below I append a series of images from coronal sections through the African elephant brainstem stained for Nissl, myelin, and immunostained for calretinin. These sections are labelled according to standard mammalian nomenclature. In these complete sections of the elephant brainstem, we do not see a serrated appearance of the IOM (as described previously and in the current study by the authors). Rather the principal nucleus of the IOM appears to be bulbous in nature. In the current study, no image of myelin staining in the IOM/VsensR is provided by the authors. However, in the images I provide, we do see the reported myelin stripes in all stains - agreement between the authors and reviewer on this point. The higher magnification image to the bottom left of the plate shows one of the IOM/VsensR myelin stripes immunostained for calretinin, and within the myelin stripes axons immunopositive for calretinin are seen (labelled with an arrow). The climbing fibres of the elephant cerebellar cortex are similarly calretinin immunopositive (10.1159/000345565). In contrast, although not shown at high magnification, the fibres forming the Sp5 in the elephant (in the Maseko description, unnamed in the description of the authors) show no immunoreactivity to calretinin.

Decision letter image 6.

Peripherin Immunostaining

In their revised manuscript the authors present immunostaining of peripherin in the elephant brainstem. This is an important addition (although it does replace the only staining of myelin provided by the authors which is unusual as the word myelin is in the title of the paper) as peripherin is known to specifically label peripheral nerves. In addition, as pointed out by the authors, peripherin also immunostains climbing fibres (Errante et al., 1998). The understanding of this staining is important in determining the identification of the IO and Vsens in the elephant, although it is not ideal for this task as there is some ambiguity. Errante and colleagues (1998; Fig. 1) show that climbing fibres are peripherin-immunopositive in the rat. But what the authors do not evaluate is the extensive peripherin staining in the rat Sp5 in the same paper (Errante et al, 1998, Fig. 2). The image provided by the authors of their peripherin immunostaining (their new Figure 2) shows what I would call the Sp5 of the elephant to be strongly peripherin immunoreactive, just like the rat shown in Errant et al (1998), and more over in the precise position of the rat Sp5! This makes sense as this is where the axons subserving the "extraordinary" tactile sensitivity of the elephant trunk would be found (in the standard model of mammalian brainstem anatomy). Interestingly, the peripherin immunostaining in the elephant is clearly lamellated...this coincides precisely with the description of the trigeminal sensory nuclei in the elephant by Maskeo et al (2013) as pointed out by the authors in their rebuttal. Errante et al (1998) also point out peripherin immunostaining in the inferior olive, but according to the authors this is only "weakly present" in the elephant IOM/VsensR. This latter point is crucial. Surely if the elephant has an extraordinary sensory innervation from the trunk, with 400 000 axons entering the brain, the VsensR/IOM should be highly peripherin-immunopositive, including the myelinated axon bundles?! In this sense, the authors argue against their own interpretation - either the elephant trunk is not a highly sensitive tactile organ, or the VsensR is not the trigeminal nuclei it is supposed to be.

Summary:

(1) Comparative data of species closely related to elephants (Afrotherians) demonstrates that not all mammals exhibit the "serrated" appearance of the principal nucleus of the inferior olive.

(2) The location of the IO and Vsens as reported in the current study (IOR and VsensR) would require a significant, and unprecedented, rearrangement of the brainstem in the elephants independently. I argue that the underlying molecular and genetic changes required to achieve this would be so extreme that it would lead to lethal phenotypes. Arguing that the "switcheroo" of the IO and Vsens does occur in the elephant (and no other mammals) and thus doesn't lead to lethal phenotypes is a circular argument that cannot be substantiated.

(3) Myelin stripes in the subnuclei of the inferior olivary nuclear complex are seen across all related mammals as shown above. Thus, the observation made in the elephant by the authors in what they call the VsensR, is similar to that seen in the IO of related mammals, especially when the IO takes on a more bulbous appearance. These myelin stripes are the origin of the olivocerebellar pathway, and are indeed calretinin immunopositive in the elephant as I show.

(4) What the authors see aligns perfectly with what has been described previously, the only difference being the names that nuclear complexes are being called. But identifying these nuclei is important, as any functional sequelae, as extensively discussed by the authors, is entirely dependent upon accurately identifying these nuclei.

(4) The peripherin immunostaining scores an own goal - if peripherin is marking peripheral nerves (as the authors and I believe it is), then why is the VsensR/IOM only "weakly positive" for this stain? This either means that the "extraordinary" tactile sensitivity of the elephant trunk is non-existent, or that the authors have misinterpreted this staining. That there is extensive staining in the fibre pathway dorsal and lateral to the IOR (which I call the spinal trigeminal tract), supports the idea that the authors have misinterpreted their peripherin immunostaining.

(5) Evolutionary expediency. The authors argue that what they report is an expedient way in which to modify the organisation of the brainstem in the elephant to accommodate the "extraordinary" tactile sensitivity. I disagree. As pointed out in my first review, the elephant cerebellum is very large and comprised of huge numbers of morphologically complex neurons. The inferior olivary nuclei in all mammals studied in detail to date, give rise to the climbing fibres that terminate on the Purkinje cells of the cerebellar cortex. It is more parsimonious to argue that, in alignment with the expansion of the elephant cerebellum (for motor control of the trunk), the inferior olivary nuclei (specifically the principal nucleus) have had additional neurons added to accommodate this cerebellar expansion. Such an addition of neurons to the principal nucleus of the inferior olive could readily lead to the loss of the serrated appearance of the principal nucleus of the inferior olive, and would require far less modifications in the developmental genetic program that forms these nuclei. This type of quantitative change appears to be the primary way in which structures are altered in the mammalian brainstem.

Reviewer #2 (Public Review):

Summary:

The manuscript by Gitanjali Roy et al. applies deep transfer learning (DEGAS) to assign patient-level disease attributes (metadata) to single cells of T2D and non-diabetic patients, including obese patients. This led to the identification of a singular cluster of T2D-associated β-cells; and two subpopulations of obese- β-cells derived from either non-diabetic or T2D donors. The objective was to identify novel and established genes implicated in T2D and obesity. Their final goal is to validate their findings at the protein level using immunohistochemistry of pancreas tissue from non-diabetic and T2D organ donors.

Strengths:

This paper is well-written, and the findings are relevant for β-cell heterogeneity in T2D and obesity.

Weaknesses:

The validation they provide is not sufficiently strong: no DLK1 immunohistochemistry is shown of obese patient-derived sections. Additional presumptive relevant candidates from this transcriptomic analysis should be screened for, at the protein level.

Reviewer #2 (Public Review):

Summary:

To design proteins and predict disease, we want to predict the effects of mutations on the function of a protein. To make these predictions, biologists have long turned to statistical models that learn patterns that are conserved across evolution. There is potential to improve our predictions however by incorporating structure. In this paper, the authors build a denoising auto-encoder model that incorporates sequence and structure to predict mutation effects. The model is trained to predict the sequence of a protein given its perturbed sequence and structure. The authors demonstrate that this model is able to predict the effects of mutations better than sequence-only models.

As well, the authors curate a set of assays measuring the effect of mutations on thermostability. They demonstrate their model also predicts the effects of these mutations better than previous models and make this benchmark available for the community.

Strengths:

The authors describe a method that makes accurate mutation effect predictions by informing its predictions with structure.

Weaknesses:

It is unclear how this model compares to other methods of incorporating structure into models of biological sequences, most notably SaProt (https://www.biorxiv.org/content/10.1101/2023.10.01.560349v1.full.pdf).

ProteinGym is largely made of deep mutational scans, which measure the effect of every mutation on a protein. These new benchmarks contain on average measurements of less than a percent of all possible point mutations of their respective proteins. It is unclear what sorts of protein regions these mutations are more likely to lie in; therefore it is challenging to make conclusions about what a model has necessarily learned based on its score on this benchmark. For example, several assays in this new benchmark seem to be similar to each other, such as four assays on ubiquitin performed at pH 2.25 to pH 3.0.

Reviewer #2 (Public Review):

Summary:

This work elaborates on a combined therapeutic approach comprising ionizing radiation and CCR5i/αPD1 immunotherapy as a promising strategy in pancreatic cancer. Previous research has established that NK cell-derived CCL5 and XCL1 play a crucial role in recruiting cDC1 cells to the tumor microenvironment, contributing to tumor control. In this study, by using a murine pancreatic cancer model, the authors propose that the addition of radiation therapy to CCR5i and αPD1 immunotherapy could upregulate CD8+ T cells and a subgroup of NK cells within the tumor and result in better tumor control. They further analyzed human single-cell sequencing data from pancreatic cancer patients and identified one subgroup of NK cells (NK C1) with tissue-resident features. Subsequent cell-cell contact analysis reveals the NK-cDC1-CD8 cell axis in pancreatic cancer. By analyzing TCGA data, they found that high NK C1 signature levels were associated with better survival in pancreatic cancer patients. Thus, radiotherapy could benefit the outcome of patients bearing low NK C1 signatures. Importantly, the positive correlation between NK C1 score with survival extends beyond pancreatic cancer, showing potential applicability across various solid cancers.

Strengths:

This study could add new insight into the clinical practice by introducing such novel combined therapy and shed light on the underlying immune cell dynamics. These findings hold potential for more effective and targeted treatment in the future. Mouse experiments nicely confirmed that such combined therapy could significantly reduce tumor volume. The elegant use of single-cell sequencing analysis and human database examination enriches the narrative and strengthens the study's foundation. Additionally, the notion that NK C1 signature correlates with patient survival in various solid cancers is of high interest and relevance.

Weaknesses:

The authors have addressed some of my concerns. However, others remain and should be discussed.

(1) The role of CCR5i requires further clarification/ discussion. While the authors demonstrated its capacity to reduce Treg in murine tumors, its impact on other cell populations, including NK cells and CD8+ T cells, was not observed. Nevertheless, the effect of CCR5i on tumor growth in Figure 2B seems pathogenic. If the combination of radiotherapy and αPD1 already can achieve good outcomes as shown in Figure 3A, the necessity to include CCR5i is questioned. Overall, a more comprehensive elucidation of the roles of CCL5 and CCR5i in this context would be good. Alternatively, this limitation should be discussed.<br /> (2) In line with this, spatial plots in Figure 4 did not include the group with only radiotherapy and αPD1. This inclusion would facilitate a clearer comparison and better highlight the essential role of CCR5i.<br /> (3) Human database analysis showed a positive correlation between NK C1 score and CCL5 in pancreatic cancer. Furthermore, radiotherapy could benefit the outcome of patients bearing low NK C1 scores. It would be interesting to test, if radiotherapy could also benefit patients with low CCL5 levels in this cohort. This is a key question since the role of CCL5/CCR5i is not well verified. Alternatively, this point could be mentioned and discussed.

Reviewer #2 (Public Review):

He and colleagues aimed to elucidate the role of the transcription factor Prdm1 in liver Type 1 ILCs (innate lymphoid cells), focusing on its regulatory mechanisms and potential implications for developing innovative immune therapy strategies against liver cancer​.

Strengths:

The study effectively integrates omics analyses and cytometry to explore Prdm1's impact on the cellular composition and immune regulation within the liver, providing a comprehensive view of its biological role​. Employing a conditional knockout mouse model adds specificity to their experiments, allowing for precise manipulation of the Prdm1 gene​​.

Weaknesses:

The study predominantly relies on limited mouse models, which may not fully represent the complexity of Type 1 ILC behavior across different cancer types. Some experimental designs, such as the limited in vitro killing assessments, and additional human data could be expanded to strengthen the findings and their interpretation​​.

The authors have demonstrated that Prdm1 plays a critical role in the function of NK cells and ILC1s within the liver, particularly in the context of tumor resistance. However, due to the use of specific disease models and lack of direct human data, the application of these findings to clinical settings remains speculative​​. While the study advances our understanding of liver ILC biology, further research is necessary to validate these effects in human systems and across more diverse cancer models​.

​Discussion on impact and utility:

This study contributes significantly to the field of immunology and cancer therapy by revealing potential new targets for immunotherapy of liver cancer. The methods and data provided could serve as a valuable resource for further research aimed at enhancing immune-based cancer treatments​.

​Additional Context for Interpretation:

Understanding the role of Prdm1 in the broader context of immune cell regulation and its interaction with other cellular components in the tumor microenvironment could be crucial. Further studies should explore the dynamic between Prdm1 expression, NK cell functionality, and tumor resistance mechanisms to fully harness the therapeutic potential of targeting this pathway in liver cancer​.

Reviewer #2 (Public Review):

The study by Drougard et al. aimed to answer a critical question on how high-fat diets trigger metabolic issues like obesity and diabetes. Their study revealed that an acute response by microglial cells in the brain to high-fat intake surprisingly benefits metabolism and cognitive function by rapidly metabolizing harmful fatty acids into alternative energy substrates like lactate and itaconate. Thus, short-term HFD intake seems to prompt a distinct beneficial response, suggesting a need for further exploration into the transition from acute to chronic effects.

Reviewer #2 (Public Review):

The authors characterized the recombinase-based cumulative fate maps for vesicular glutamate transporters (Vglut1, Vglut2 and Vglut3) expression and compared those maps to their real-time expression profiles in central NA neurons by RNA in situ hybridization in adult mice. Authors have revealed a new and intriguing expression pattern for Vglut2, along with an entirely uncharted co-expression domain for Vglut3 within central noradrenergic neurons. Interestingly, and in contrast to previous studies, the authors demonstrated that glutamatergic signaling in central noradrenergic neurons does not exert any influence on breathing and metabolic control either under normoxic/normocapnic conditions or after chemoreflex stimulation. Also, they showed for the first-time the Vglut3-expressing NA population in C2/A2 nuclei. In addition, they were also able to demonstrate Vglut2 expression in anterior NA populations, such as LC neurons, by using more refined techniques, unlike previous studies.

A major strength of the study is the use of a set of techniques to investigate the participation of NA-based glutamatergic signaling in breathing and metabolic control. The authors provided a full characterization of the recombinase-based cumulative fate maps for Vglut transporters. They performed real-time mRNA expression of Vglut transporters in central NA neurons of adult mice. Further, they evaluated the effect of knocking down Vglut2 expression in NA neurons using a DBH-Cre; Vglut2cKO mice on breathing and control in unanesthetized mice. Finally, they injected the AAV virus containing Cre-dependent Td tomato into LC of v-Glut2 Cre mice to verify the VGlut2 expression in LC-NA neurons. A very positive aspect of the article is that the authors combined ventilation with metabolic measurements. This integration holds particular significance, especially when delving into the exploration of respiratory chemosensitivity. Furthermore, the sample size of the experiments is excellent.<br /> Despite the clear strengths of the paper, some weaknesses exist. It is not clear in the manuscript if the experiments were performed in males and females and if the data were combined. I believe that the study would have benefited from a more comprehensive analysis exploring the sex specific differences. The reason I think this is particularly relevant is the developmental disorders mentioned by the authors, such as SIDS and Rett syndrome, which could potentially arise from disruptions in central noradrenergic (NA) function, exhibit varying degrees of sex predominance. Moreover, some of the noradrenergic cell groups are sexually dimorphic. For instance, female Wistar rats exhibit a larger LC size and more LC-NA neurons than male subjects (Pinos et al., 2001; Garcia-Falgueras et al., 2005). More recently, a detailed transcriptional profiling investigation has unveiled the identities of over 3,000 genes in the LC. This revelation has highlighted significant sexual dimorphisms, with more than 100 genes exhibiting differential expression within LC-NA neurons at the transcript level. Furthermore, this investigation has convincingly showcased that these distinct gene expression patterns have the capacity to elicit disparate behavioral responses between sexes (Mulvey et al., 2018). Therefore, the authors should compare the fate maps, Vglut transporters in males and females, at least considering LC-NA neurons. Even in the absence of identified sex differences, this information retains significant importance.<br /> An important point well raised by the authors is that although suggestive, these experiments do not definitively rule out that NA-Vglut2 based glutamatergic signaling has a role in breathing control. Subsequent experiments will be necessary to validate this hypothesis.

An improvement could be made in terms of measuring body temperature. Opting for implanted sensors over rectal probes would circumvent the need to open the chamber, thereby preventing alterations in gas composition during respiratory measurements. Further, what happens to body temperature phenotype in these animals under different gas exposures? These data should be included in the Tables.

Is it plausible that another neurotransmitter within NA neurons might be released in higher amounts in DBH-Cre; Vglut2 cKO mice to compensate for the deficiency in glutamate and prevent changes in ventilation?

Continuing along the same line of inquiry is there a possibility that Vglut2 cKO from NA neurons not only eliminates glutamate release but also reduces NA release? A similar mechanism was previously found in VGLUT2 cKO from DA neurons in previous studies (Alsio et al., 2011; Fortin et al., 2012; Hnasko et al., 2010). Additionally, does glutamate play a role in the vesicular loading of NA? Therefore, could the lack of effect on breathing be explained by the lack of noradrenaline and not glutamate?

Reviewer #2 (Public Review):

Summary:

The authors developed DNL343, a CNS-penetrant small molecule integrated stress response (ISR) inhibitor, to treat neurodegenerative diseases caused by ISR.

Strengths:

DNL343 is an investigational CNS-penetrant small molecule integrated stress response (ISR) inhibitor designed to activate the eukaryotic initiation factor 2B (eIF2B) and suppress aberrant ISR activation. The therapeutic efficacy of DNL343 has been extensively characterized in two animal models. Importantly, plasma biomarkers of neuroinflammation and neurodegeneration can be reversed with DNL343 treatment. Remarkably, several of these biomarkers show differential levels in CSF and plasma from patients with vanishing white matter disease (VWMD) upon DNL343 treatment. Overall, this study is very exciting that targets ISR for therapeutic interventions.

Weaknesses:

My main questions center around the characterization of DNL343.

(1) Is there any biochemical evidence showing DNL343 activates eIF2B, such as binding and in vitro biochemical activity assays? A conference presentation was cited. "Osipov, M. (2022). Discovery of DNL343: a Potent Selective and Brain-penetrant eIF2B Activator Designed for the Treatment of Neurodegenerative Diseases. Medicinal Chemistry Gordon Research Conference. New London, NH." However, there is no public information about this presentation.<br /> (2) How was the selectivity of DNL343 demonstrated? What are the off-targets of DNL343, particularly when DNL343 is administered at a high dose? Thermal-proteasome profiling or photoaffinity labeling experiments could be considered.<br /> (3) What are the total drug concentrations in the brain and plasma? What are the unbound ratios?<br /> (4) If DNL343 is given intravenously, what are the concentrations in the brain and plasma after 5 minutes and 1 h or longer time points? In other words, does DNL343 cross BBB through passive diffusion or an active process?<br /> (5) What is the full PK profile of DNL343 for intravenous and oral dosing?<br /> (6) Are there any major drug metabolites that could be concerns?

Review for Revision:

The companion JMC paper, doi.org/10.1021/acs.jmedchem.3c02422, addressed most of my questions. However, I was unable to find the total concentrations of DNL343 in the brain and plasma or the raw data for the full PK in the JMC paper. Otherwise, the JMC publication addressed all my questions.

Reviewer #2 (Public Review):

Summary:

Gil & Lim et al. applied mouse genetic models to study the roles of chromatin regulator KDM6B in regulating the development of the hippocampal dentate gyrus (DG), as well as the establishment and maintenance of DG NSCs. KDM6B is expressed in postnatal DGs. Importantly, conditional knockout of Kdm2b in embryonic DG progenitors leads to a significantly smaller DG with loss of DG NSCs. Hippocampal-dependent behaviors are defective in Kdm6b-cKO mice. Deletion of Kdm6b results in precocious neuronal differentiation and loss of the NSC population in both postnatal and adult DGs. Single-cell RNA-seq reveals disrupted stem cell maintenance gene signature in Kdm6b-deleted NSCs. Moreover, CUT&RUN studies showed that Kdm6b deletion increases H3K27me3 levels at a few NSC maintenance genes.

Strengths:

The conclusions of this paper are mostly well supported by data. The discussion is thorough.

Weaknesses:

I concur with the two reviewing editors who noted that the paper lacks insights into how KDM6B regulates the expression of NSC genes in DG precursors. Additionally, the authors did not provide evidence regarding whether the function of KDM6B is enzymatically dependent.

The Kdm6b-cKO brain exhibited apparently smaller DGs, indicating compromised neurogenesis. While the authors observed an increased number of IPCs in the E17.5 DGs (Figure 4B-4C) and an increased number of BrdU+TBR2+PROX1+ cells in the P0.5 DGs (Figure 5B-5C), it is perplexing why this does not lead to an increased number of PROX1+ DG neurons? Further investigation into the cellular mechanisms underlying these events would enhance the understanding of Kdm6b's role in neurogenesis.

Many data were not of sufficient quality and should be improved.

Reviewer #2 (Public Review):

Summary:

Bloch et al. investigate the relationships between aerial foragers (little swifts) tracked with an automated radio-telemetry system (Atlas) and their prey (flying insects) monitored with a small-scale vertical-looking radar device (BirdScan MR1). The aim of the study was to test whether little swifts optimise their foraging with the abundance of their prey. However, the results provided little evidence of optimal foraging behaviour.

Strengths:

This study addresses fundamental knowledge gaps on the prey-predator dynamics in the airspace. It describes the coincidence between the abundance of flying insects and features derived from tracking individual swifts.

Weaknesses:

The article uses hypotheses broadly derived from optimal foraging theory, but mixes the form of natural selection: parental energetics, parental survival (predation risks), nestling foraging, and breeding success. Results are partly incoherent (e.g., "Thus, even when the birds foraged close to the colony under optimal conditions, the shorter traveling distance is not thought to not confer lower flight-related energetic expenditure because more return trips were made.", L285-287), and confounding factors (e.g., brooding vs. nestling phase) are ignored. Some limits are clearly recognised by the authors (L329 and ff). To illustrate potential confounding effects, the daily flight duration (Prediction 4) should decrease with prey abundance, but how far does the daily flight duration coincide with departure and arrival at sunrise and sunset (note that day length increases between March and May), respectively, and how much do parents vary in the duration of nest attendance during the day across chick ages? To conclude, insufficient analyses are performed to rigorously assess whether little swifts optimize their foraging.

Filters applied on tracking data are necessary but may strongly influence derived features based on maximum or mean values. Providing sensitivity tests or using features less dependent on extreme values may provide more robust results.

Radar insect monitoring is incomplete and strongly size-dependent. What is the favourite prey size of swifts? How does it match with BirdScan MR1 monitoring capability?

Reviewer #2 (Public Review):

Summary:

Forconi et al explore the heterogeneity of circulating Tfh cell responses in children and adults from malaria-endemic Kenya, and further compare such differences following stimulation with two malaria antigens. In particular, the authors also raised an important consideration for the study of Tfh cells in general, which is the hidden diversity that may exist within the current 'standard' gating strategies for these cells. The utility of multiparametric flow cytometry as well as unbiased clustering analysis provides a potentially potent methodology for exploring this hidden depth. However, the current state of analysis presented does not aid the understanding of this heterogeneity. This main goal of the study could hopefully be achieved by putting all the parameters used in one context, before dissecting such differences into their specific clinical contexts.

Strengths:

Understanding the full heterogeneity of Tfh cells in the context of infection is an important topic of interest to the community. The study included clinical groupings such as age group differences and differences in response to different malaria antigens to further highlight context-dependent heterogeneity, which offers new knowledge to the field. However, improvements in data analyses and presentation strategies should be made in order to fully utilize the potential of this study.

Weaknesses:

In general, most studies using multiparameter analysis coupled with an unbiased grouping/clustering approach aim to describe differences between all the parameters used for defining groupings, prior to exploring differences between these groupings in specific contexts. However, the authors have opted to separate these into sections using "subset chemokine markers", "surface activation markers" and then "cytokine responses", yet nuances within all three of these major groups were taken into account when defining the various Tfh identities. Thus, it would make sense to show how all of these parameters are associated with one another within one specific context to first logically establish to the readers how can we better define Tfh heterogeneity. When presented this way, some of the identities such as those that are less clear such as "MC03/MC04/ MC05/ MC08" may even be better revealed. once established, all of these clusters can then be subsequently explored in further detail to understand cluster-specific differences in children vs adults, and in the various stimulation conditions. Since the authors also showed that many of the activation markers were not significantly altered post-stimulation thus there is no real obstacle for merging the entire dataset for the first part of this study which is to define Tfh heterogeneity in an unbiased manner regardless of age groups or stimulation conditions. Other studies using similar approaches such as Mathew et al 2020 (doi: 10.1126/science.abc8) or Orecchioni et al 2017 (doi: 10.1038/s41467-017-01015-3) can be referred to for more effective data presentation strategies.

Accordingly, the expression of cytokines and transcription factors can only be reliably detected following stimulation. However, the underlying background responses need to be taken into account for understanding "true" positive signals. The only raw data for this was shown in the form of of heatmap where no proper ordering was given to ensure that readers can easily interpret the expression of these markers following stimulation relative to no stimulation. Thus, it is difficult to reliably interpret any real differences reported without this. Finally, the authors report differences in either cluster abundance or cluster-specific cytokine/ transcription factor expression in Tfh cell subsets when comparing children vs adults, and between the two malaria antigens. The comparisons of cytokine/transcription factor between groups will be more clearly highlighted by appropriately combining groupings rather than keeping them separate as in Figures 6 and 7.

Reviewer #2 (Public Review):

Summary:

This study investigated the role of the Caspar (Casp) gene, a Drosophila homolog of human Fas-associated factor-1. It revealed that maternal loss of Casp led to centrosomal and cytoskeletal abnormalities during nuclear cycles in Drosophila early embryogenesis, resulting in defective gastrulation. Moreover, Casp regulates PGC numbers, likely by regulating the levels of Smaug and then Oskar. They demonstrate that Casp protein levels are linearly correlated to the PGC number. The partner protein TER94, an ER protein, shows similar but slightly distinct phenotypes. Based on the deletion mutant analysis, TER94 seems functionally relevant for the observed Casp phenotype. Additionally, it is likely involved in regulating protein degradation during PGC specification.

Strengths:

The paper reveals an unexpected function of the maternally produced Casp gene, previously implicated in immune response regulation and NF-kB signaling inhibition, in nuclear division and PGC formation in early fly embryos. Experiments are properly conducted and strongly support the conclusion. The rescue experiment using deletion mutant form is particularly informative as it suggests the requirement of each domain function.

Weaknesses:

Functional relationships among molecules shown here (and other genes known to regulate these processes) are still unclear.

Reviewer #2 (Public Review):

Summary:

Nagarajan et al. investigate the role of the anterior cingulate cortex (ACC) in vocal development of infant marmoset monkeys using lesions in this brain area. Many previous studies show that ACC plays an important role in volitional and emotion-driven vocal behavior in mammals. The experiments Nagarajan et al. performed strengthen the long-standing hypothesis that ACC influences the development of social-vocal behavior in non-human primates. Furthermore, their anatomical studies support the idea of cortical structures exerting cognitive control over subcortical networks for innate vocalization, and thus, enabling mammals to perform flexible social-vocal communication.

Strengths:

Many invasive behavioral studies in monkeys often times use 2-3 animals. The authors used a sufficiently high number of animals for their experiments. This increases the power of their conclusions.<br /> The study also investigates the impact of ACC lesions on downstream areas important for innate vocal production. This adds further evidence to the role of ACC in influencing these subcortical regions during vocal development and vocal behavior in general.

Weaknesses:

The authors state that the integrity of white matter tracts at the injection site was impacted but do not show data.

The study only provides data up to the 6th week after birth. Given the plasticity of the cortex, it would be interesting to see if these impairments in vocal behavior persist throughout adulthood or if the lesioned marmosets will recover their social-vocal behavior compared to the control animals.

Even though this study focuses entirely on the development of social vocalizations, providing data about altered social non-vocal behaviors that accompany ACC lesions is missing. This data can provide further insights and generate new hypotheses about the exact role of ACC in social-vocal development. For example, do these marmosets behave differently towards their conspecifics or family members and vice versa, and is this an alternate cause for the observed changes in social-vocal development?

Reviewer #2 (Public Review):

Summary

This manuscript by Petty and Bruno delves into the still poorly understood role of higher-order thalamic nuclei in the encoding of sensory information by examining the activity in the Pom and LP cells in mice performing an associative learning task. They developed an elegant paradigm in which they conditioned head-fixed mice to attend to a stimulus of one sensory modality (visual or tactile) and ignore a second stimulus of the other modality. They recorded simultaneously from POm and LP, using 64-channel electrode arrays, to reveal the context-dependency of the firing activity of cells in higher-order thalamic nuclei. They concluded that behavioral training reshapes activity in these secondary thalamic nuclei. I have no major concerns with the manuscript's conclusions, but some important methodological details are lacking and I feel the manuscript could be improved with the following revisions.

Strengths

The authors developed an original and elegant paradigm in which they conditioned head-fixed mice to attend to a stimulus of one sensory modality, either visual or tactile, and ignore a second stimulus of the other modality. As a tactile stimulus, they applied gentle air puffs on the distal part of the vibrissae, ensuring that the stimulus was innocuous and therefore none aversive which is crucial in their study.

It is commonly viewed that the first-order thalamus performs filtering and re-encoding of the sensory flow; in contrast, the computations taking place in high-order nuclei are poorly understood. They may contribute to cognitive functions. By integrating top-down control, high-order nuclei may participate in generating updated models of the environment based on sensory activity; how this can take place is a key question that Petty and Bruno addressed in the present study.

Weaknesses

(1) Overall, methods, results, and discussion, involving sensory responses, especially for the Pom, are confusing. I have the feeling that throughout the manuscript, the authors are dealing with the sensory and non-sensory aspects of the modulation of the firing activity in the Pom and LP, without a clear definition of what they examined. Making subsections in the results, or a better naming of what is analyzed could convey the authors' message in a clearer way, e.g., baseline, stim-on, reward.

In line #502 in Methods, the authors defined "Sensory Responses. We examined each cell's putative sensory response by comparing its firing rate during a "stimulus period" to its baseline firing rate. We first excluded overlapping stimuli, defined as any stimulus occurring within 6 seconds of a stimulus of a different type. We then counted the number of spikes that occurred within 1 second prior to the onset of each stimulus (baseline period) and within one second of the stimulus onset (stimulus period). The period within +/-50ms of the stimulus was considered ambiguous and excluded from analysis."

Considering that the responses to whisker deflection, while weak and delayed, were shown to occur, when present, before 50 ms in the Pom (Diamond et al., 1992), it is not clear what the authors mean and consider as "Sensory Responses"?

Precise wording may help to clarify the message. For instance, line #134: "Of cells from tactilely conditioned mice, 175 (50.4%) significantly responded to the air puff, as defined by having a firing rate significantly different from baseline within one second from air puff onset (Figure 3d, bottom)", could be written "significantly responded to the air puff" should be written "significantly increased (or modified if some decreased) their firing rate within one second after the air puff onset (baseline: ...)". This will avoid any confusion with the sensory responses per se.

(2) To extend the previous concern, the latency of the modulation of the firing rate of the Pom cells for each modality and each conditioning may be an issue. This latency, given in Figure S2, is rather long, i.e. particularly late latencies for the whisker system, which is completely in favor of non-sensory "responses" per se and the authors' hypothesis that sensory-, arousal-, and movement-evoked activity in Pom are shaped by associative learning. Latency is a key point in this study.

Therefore,<br /> - latencies should be given in the main text, and Figure S2 could be considered for a main figure, at least panels c, d, and e, could be part of Figure 3.

- the Figure S2b points out rather short latency responses to the air puff, at least in some cells, in addition to late ones. The manuscript would highly benefit from an analysis of both early and late latency components of the "responses" to air puffs and drafting grating in both conditions. This analysis may definitely help to clarify the authors' message. Since the authors performed unit recordings, these data are accessible.

- it would be highly instructive to examine the latency of the modulation of Pom cells firing rate in parallel with the onset of each behavior, i.e. modification of pupil radius, whisking amplitude, lick rate (Figures 1e, g and 3a, b). The Figure 1 does not provide the latency of the licks in conditioned mice.

- the authors mention in the discussion low-latency responses, e.g., line #299: "In both tactilely and visually conditioned mice, movement could not explain the increased firing rate at air puff onset. These low-latency responses across conditioning groups is likely due in part to "true" sensory responses driven by S1 and SpVi."; line #306: "Like POm, LP displayed varied stimulus-evoked activity that was heavily dependent on conditioning. LP responded to the air puff robustly and with low latency, despite lacking direct somatosensory inputs."<br /> But which low-latency responses do the authors refer to? Again, this points out that a robust analysis of these latencies is missing in the manuscript but would be helpful to conclude.

(3) Anatomical locations of recordings in the dorsal part of the thalamus. Line #122 "Our recordings covered most of the volume of POm but were clustered primarily in the anterior and medial portions of LP (Figure 2d-f). Cells that were within 50 µm of a region border were excluded from analysis."<br /> How did the authors distinguish the anterior boundary of the LP with the LD nucleus just more anterior to the LP, another higher-order nucleus, where whisker-responsive cells have been isolated (Bezdudnaya and Keller, 2008)?

(4) The mention in the Methods about the approval by an ethics committee is missing.<br /> All the surgery (line #381), i.e., for the implant, the craniotomy, as well as the perfusion, are performed under isoflurane. But isoflurane induces narcosis only and not proper anesthesia. The mention of the use of analgesia is missing.

Reviewer #2 (Public Review):

Summary:<br /> The core findings demonstrate that the neuropeptide-like protein FLP-2, released from the intestine of C. elegans, is essential for activating the intestinal oxidative stress response. This process is mediated by endogenous hydrogen peroxide (H2O2), which is produced in the mitochondrial matrix by superoxide dismutases SOD-1 and SOD-3. H2O2 facilitates FLP-2 secretion through the activation of protein kinase C family member pkc-2 and the SNAP25 family member aex-4. The study further elucidates that FLP-2 signaling potentiates the release of the antioxidant FLP-1 neuropeptide from neurons, highlighting a bidirectional signaling mechanism between the intestine and the nervous system.

Strengths:

This study presents a significant contribution to the understanding of the gut-brain axis and its role in oxidative stress response and significantly advances our understanding of the intricate mechanisms underlying the gut-brain axis's role in oxidative stress response. By elucidating the role of FLP-2 and its regulation by H2O2, the study provides insights into the molecular basis of inter-tissue communication and antioxidant defense in C. elegans. These findings could have broader implications for understanding similar pathways in more complex organisms, potentially offering new targets for therapeutic intervention in diseases related to oxidative stress and aging.

Weaknesses:

（1）The experimental techniques employed in the study were somewhat simple and could benefit from the incorporation of more advanced methodologies.

（2）The weak identification of the key receptors mediating the interaction between FLP-2 and AIY neurons, as well as the receptors in the gut that respond to FLP-1.

（3）The study could be improved by incorporating a sensor for the direct measurement of hydrogen peroxide levels.

Reviewer #2 (Public Review):

Summary:

Franziska Auer et al. investigate the role of cerebellar Purkinje cells in controlling posture in larval zebrafish using the chemogenetic tool TRPV1/capsaicin to bidirectionally manipulate (i.e., activate or ablate) these cells. This tool has been developed for zebrafish previously but has not been applied to Purkinje cells.

High-throughput behavioral experiments are presented to monitor how body posture is affected by these perturbations. The analysis of postural control focuses on a specific subaspect of posture: the body tilt-angle relative to horizontal just before a swim bout is executed, quantified separately for pre-ascent and pre-dive bouts. They report a broad bimodal distribution of pre-ascent bout posture ranging from -20 to +40 degrees, while the pre-dive bout posture was more Gaussian, ranging between -40 and 0 degrees. The treatment effect is quantified as the change in the median of these distributions.

Purkinje cell activation and ablation in 7 days post-fertilization (dpf) fish shifted the median of the ascending bout posture distributions to positive values. The authors hypothesize that the stochastic nature of the activation process might desynchronize Purkinje cell activity, thus abolishing Purkinje cells' role in postural control, similar to ablation. However, this does not explain why dive bout posture decreased upon activation but was unaffected by ablation.

To test whether the role of Purkinje cells in postural control matures over development, the authors repeated the ablation experiments at 14 dpf. They state that "at 14 dpf, the effects of Purkinje cell lesions on posture were more widespread than at 7 dpf." However, this effect size is comparable to that observed at 7 dpf, suggesting no further maturation of the role of Purkinje cells in pre-ascending bout postural control. The median pre-dive bout posture decreased at 14 dpf, contrasting with no effect at 7 dpf, yet this change was comparable in effect size to the activation effect on Purkinje cells at 7 dpf. The current data breadth may not be sufficient to conclude that signatures of emerging cerebellar control of posture across early development were uncovered.

The study's exploration of activating Purkinje cells in freely swimming fish using TRPV1/capsaicin is of special interest, but the practicability of this method is unclear from the current presentation. It would be beneficial to present the distribution of the percentage of activatable Purkinje cells across animals and time points to provide insight into the method's efficiency. Discussing this limitation and potential improvements would aid in evaluating the method, especially since the authors report that the activation experiments were labor-intensive, limiting repeat experiments. This may explain why the activation experiment at 7 dpf is the only data presented with cell activation, with other analyses performed using the cell ablation capabilities of the TRPV1/capsaicin method. Another data point at 14dpf would significantly strengthen the conclusions.

The authors analyze Purkinje cell-controlled fin-trunk coordination by examining ascending bout posture across different swim bout speeds. They make the important finding that pectoral fin movements contribute significant lift for median and fast swim bouts but not for slow ones, and that Purkinje cell ablation disrupts lift generation at all speeds.

Finally, the authors examined whether Purkinje cell activity encodes postural tilt-angle by performing calcium imaging on 31 cells from 8 fish using their Tilt In Place Microscope (TIPM). They report that they could decode the tilt-angle from individual neurons with a highly tuned response, and also from neurons that were not obviously tuned when pooling them and analyzing the population response. However, due to the non-simultaneous recordings across animals, definitive conclusions about population-level encoding should be made cautiously, it might be better to suggest potential population encoding that needs confirmation with more targeted experiments involving simultaneous recordings.

Strengths:

- The study introduces a novel application of the chemogenetic tool TRPV1/capsaicin to study cerebellar function in zebrafish.

- High-throughput behavioral experiments provide detailed analysis of postural control.

- The further investigation of Purkinje cell-controlled fin-trunk coordination offers new insights into motor control mechanisms.

- The use of calcium imaging to decode postural tilt-angle from Purkinje cell activity presents interesting preliminary results on neuronal population encoding.

Weaknesses:

- The term "disruption" for postural control effects may lead to misleading expectations.

- The supporting data show only subtle median shifts in postural angle, raising questions about the significance of observed effects. Statistical methods that account for the hierarchical structure of the data might be required to support the conclusions.

- The study's data breadth may not be sufficient to conclude emerging cerebellar postural control across early development.

- The current presentation does not adequately detail the practicability and efficiency of the TRPV1/capsaicin method for activating Purkinje cells, and the labor-intensive nature of these experiments constrains the ability to replicate and validate the findings.

- Non-simultaneous recordings in calcium imaging necessitate cautious interpretation of population-level encoding results.

Reviewer #2 (Public Review):

Summary:

Li et al.'s goal is to understand the mechanisms of audiovisual temporal recalibration. This is an interesting challenge that the brain readily solves in order to compensate for real-world latency differences in the time of arrival of audio/visual signals. To do this they perform a 3-phase recalibration experiment on 9 observers that involves a temporal order judgment (TOJ) pretest and posttest (in which observers are required to judge whether an auditory and visual stimulus were coincident, auditory leading or visual leading) and a conditioning phase in which participants are exposed to a sequence of AV stimuli with a particular temporal disparity. Participants are required to monitor both streams of information for infrequent oddballs, before being tested again in the TOJ, although this time there are 3 conditioning trials for every 1 TOJ trial. Like many previous studies, they demonstrate that conditioning stimuli shift the point of subjective simultaneity (pss) in the direction of the exposure sequence.

These shifts are modest - maxing out at around -50 ms for auditory leading sequences and slightly less than that for visual leading sequences. Similar effects are observed even for the longest offsets where it seems unlikely listeners would perceive the stimuli as synchronous (and therefore under a causal inference model you might intuitively expect no recalibration, and indeed simulations in Figure 5 seem to predict exactly that which isn't what most of their human observers did). Overall I think their data contribute evidence that a causal inference step is likely included within the process of recalibration.

Strengths:

The manuscript performs comprehensive testing over 9 days and 100s of trials and accompanies this with mathematical models to explain the data. The paper is reasonably clearly written and the data appear to support the conclusions.

Weaknesses:

While I believe the data contribute evidence that a causal inference step is likely included within the process of recalibration, this to my mind is not a mechanism but might be seen more as a logical checkpoint to determine whether whatever underlying neuronal mechanism actually instantiates the recalibration should be triggered.

The authors' causal inference model strongly predicts that there should be no recalibration for stimuli at 0.7 ms offset, yet only 3/9 participants appear to show this effect. They note that a significant difference in their design and that of others is the inclusion of longer lags, which are unlikely to originate from the same source, but don't offer any explanation for this key difference between their data and the predictions of a causal inference model.

I'm also not completely convinced that the causal inference model isn't 'best' simply because it has sufficient free parameters to capture the noise in the data. The tested models do not (I think) have equivalent complexity - the causal inference model fits best, but has more parameters with which to fit the data. Moreover, while it fits 'best', is it a good model? Figure S6 is useful in this regard but is not completely clear - are the red dots the actual data or the causal inference prediction? This suggests that it does fit the data very well, but is this based on predicting held-out data, or is it just that by having more parameters it can better capture the noise? Similarly, S7 is a potentially useful figure but it's not clear what is data and what are model predictions (what are the differences between each row for each participant; are they two different models or pre-test post-test or data and model prediction?!).

I'm not an expert on the implementation of such models but my reading of the supplemental methods is that the model is fit using all the data rather than fit and tested on held-out data. This seems problematic.

I would have liked to have seen more individual participant data (which is currently in the supplemental materials, albeit in a not very clear manner as discussed above).

The way that S3 is described in the text (line 141) makes it sound like everyone was in the same direction, however, it is clear that 2 /9 listeners show the opposite pattern, and 2 have confidence intervals close to zero (albeit on the -ve side).

Reviewer #2 (Public Review):

Naud et al investigate whether single spikes and bursts encode different information in behavior. To do this, they reanalyze juxtasomal recordings of deep-layer cortical neurons from behaving rats collected in two previous studies by Doron et al. Rats were trained (in a Go-NoGo design) to lick a spout for a water reward in response to electrical microstimulation of the primary somatosensory cortex, which rats quickly learn to do in a single day. Juxtasomal recordings near the site of micro stimuli are then divided up into single spikes ("events") versus high-frequency bursts ("bursts"). Training results in the appearance of bursts, which do not seem to correlate with the rate of events, suggesting that bursts and events carry different information. While the fraction of bursts is elevated during Hit trials, errors appear to uniquely trigger additional bursts. The distribution of burst times appears to shift from long after the stimulus (early in training) to shortly after the stimulus (later in training). Bursts of layer 5 pyramidal neurons in particular are associated with apical tuft activity that could enhance plasticity. The observed increased bursting is therefore suggestive of a potential mechanism by which errors engage plasticity.

This paper has substantial strengths: the experiments appear to be well performed, the dataset is substantial, and the questions and phenomena are interesting.

The exclusion of fast-spike (inhibitory) data, which the experiments seem to have generated, is a weakness as these data could have provided an important control. If the bursts here reflect apical dendrite activity, the same phenomena might be absent in inhibitory cells as they lack apical tufts.

Another weakness is the need to better control movement, which could be an alternative explanation to the top-down modulation of apicals that the authors suspect. For example, the bursts on error trials could be due to the animals moving more when an error occurs. Layer 5 of the somatosensory cortex has increased activity during whisking or body movements. If the mouse fidgets out of frustration that the reward has not occurred or whisks more, bursts are highly likely due to less exotic purely bottom-up inputs.

Reviewer #2 (Public Review):

Summary:

The authors address three primary questions:

(1) how FGF13 variants confer seizure susceptibility,<br /> (2) the specific cell types involved, and<br /> (3) the underlying mechanisms, particularly regarding Nav dysfunction.

They use different Cre drivers to generate cell type-specific knockouts (KOs). First, using Nestin-Cre to create a whole-brain Fgf13 KO, they observed spontaneous seizures and premature death. While KO of Fgf13 in excitatory neurons does not lead to spontaneous seizures, KO in inhibitory neurons recapitulates the seizures and premature death observed in the Nestin-Cre KO. They further narrow down the critical cell type to MGE-derived interneurons (INs), demonstrating that MGE-neuron-specific KO partially reproduces the observed phenotypes. "All interneuron" KOs exhibit deficits in synaptic transmission and interneuron excitability, not seen in excitatory neuron-specific KOs. Finally, they rescue the defects in the interneuron-specific KO by expressing specific Fgf13 isoforms. This is an elegant and important study adding to our knowledge of mechanisms that contribute to seizures.

Strengths

• The study provides much-needed cell type-specific KO models.<br /> • The authors use appropriate Cre lines and characterize the phenotypes of the different KOs.<br /> • The metabolomic analysis complements the rest of the data effectively.<br /> • The study confirms and extends previous research using improved approaches (KO lines vs. in vitro KD or antibody infusion).<br /> • The methods and analyses are robust and well-executed.

Weaknesses

• One weakness lies in the use of the Nkx2.1 line (instead of Nkx2.1CreER) in the paper. As a result, some answers to key questions are incomplete. For instance, it remains unclear whether the observed effects are due to Chandelier cells or NGFCs, potentially both MGE and CGE derived, explaining why Nkx2.1 alone does not fully replicate the overall inhibitory KO. Using Nkx2.1CreER could have helped address the cell specificity. With the Nkx2.1 line used in the paper, the answer is partial.

• While the mechanism behind the reduced inhibitory drive in the IN-specific KO is suggested to be presynaptic, the chosen method does not allow them to exactly identify the mechanisms (spontaneous vs mEPSC/mIPSC), and whether it is a loss of inhibitory synapses (potentially axo-axonic) or release probability.

• Some supporting data (e.g. Supplemental Figure 7 and 8) appear to come from only one (or two) WT and one (or two) KO mice. Supplementary data, like main data, should come from at least three mice in total to be considered complete/solid (even if the statistical analysis is done with cells).

General Assessment

The general conclusions of this paper are supported by data. As it is, the claim that "these results enhance our understanding of the molecular mechanisms that drive the pathogenesis of Fgf13-related seizures" is partially supported. A more cautious term may be more appropriate, as the study shows the mechanism is not Nav-mediated and suggests alternative mechanisms without unambiguously identifying them. The conclusion that the findings "expand our understanding of FGF13 functions in different neuron subsets" is supported, although somewhat overstated, as the work is not conclusive about the exact neuron subtypes. However, it does indeed show differential functions for specific neuronal classes, which is a significant result.

Impact and Utility

This paper is undoubtedly valuable. Understanding that excitatory neurons are not the primary contributors to the observed phenotypes is crucial. The finding that the effects are not MGE-unique is also important. This work provides a solid foundation for further research and will be a useful resource for future studies.

Reviewer #2 (Public Review):

Summary:

The authors aimed to develop and validate a machine-learning-driven neural network capable of automatic scoring of the Rey-Osterrieth Complex Figure. They aimed to further assess the robustness of the model to various parameters such as tilt and perspective shift in real drawings. The authors leveraged the use of a huge sample of lay workers in scoring figures and also a large sample of trained clinicians to score a subsample of figures. Overall, the authors found their model to have exceptional accuracy and perform similarly to crowdsourced workers and clinicians with, in some cases, less degree of error/score dispersion than clinicians.

Strengths:

The authors used very large data; including a large number of Rey-Osterrieth Complex Figures, a huge crowdsourced human worker sample, and a large clinician sample.

The authors deeply describe their model in relatively accessible terms.

The writing style of the paper is accessible, scientific, and thorough.

Pre-registration of the prospectively collected new data was acceptable.

Weaknesses:

There is no detail on how the final scoring app can be accessed and whether it is medical device-regulated.

No discussion on the difference in sample sizes between the pre-registration of the prospective study and the results (e.g., aimed for 500 neurological patients but reported data from 288).

Details in pre-registration and paper regarding samples obtained in the prospective study were lacking.

Demographics for the assessment of the representation of healthy and non-healthy participants were not present.

The authors achieved their aims and their results and conclusions are supported by strong methods and analyses. The resulting app produced in this work, if suitable for clinical practice, will have impact in automated scoring, which many clinicians will be exceptionally happy with.

Reviewer #2 (Public Review):

Summary:

Both human and non-human animals modulate the frequency of their vocalizations to communicate important information about context and internal state. While regulation of the size of the laryngeal opening is a well-established mechanism to regulate vocal pitch, the contribution of expiratory airflow to vocal pitch is less clear. To consider this question, this study first characterizes the relationship between the dominant frequency contours of adult mouse ultrasonic vocalizations (USVs) and expiratory airflow using whole-body plethysmography. The authors also include data from a single mouse that combines EMG recordings from the diaphragm and larynx with plethysmography to provide evidence that the respiratory central pattern generator can be re-engaged to drive "mini-breaths" that occur during the expiratory phase of a vocal breath. Next, the authors build off of their previous work characterizing intermediate reticular oscillator (iRO) neurons in mouse pups to establish the existence of a genetically similar population of neurons in adults and show that artificial activation of iRO neurons elicits USV production in adults. Third, the authors examine the acoustic features of USV elicited by optogenetic activation of iRO and find that a majority of natural USV types (as defined by pitch contour) are elicited by iRO activation and that these artificially elicited USVs are more likely than natural USVs to be marked by positive intonation (positive relationship between USV dominant frequency and expiratory airflow).

Strengths:

Strengths of the study include the novel consideration of expiratory airflow as a mechanism to regulate vocal pitch and the use of intersectional methods to identify and activate the iRO in adult mice. The establishment of iRO neurons as a brainstem population that regulates vocal production across development is an important finding.

Weaknesses:

The conclusion that the respiratory CPG is re-engaged during "mini-breaths" throughout a given vocal breath would be strengthened by including analyses from more than one mouse.

Reviewer #2 (Public Review):

This work by Bray et al. presented a customized way to induce small electrolytic lesions in the brain using chronically implanted intracortical multielectrode arrays. This type of lesioning technique has the benefit of high spatial precision and low surgical complexity while allowing simultaneous electrophysiology recording before, during, and after the lesion induction. The authors have validated this lesioning method with a Utah array, both ex vivo and in vivo using pig models and awake-behaving rhesus macaques. Given its precision in controlling the lesion size, location, and compatibility with multiple animal models and cortical areas, the authors believe this method can be used to study cortical circuits in the presence of targeted neuronal inactivation or injury and to establish causal relationships before behavior and cortical activity.

Strengths:

- Overall the techniques, parameters, and data analysis methods are better described in the revised version.

- The authors added the section "Relationship Between Applied Current and Lesion Volume" as well as Figure 4 and 5 to address our comments regarding parameter testing. Multiple combinations of current amplitude and duration were tested and the induced lesion volumes were estimated, providing a better picture of why certain parameters were chosen for in vivo studies.

- The authors added Figure 7 which addressed our comment "more evidence is needed to suggest robust neuronal inactivation or termination in rhesus macaques after electrolytic lesioning." They went into more details to explain the observed changes in pairwise comparisons of spike waveforms (difference in projected radii). Particularly in Fig 7C, they identified a new cluster from the pre-post lesioning group, which effectively represented neuronal loss from the<br /> recorded population.

- The authors discussed their method in the context of other literature and stating its strength and limitation.

Major comments:

-The lack of histology limits the validation of lesion induction, ideally cell loss and neuronal loss in vivo needs to be quantified. In addition based on the lack of access to histology, it is not clear how the lesion volumes are calculated which also impacts the scientific rigor of the work. The authors mention that layers 2/3 and maybe 4 have been impacted. The lack of information on the extent of the lesion severely limits the use of their technique for neuroscience experiments.

-The lack of histology in combination with behavioral measures still limits the impact of the paper in the context of NHP research.

- Figure 5 involves fitting an exponential model to the generated lesion volume given the applied current amplitude and duration. However, the data from ex vivo sheep and pig cortex with the same current amplitude & three durations showed very large variability in lesion volume at Time = 2min (larger than the difference from 2 to ~2.2min). Very limited data points exist for the other two parameter combinations. These may suggest that the exponential fit is not the best model in this scenario.

- Regarding the comment on neuronal inactivation, the authors still did not show any evidence of single unit activity loss or changes in local field potential/multi-unit activity from the region being lesioned.

- Regarding this comment "The lesioning procedure was performed in Monkey F while sedated, but no data was presented for Monkey F in terms of lesioning parameters, lesion size, recorded electrophysiology, histological, or behavioral outcomes. It is also unclear if Monkey F was in a terminal study" the authors explained that "a lesion was performed on a sedated rhesus macaque (monkey F) who was subsequently euthanized due to unrelated health complications, in order to further verify safety before use in awake-behaving rhesus" but still no histology data is shown regarding monkey F to demonstrate this verification. Given that NHPs are highly valuable resources, it's important to make use of all collected data and to show that the induced lesion is comparable to those in the pig cortex.

Reviewer #2 (Public Review):

Summary:

Last et al. present Ais, a new deep learning-based software package for the segmentation of cryo-electron tomography data sets. The distinguishing factor of this package is its orientation to the joint use of different models, rather than the implementation of a given approach. Notably, the software is supported by an online repository of segmentation models, open to contributions from the community.

The usefulness of handling different models in one single environment is showcased with a comparative study on how different models perform on a given data set; then with an explanation of how the results of several models can be manually merged by the interactive tools inside Ais.

The manuscripts present two applications of Ais on real data sets; one is oriented to showcase its particle-picking capacities on a study previously completed by the authors; the second one refers to a complex segmentation problem on two different data sets (representing different geometries as bacterial cilia and mitochondria in a mouse neuron), both from public databases.

The software described in the paper is compactly documented on its website, additionally providing links to some YouTube videos (less than an hour in total) where the authors videocapture and comment on major workflows.

In short, the manuscript describes a valuable resource for the community of tomography practitioners.

Strengths:

A public repository of segmentation models; easiness of working with several models and comparing/merging the results.

Weaknesses:

A certain lack of concretion when describing the overall features of the software that differentiate it from others.

Reviewer #2 (Public Review):

Summary:

This study analyses camera trapping information on the occurrence of forest mammals along a gradient of human modification of the environment. The key hypotheses are that human disturbance squeezes wildlife into a smaller area or their activity into only part of the day, leading to increased co-occurrence under modification. The method used is joint species distribution modelling (JSDM).

Strengths:

The data source seems to be very nice, although since very little information is presented, this is hard to be sure of. Also, the JSDM approach is, in principle, a nice way of simultaneously analysing the data.

Weaknesses:

The manuscript suffers from a mismatch of hypotheses and methods at two different levels.

(1) At the lower level, we would need to better understand what the individual species do and "like" (their environmental niche).

(2) The hypothesis clearly asks for an analysis of the statistical interaction between human disturbance and co-occurrence. Yet, the study is not set up in a way to test this directly.

The hypotheses point towards presenting the spatial and the temporal niche, and how it changes, species for species, under human disturbance. To this, one could then add the layer of interspecific associations.

The change in activity and space use could be analysed by looking at the activity times and spatial distribution directly. If biotic interactions change along the disturbance gradient, then observed data are already the outcome of such changed interactions. We thus cannot use the data to infer them! But we can show, for each species, that the habitat preferences change along the disturbance gradient - or not, as the case may be.

The per-species models are simplistic: the predictors are only linear, and there are no statistical interactions. It is unclear how spatial autocorrelations of residuals were treated, although they form the basis for the association analysis. Why are times of day and day of the year not included as predictors IN INTERACTION with niche predictors and human disturbance, since they represent the temporal dimension on which niches are hypothesised to change?

The discussion has little to add to the results. The complexity of the challenge (understanding a community-level response after accounting for species-level responses) is not met, and instead substantial room is given to general statements of how important this line of research is. What is the advance in ecological understanding at the community level?

Reviewer #2 (Public Review):

Summary:

The authors provided benchmarking study results on tRNA-seq in terms of read alignment and quantification software with optimal parameterization. This result can be a useful guideline for choosing optimal parameters for tRNA-seq read alignment and quantification.

Strengths:

Benchmarking results for read alignment can be a useful guideline to choose optimal parameters and mapping strategy (mapping to amino acid) for various tRNAseq.

Weaknesses:

The topic is highly specific, and the novelty of the analysis might not be widely useful for general readers.

Some details of the sequencing data analysis pipeline are not clear for general readers:

(1) The explanation of the parameter D for bowtie2 sounds ambiguous. "How much effort to expend" needs to be explained in more detail.

(2) Please provide optimal parameters (L and D) for tRNA-seq alignment.

(3) I think the authors chose L=10 and D=100 based on Figure 1A. Which dataset did you choose for this parameterization among ALL-tRNAseq, DM-tRNAseq, mim-tRNAseq, QuantM-tRNA-seq, and YAMAT-seq?

(4) Salmon does not need a read alignment process such as Bowtie2. Hence, it is not clear "Only results from alignment with bowtie2" in Figure legend for Figure 4a.

Reviewer #2 (Public Review):

Summary:

In this study, Swarang and colleagues identified the lipid metabolite 15d-PGJ2 as a potential component of senescent myoblasts. They proposed that 15d-PGJ2 inhibits myoblast proliferation and differentiation by binding and regulating HRas, suggesting its potential as a target for restoring muscle homeostasis post-chemotherapy.

Strengths:

The regulation of HRas by 15d-PGJ2 is well controlled.

Weaknesses:

(1) I still think the novelty is limited by previous published findings. The authors themselves noted that the accumulation of 15d-PGJ2 in senescent cells has been reported in various cell types, including human fibroblasts, HEPG2 hepatocellular carcinoma cells, and HUVEC endothelial cells (PMCID: PMC8501892). Although the current study observed similar activation of 15d-PGJ2 in myoblasts, it appears to be additive rather than fundamentally novel. The covalent adduct of 15d-PGJ2 with Cys-184 of H-Ras was reported over 20 years ago (PMID: 12684535), and the biochemical principles of this interaction are likely universal across different cell types. The regulation of myogenesis by both HRas and 15d-PGJ2 has also been previously extensively reported (PMID: 2654809, 1714463, 17412879, 20109525, 11477074). The main conceptual novelty may lie in the connection between these points in myoblasts. But as discussed in another comment, the use of C2C12 cells as a model for senescence study is questionable due to the lack of the key regulator p16. The findings in C2C12 cells may not accurately represent physiological-relevant myoblasts. It is recommended that these findings be validated in primary myoblasts to strengthen the study's conclusions.

(2) The C2C12 cell line is not an ideal model for senescence study.<br /> C2C12 cells are a well-established model for studying myogenesis. However, their suitability as a model for senescence studies is questionable. C2C12 cells are immortalized and do not undergo normal senescence like primary cells as C2C12 cells are known to have a deleted p16/p19 locus, a crucial regulator of senescence (PMID: 20682446). The use of C2C12 cells in published studies does not inherently validate them as a suitable senescence model. These studies may have limitations, and the appropriateness of the C2C12 model depends on the specific research goals.<br /> In the study by Moustogiannis et al. (PMID: 33918414), they claimed to have aged C2C12 cells through multiple population doublings. However, the SA-β-gal staining in their data, which is often used to confirm senescence, showed almost fully confluent "aged" C2C12 cells. This confluent state could artificially increase SA-β-gal positivity, suggesting that these cells may not truly represent senescence. Moreover, the "aged" C2C12 cells exhibited normal proliferation, which contradicts the definition of senescence. Similar findings were reported in another study of C2C12 cells subjected to 58 population doublings (PMID: 21826704), where even at this late stage, the cells were still dividing every 2 or 3 days, similar to younger cells at early passages. More importantly, I do know how the p16 was detected in that paper since the locus was already mutated. In terms of p21, there was no difference in the proliferative C2C12 cells at day 0.<br /> In the study by Moiseeva et al. in 2023 (PMID: 36544018), C2C12 cells were used for senescence modeling for siRNA transfection. However, the most significant findings were obtained using primary satellite cells or confirmed with complementary data.<br /> In conclusion, while molecular changes observed in studies using C2C12 cells may be valid, the use of primary myoblasts is highly recommended for senescence studies due to the limitations and questionable senescence characteristics of the C2C12 cell line.

(3) Regarding source of increased PGD in the conditioned medium, I want to emphasize that it's unclear whether the PGD or its metabolites increase in response to DNA damage or the senescence state. Thus, using a different senescent model to exclude the possibility of DNA damage-induced increase will be crucial.

(4) Similarly for the in vivo Doxorubicin (Doxo) injection, both reviewers have raised concerns about the potential side effects of Doxo, including inflammation, DNA damage, and ROS generation. These effects could potentially confound the results of the study. The physiological significance of this study will heavily rely on the in vivo data. However, the in vivo senescence component is confounded by the side effects of Doxo.

(5) Figure 2A lacks an important control from non-senescent cells during the measurement of C2C12 differentiation in the presence of conditioned medium. The author took it for granted that the conditioned medium from senescent cells would inhibit myogenesis, relying on previous publications (PMID: 37468473). However, that study was conducted in the context of myotonic dystrophy type 1. To support the inhibitory effect in the current experimental settings, direct evidence is required. It would be necessary to include another control with conditioned medium from normal, proliferative C2C12 cells.

(6) Statistical analyses problems.<br /> Only t-test was used throughout the study even when there are more than two groups. Please have a statistician to evaluate the replicates and statistical analyses used.<br /> For the 15d-PGJ2/cell concentration measurements in Figure 1F, there were only two replicates, which was provided in the supplementary table after required. Was that experiment repeated with more biological replicates?<br /> For figure 1C, Fig 1F, 1G, 1J, 2C, 2E, 3A, 3E, 3F, 4D, 4E, please include each data points in bar graphs as used in Fig 1D, or at least provide how many biological replicates were used for each experiment?<br /> There is no error bar in a lot of control groups (Fig 2C, 2E, 3EF, 4E, S4B).<br /> For qPCR data in Figure 1C, the author responded in that the data in was plotted using 2-ΔCT instead of 2-ΔΔCT to show the variability in the expression of mRNAs isolated from animals treated with Saline. This statement does not align with the method section. Please revise.

(7) For Figure 1, the title may not be appropriate as there is insufficient data to support the inhibition of myoblast differentiation.

Reviewer #2 (Public Review):

Summary:

Eaton and colleagues use targeted protein degradation coupled with nascent transcription mapping to highlight a role for the integrator component INST11 in terminating antisense transcription. They find that upon inhibition of CDK9, INST11 can terminate both antisense and sense transcription - leading to a model whereby INST11 can terminate antisense transcription and the activity of CDK9 protects sense transcription from INST11-mediated termination. They further develop a new method called sPOINT which selectively amplifies nascent 5' capped RNAs and find that transcription initiation is more efficient in the sense direction than in the antisense direction. This is an excellent paper which uses elegant experimental design and innovative technologies to uncover a novel regulatory step in the control of transcriptional directionality.

Strengths:

One of the major strengths of this work is that the authors endogenously tag two of their proteins of interest - RBBP6 and INST11. This tag allows them to rapidly degrade these proteins - increasing the likelihood that any effects they see are primary effects of protein depletion rather than secondary effects. Another strength of this work is that the authors immunoprecipitate RNAPII and sequence extracted full length RNA (POINT-seq) allowing them to map nascent transcription. A technical advance from this work is the development of sPOINT which allows the selective amplification of 5' capped RNAs < 150 nucleotides, allowing the direction of transcription initiation to be resolved.

Weaknesses:

While the authors provide strong evidence that INST11 and CDK9 play important roles in determining promoter directionality, their data suggests that when INST11 is degraded and CDK9 is inhibited there remains a bias in favour of sense transcription (Figure 4B and C). This suggests that there are other unknown factors that promote sense transcription over antisense transcription and future work could look to identify these.

Reviewer #2 (Public Review):

The authors identified new target elements for prostaglandin E2 (PGE2) through which insulin release can be regulated in pancreatic beta cells under physiological conditions. In vitro extracellular exposure to PGE2 could directly and dose-dependently inhibit the potassium channel Kv2.2. In vitro pharmacology revealed that this inhibition occurs through the EP2/4 receptors, which activate protein kinase A (PKA). By screening specific sites of the Kv2.2 channel, the target phosphorylation site (S448) for PKA regulation was found. The physiological relevance of the described signaling cascade was investigated and confirmed in vivo, using a Kv2.2 knockdown mouse model.

The strength of this manuscript is the novelty of the (EP2/4-PKA-Kv2.2 channel) molecular pathway described and the comprehensive methodological toolkit the authors have relied upon.

The introduction is detailed and contains all the information necessary to place the claims in context. Although the dataset is comprehensive and a logical lead is consistently built, there is one important point to consider: to clarify that the described signaling pathway is characteristic of normal physiological conditions and thus differs from pathological changes. It would be useful to carry out basic experiments in a diabetes model (regardless of whether this is in mice or rats).

Reviewer #2 (Public Review):

Summary:

The authors report here interesting data on the interactions mediated by the SH3 domain of BIN1 that expand our knowledge on the role of the SH3 domain of BIN1 in terms of mediating specific interactions with a potentially high number of proteins and how variants in this region alter or prevent these protein-protein interactions. These data provide useful information that will certainly help to further dissect the networks of proteins that are altered in some human myopathies as well as the mechanisms that govern the correct physiological activity of muscle cells.

Strengths:

The work is mostly based on improved biochemical techniques to measure protein-protein interaction and provide solid evidence that the SH3 domain of BIN1 can establish an unexpectedly high number of interactions with at least a hundred cellular proteins, among which the authors underline the presence of other proteins known to be causative of skeletal muscle diseases and not known to interact with BIN1. This represents an unexpected and interesting finding relevant to better define the network of interactions established among different proteins that, if altered, can lead to muscle disease. An interesting contribution is also the detailed identification of the specific sites, namely the Proline-Rich Motifs (PRMs) that in the interacting proteins mediate binding to the BIN1 SH3 domain.

Weaknesses:

Less convincing, or too preliminary in my opinion, are the data supporting BIN1 co-localization with PRC1. Indeed, the affinity of PRC1 is significantly lower than that of DNM2, an established BIN1 interacting protein. Thus, this does not provide compelling evidence to support PRC1 as a significant interactor of BIN1. Similarly, the localization data appears somewhat preliminary to substantiate a role of BIN1 in mitotic processes. These findings may necessitate additional experimental work to be more convincing.

Reviewer #2 (Public Review):

This study reports a set of experiments and subsequent analyses focusing on the role of Drosophila boundary elements in shaping 3D genome structure and regulating gene expression. The authors primarily focus on the region of the fly genome containing the even skipped (eve) gene; eve is expressed in a canonical spatial pattern in fly embryos and its locus is flanked by the well-characterized neighbor of homie (nhomie) and homie boundary elements. The main focus of the investigation is the orientation dependence of these boundary elements, which had been observed previously using reporter assays. In this study, the authors use Crispr/Cas9 editing followed by recombination-mediated cassette exchange to create a series of recombinant fly lines in which the nhomie boundary element is either replaced with exongenous sequence from phage 𝝀, an inversion of nhomie, or a copy of homie that has the same orientation as the endogenous homie sequence. The nhomie sequence is also regenerated in its native orientation to control for effects introduced by the transgenesis process.

The authors then perform high-resolution Micro-C to analyze 3D structure and couple this with fluorescent and colorimetric RNA in situ hybridization experiments to measure the expression of eve and nearby genes during different stages of fly development. The major findings of these experiments are that total loss of boundary sequence (replacement with 𝝀 DNA) results in major 3D structure changes and the most prominent observed gene changes, while inversion of the nhomie boundary or replacement with homie resulted in more modest effects in terms of 3D structure and gene expression changes and a distinct pattern of gene expression change from the 𝝀 DNA replacement. As the samples in which the nhomie boundary is inverted or replaced with homie have similar Micro-C profiles at the eve locus and show similar patterns of a spurious gene activation relative to the control, the observed effects appear to be driven by the relative orientation of the nhomie and homie boundary elements to one another.

Collectively, the findings reported in the manuscript are of broad interest to the 3D genome field. Although extensive work has gone into characterizing the patterns of 3D genome organization in a whole host of species, the underlying mechanisms that structure genomes and their functional consequences are still poorly understood. The perhaps best understood system, mechanistically, is the coordinated action of CTCF with the cohesin complex, which in vertebrates appears to shape 3D contact maps through a loop extrusion-pausing mechanism that relies on orientation-dependent sequence elements found at the boundaries of interacting chromatin loops. Despite having a CTCF paralog and cohesin, the Drosophila genome does not appear to be structured by loop extrusion-pausing. The identification of orientation-dependent elements with pronounced structural effects on genome folding thus may shed light on alternative mechanisms used to regulated genome structure, which in turn may yield insights into the significance of particular folding patterns.

On the whole, this study is comprehensive and represents a useful contribution to the 3D genome field. The transgenic lines and Micro-C datasets generated in the course of the work will be valuable resources for the research community. Moreover, the manuscript, while dense in places, is generally clearly written and comprehensive in its description of the work. However, I have a number of comments and critiques of the manuscript, mainly centering on the framing of the experiments and presentation of the Micro-C results and on the manner in which the data are analyzed and reported.

As this document now reflects my review of a revised version of the initial preprint, I will begin to add the new content at this point. As discussed in detail in the following paragraphs, my initial impression of the manuscript has not changed, so I have accordingly left the above text unaltered.

In my initial review, I provided a number of suggestions to improve the quality of the manuscript. These suggestions, which took the form of six major and three minor points, largely focused on 1) altering the writing in certain places to make the story more broadly accessible to the readership and 2) the inclusion of key, missing methodological detail to increase the rigor and reproducibility of the study. No new experiments were requested, and all of the points could be readily addressed with rather straightforward textual changes.

In their revised manuscript, the authors elected to directly address one of the major points and two of the minor points (major point 4, minor points 1 and 3). The remainder of my suggestions remain entirely unaddressed. A similar level of responsiveness was afforded to the very reasonable critiques of the other Reviewer and the Reviewing Editor. The authors have instead largely chosen to respond to the points raised exclusively in the rebuttal document. This document sprawls across >22 pages, includes numerous in-line figures, and cites dozens of references. The tone of this document, in many places, is at best forceful. In a less generous interpretation, many sections are combative, dismissive, and borderline unprofessional.

It is my opinion that the authors are doing the scientific community a disservice with their response. While it is my understanding that readers will be able see the rebuttal letter, I find that end result far from satisfying. How many readers will take the trouble to access that file, versus the manuscript itself? Skirting the review critiques places an unfair burden on readers, who are expecting peer-reviewed science, to dig into the accessory files to follow the critique and response, rather than seeing in reflected in the final product as they accustomed. Intentionally or not, the tactics the authors have chosen detract from what is otherwise a novel and well-intentioned new publishing model. It is also worth pointing out that peer review is done as an act of service to the scientific community, as the senior authors are doubtless aware. The other reviewer, the Reviewing Editor, and I have all taken time away from advancing our own careers and those of our trainees to offer the thoughtful critiques that were so pointedly dismissed.

In summary, as the vast majority of my critiques remain unaddressed, I have simply reproduced them below.

Major Points:

(1) The authors motivate much of the introduction and results with hypothetical "stem loop" and "circle loop" models of chromosome confirmation, which they argue are reflected in the Micro-C data and help to explain the observed ISH patterns. While such structures may possibly form, the support for these specific models vs. the many alternatives is not in any way justified. For instance, no consideration is given to important biophysical properties such as persistence length, packing/scaling, and conformational entropy. As the biophysical properties of chromatin are a very trafficked topic both in terms of experimentation and computational modeling and generally considered in the analysis of chromosome conformation data, the study would be strengthened by acknowledgement of this body of work and more direct integration of its findings.

(2) Similar to Point 1, while there is a fair amount of discussion of how the observed results are or are not consistent with loop extrusion, there is no discussion of the biophysical forces that are thought to underly compartmentalization such as block-polymer co-segregation and their potential influence. I found this absence surprising, as it is generally accepted that A/B compartmentalization essentially can explain the contact maps observed in Drosophila and other non-vertebrate eukaryotes (Rowley, ..., Corces 2017; PMID 28826674). The manuscript would be strengthened by consideration of this phenomenon.

(3) The contact maps presented in the study represent many cells and distinct cell types. It is clear from single-cell Hi-C and multiplexed FISH experiments that chromosome conformation is highly variable even within populations of the same cell, let alone between cell types, with structures such as TADs being entirely absent at the single cell level and only appearing upon pseudobulking. It is difficult to square these observations with the models of relatively static structures depicted here. The authors should provide commentary on this point.

(4) Related to Point 4, the lack of quantitative details about the Micro-C data make it difficult to evaluate if the changes observed are due to biological or technical factors. It is essential that the authors provide quantitative means of controlling for factors like sampling depth, normalization, and data quality between the samples.

(5) The ISH effects reported are modest, especially in the case of the HCR. The details provided for how the imaging data were acquired and analyzed are minimal, which makes evaluating them challenging. It would strengthen the study to provide much more detail about the acquisition and analysis and to include depiction of intermediates in the analysis process, e.g. the showing segmentation of stripes.

Reviewer #2 (Public Review):

The authors have engaged constructively with some of the points raised. In particular the addition of more details about the experimental cryo-EM procedures has strengthened the manuscript.

I do worry that the FSC values of model-vs-map appear to be higher than expected from the corresponding FSCs between the half-maps (e.g. see Fig 13). The implication of this observation is that the atomic models may have been overfitted in the maps, which would have led to a deterioration of their geometry. A table with rmsd on bond lengths, angles, etc would probably show this. In addition, to check for overfitting, the atomic model for each data set could be refined in one of the half-maps, and then that same model could be used to calculate 2 FSC model-vs-map curves: one against the half-map it was refined in and one against the other half-map. Deviations between these two curves are an indication of overfitting.

In addition, the sudden drop in the FSC curves in Figure 16 shows that something unexpected has happened to this refinement. Are the authors sure that only the procedures outlined in the Methods were used to create these curves? The unexpected nature of the FSC curve for this type (2A) raises doubts about the correctness of the reconstruction.

Reviewer #2 (Public Review):

Summary:

The authors provided a novel antigen delivery system that showed remarkable efficacy in transporting antigens to develop cancer therapeutic vaccines.

Strengths:

This manuscript was innovative, meaningful, and had a rich amount of data.

Weaknesses:

There are still some issues that need to be addressed and clarified.

(1) The format of images and data should be unified. Specifically, as follows: a. The presentation of flow cytometry results; b, The color schemes for different groups of column diagrams.

(2) The P-value should be provided in Figures, including Figure 1F, 1H, 3C, 3D, and 3E.

(3) The quality of Figure 1C was too low to support the conclusion. The author should provide higher-quality images with no obvious background fluorescent signal. Meanwhile, the fluorescent image results of "Egfp+VSVg" group were inconsistent with the flow cytometry data. Additionally, the reviewer recommends that the authors use a confocal microscope to repeat this experiment to obtain a more convincing result.

(4) The survival situation of the mouse should be provided in Figure 5, Figure 6, and Figure 7 to support the superior tumor therapy effect of ePAC.

(5) To demonstrate that ePAC could trigger a strong immune response, the positive control group in Figure 4K should be added.

(6) In Figure 6G-I and other figures, the author should indicate the time point of detection. Meanwhile, there was no explanation for the different numbers of mice in Figure 6G-I. If the mouse was absent due to death, it may be necessary to advance the detection time to obtain a more convincing result.

(7) In Figure 6B, the rainbow color bar with an accurate number of maximum and minimum fluorescence intensity should be provided. In addition, the corresponding fluorescence intensity in Figure 6B should be noted.

(8) The quality of images in Figure 1D and Figure S1B could not support the author's conclusion; please provide higher-quality images.

(9) In Figure 2F, the bright field in the overlay photo may disturb the observation. Meanwhile, the scale bar should be provided in enlarged images.

Reviewer #2 (Public Review):

Summary:<br /> The authors investigate the phosphotransfer capacity of Ser/Thr kinase IκB kinase (IKK), a mediator of cellular inflammation signaling. Canonically, IKK activity is promoted by activation loop phosphorylation at Ser177/Ser181. Active IKK can then unleash NF-κB signaling by phosphorylating repressor IκBα at residues Ser32/Ser26. Noting the reports of other IKK phosphorylation sites, the authors explore the extent of autophosphorylation.

Semi-phosphorylated IKK purified from Sf9 cells, exhibits the capacity for further autophosphorylation. Anti-phosphotyrosine immunoblotting indicated unexpected tyrosine phosphorylation. Contaminating kinase activity was tested by generating a kinase-dead K44M variant, supporting the notion that the unexpected phosphorylation was IKK-dependent. In addition, the observed phosphotyrosine signal required phosphorylated IKK activation loop serines.

Two candidate IKK tyrosines were examined as the source of the phosphotyrosine immunoblotting signal. Activation loop residues Tyr169 and Tyr188 were each rendered non-phosphorylatable by mutation to Phe. The Tyr variants decreased both autophosphorylation and phosphotransfer to IκBα. Likewise, Y169F and Y188F IKK2 variants immunoprecipitated from TNFa-stimulated cells also exhibited reduced activity in vitro.

The authors further focus on Tyr169 phosphorylation, proposing a role as a phospho-sink capable of phosphotransfer to IκBα substrate. This model is reminiscent of the bacterial two-component signaling phosphotransfer from phosphohistidine to aspartate. Efforts are made to phosphorylate IKK2 and remove ATP to assess the capacity for phosphotransfer. Phosphorylation of IκBα is observed after ATP removal, although there are ambiguous requirements for ADP.

Strengths:

Ultimately, the authors draw together the lines of evidence for IKK2 phosphotyrosine and ATP-independent phosphotransfer to develop a novel model for IKK2-mediated phosphorylation of IκBα. The model suggests that IKK activation loop Ser phosphorylation primes the kinase for tyrosine autophosphorylation. With the assumption that IKK retains the bound ADP, the phosphotyrosine is conformationally available to relay the phosphate to IκBα substrate. The authors are clearly aware of the high burden of evidence required for this unusual proposed mechanism. Indeed, many possible artifacts (e.g., contaminating kinases or ATP) are anticipated and control experiments are included to address many of these concerns. Taken together, the observations are thought-provoking, and I look forward to seeing this model tested in a cellular system.

Weaknesses:

It seems that the analysis hinges on the fidelity of pan-specific phosphotyrosine antibodies.

The analysis often returns to the notion that tyrosine phosphorylation(s) (and critical active site Lys44) dictate IKK2 substrate specificity, but evidence for this seems diffuse and indirect. This is an especially difficult claim to make with in vitro assays, omitting the context of other cellular specificity determinants (e.g., localization, scaffolding, phosphatases).

Multiple phosphorylated tyrosines in IKK2 were apparently identified by mass spectrometric analyses, but the data and methods are not described. It is common to find non-physiological post-translational modifications in over-expressed proteins from recombinant sources. Are these IKK2 phosphotyrosines evident by MS in IKK2 immunoprecipitated from TNFa-stimulated cells? Identifying IKK2 phosphotyrosine sites from cells would be especially helpful in supporting the proposed model.

Reviewer #2 (Public Review):

Summary:

The paper aims to investigate the synergies between desiccation chaperones and small molecule cosolutes, and describe its mechanistic basis. The paper reports that IDP chaperones have stronger synergies with the cosolutes they coexist with, and in one case suggests that this is related to oligomerization propensity of the IDP.

Strengths:

The study uses a lot of orthogonal methods and the experiments are technically well done. They are addressing a new question that has not really been addressed previously.

Weaknesses:

The conclusions are based on a few examples and only partial correlations. While the data support mechanistic conclusions about the individual proteins studied, it is not clear that the conclusions can be generalized to the extent proposed by the authors due to small effect sizes, small numbers of proteins, and only partial correlations.

The authors pose relevant questions and try to answer them through a systematic series of experiments that are all technically well-conducted. The data points are generally interpreted appropriately in isolation, however, I am a little concerned about a tendency to over-generalize their findings. Many of the experiments give negative or non-conclusive results (not a problem in itself), which means that the overall storyline is often based on single examples. For example, the central conclusion that IDPs interact synergistically with their endogenous co-solute (Figure 2E) is largely driven by one outlier from Arabidopsis. The rest are relatively close to the diagonal, and one could equally well suggest that the cosolutes affect the IDPs equally (which is also the conclusion in 1F). Similarly, the mechanistic explanations tend to be based on single examples. This is somewhat unavoidable as biophysical studies cannot be done on thousands of proteins, but the text should be toned down to reflect the strength of the conclusions.

The central hypothesis revolves around the interplay between cosolutes and IDP chaperones comparing chaperones from species with different complements of cosolutes. In Table 1, it is mentioned that Arabidopsis uses both trehalose and sucrose as a cosolute, yet experiments are only done with either of these cosolutes and Arabidopsis is counted in the sucrose column. While it makes sense to compare them separately from a biophysical point of view, the ability to test the co-evolution of these systems is somewhat diminished by this. At least it should be discussed clearly.

It would be helpful if the authors could spell out the theoretical basis of how they quantify synergy. I understand what they are doing - and maybe there are no better ways to do it - but it seems like an approach with limitations. The authors identify one in that the calculation only works far from 100%, but to me, it seems there would be an equally strict requirement to be significantly above 0%. This would suggest that it is used wrongly in Figure 6H, where there is no effect of betaine (at least as far as the color scheme allows one to distinguish the different bars). In this case, the authors cannot really conclude synergy or not, it could be a straight non-synergistic inhibition by betaine.

Reviewer #2 (Public Review):

Retroviral integration in general, and HIV integration in particular, takes place in dsDNA, not in R-loops. Although HIV integration can occur in vitro on naked dsDNA, there is good evidence that, in an infected cell, integration occurs on DNA that is associated with nucleosomes. This review will be presented in two parts. First, a summary will be provided giving some of the reasons to be confident that integration occurs on dsDNA on nucleosomes. The second part will point out some of the obvious problems with the experimental data that are presented in the manuscript.

(1) 2017 Dos Passos Science paper describes the structure of the HIV intasome. The structure makes it clear that the target for integration is dsDNA, not an R-loop, and there are very good reasons to think that structure is physiologically relevant. For example, there is data from the Cherepanov, Engelman, and Lyumkis labs to show that the HIV intasome is quite similar in its overall structure and organization to the structures of the intasomes of other retroviruses. Importantly, these structures explain the way integration creates a small duplication of the host sequences at the integration site. How do the authors propose that an R-loop can replace the dsDNA that was seen in these intasome structures?

(2) As noted above, concerted (two-ended) integration can occur in vitro on a naked dsDNA substrate. However, there is compelling evidence that, in cells, integration preferentially occurs on nucleosomes. Nucleosomes are not found in R loops. In an infected cell, the viral RNA genome of HIV is converted into DNA within the capsid/core which transits the nuclear pore before reverse transcription has been completed. Integration requires the uncoating of the capsid/core, which is linked to the completion of viral DNA synthesis in the nucleus. Two host factors are known to strongly influence integration site selection, CPSF6 and LEDGF. CPSF6 is involved in helping the capsid/core transit the nuclear pore and associate with nuclear speckles. LEDGF is involved in helping the preintegration complex (PIC) find an integration site after it has been released from the capsid/core, most commonly in the bodies of highly expressed genes. In the absence of an interaction of CPSF6 with the core, integration occurs primarily in the lamin-associated domains (LADs). Genes in LADs are usually not expressed or are expressed at low levels. Depending on the cell type, integration in the absence of CPSF6 can be less efficient than normal integration, but that could well be due to a lack of LEDGF (which is associated with expressed genes) in the LADs. In the absence of an interaction of IN with LEDGF (and in cells with low levels of HRP2) integration is less efficient and the obvious preference for integration in highly expressed genes is reduced. Importantly, LEDGF is known to bind histone marks, and will therefore be preferentially associated with nucleosomes, not R-loops. LEDGF fusions, in which the chromatin binding portion of the protein is replaced, can be used to redirect where HIV integrates, and that technique has been used to map the locations of proteins on chromatin. Importantly, LEDGF fusions in which the chromatin binding component of LEDGF is replaced with a module that recognizes specific histone marks direct integration to those marks, confirming integration occurs efficiently on nucleosomes in cells. It is worth noting that it is possible to redirect integration to portions of the host genome that are poorly expressed, which, when taken with the data on integration into LADs (integration in the absence of a CPSF6 interaction) shows that there are circumstances in which there is reasonably efficient integration of HIV DNA in portions of the genome in which there are few if any R-loops.

(3) Given that HIV DNA is known to preferentially integrate into expressed genes and that R-loops must necessarily involve expressed RNA, it is not surprising that there is a correlation between HIV integration and regions of the genome to which R loops have been mapped. However, it is important to remember that correlation does not necessarily imply causation.

If we consider some of the problems in the experiments that are described in the manuscript:

(1) In an infected individual, cells are almost always infected by a single virion and the infecting virion is not accompanied by large numbers of damaged or defective virions. This is a key consideration: the claim that infection by HIV affects R-loop formation in cells was done with a VSVg vector in experiments in which there appears to have been about 6000 virions per cell. Although most of the virions prepared in vitro are defective in some way, that does not mean that a large fraction of the defective virions cannot fuse with cells. In normal in vivo infections, HIV has evolved in ways that avoid signaling infected the cell of its presence. To cite an example, carrying out reverse transcription in the capsid/core prevents the host cell from detecting (free) viral DNA in the cytoplasm. The fact that the large effect on R-loop formation which the authors report still occurs in infections done in the absence of reverse transcription strengthens the probability that the effects are due to the massive amounts of virions present, and perhaps to the presence of VSVg, which is quite toxic. To have physiological relevance, the infections would need to be carried out with virions that contain HIV even under circumstances in which there is at most one virion per cell.

(2) Using the Sso7d version of HIV IN in the in vitro binding assays raises some questions, but that is not the real question/problem. The real problem is that the important question is not what/how HIV IN protein binds to, but where/how an intasome binds. An intasome is formed from a combination of IN bound to the ends of viral DNA. In the absence of viral DNA ends, IN does not have the same structure/organization as it has in an intasome. Moreover, HIV IN (even Sso7d, which was modified to improve its behavior) is notoriously sticky and hard to work with. If viral DNA had been included in the experiment, intasomes would need to be prepared and purified for a proper binding experiment. To make matters worse, there are multiple forms of multimeric HIV IN and it is not clear how many HIV INs are present in the PICs that actually carry out integration in an infected cell.

(3) As an extension of comment 2, the proper association of an HIV intasome/PIC with the host genome requires LEDGF and the appropriate nucleic acid targets need to be chromatinized.

(4) Expressing any form of IN, by itself, in cells to look for what IN associates with is not a valid experiment. A major factor that helps to determine both where integration takes place and the sites chosen for integration is the transport of the viral DNA and IN into the nucleus in the capsid core. However, even if we ignore that important part of the problem, the IN that the authors expressed in HeLa cells won't be bound to the viral DNA ends (see comment 2), even if the fusion protein would be able to form an intasome. As such, the IN that is expressed free in cells will not form a proper intasome/PIC and cannot be expected to bind where/how an intasome/PIC would bind.

(5) As in comment 1, for the PLA experiments presented in Figure 5 to work, the number of virions used per cell (which differs from the MOI measured by the number of cells that express a viral marker) must have a high, which is likely to have affected the cells and the results of the experiment. However, there is the additional question of whether the IN-GFP fusion is functional. The fact that the functional intasome is a complex multimer suggests that this could be a problem. There is an additional problem, even if IN-GFP is fully functional. During a normal infection, the capsid core will have delivered copies of IN (and, in the experiments reported here, the IN-GFP fusion) into the nucleus that is not part of the intasome. These "free" copies of IN (here IN-GFP) are not likely to go to the same sites as an intasome, making this experiment problematic (comment 4).

(6) In the Introduction, the authors state that the site of integration affects the probability that the resulting provirus will be expressed. Although this idea is widely believed in the field, the actual data supporting it are, at best, weak. See, for example, the data from the Bushman lab showing that the distribution of integration sites is the same in cells in which the integrated proviruses are, and are not, expressed. However, given what the authors claim in the introduction, they should be more careful in interpreting enzyme expression levels (luciferase) as a measure of integration efficiency in experiments in which they claim proviruses are integrated in different places.

(7) Using restriction enzymes to create an integration site library introduces biases that derive from the uneven distribution of the recognition sites for the restriction enzymes.

Reviewer #2 (Public Review):

Summary:

The authors aimed to functionally characterize primary human airway macrophages and monocyte-derived macrophages, correlating their glycolytic shift in metabolism. They conducted this macrophage characterization in response to type II interferon and IL-4 priming signals, followed by different stimuli of irradiated Mycobacterium tuberculosis and LPS.

Strengths:

(1) The study employs a thorough measurement of metabolic shift in metabolism by assessing extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of differentially polarized primary human macrophages using the Seahorse XFe24 Analyzer.<br /> (2) The effect of differential metabolic shift on the expression of different surface markers for macrophage activation is evaluated through immunofluorescence flow cytometry and cytokine measurement via ELISA.<br /> (3) The authors have achieved their aim of preliminarily characterizing the glycolysis-dependent cytokine profile and activation marker expression of IFN-g and IL-4 primed primary human macrophages.<br /> (4) The results of the study support its conclusion of glycolysis-dependent phenotypical differences in cytokine secretion and activation marker expression of AMs and MDMs.

Weaknesses:

(1) The data are presented in duplicates for cross-analyses.<br /> (2) The data presented supports a distinct functional profile of airway macrophages (AMs) compared to monocyte (blood)-derived macrophages (MDMs) in response to the same priming signals. However, the study does not attempt to explore the underlying mechanism for this difference.<br /> (3) The study is descriptive in nature, and the results validate IFN-g-mediated glycolytic reprogramming in primary human macrophages without providing mechanistic insights.

Reviewer #2 (Public Review):

Summary:

Glioblastoma is a common primary brain cancer, that is difficult to treat and has a low survival rate. The lack of genetically tractable and immunocompetent vertebrate animal model has prevented discovery of new therapeutic targets and limited efforts for screening of pharmaceutical agents for the treatment of the disease. Here Weiss et al., express oncogenic variants frequently observed in human glioblastoma within zebrafish lacking the tumor suppressor TP53 to generate a patient-relevant in vivo model. The authors demonstrate that loss of TP53 and overexpression of EGFR, PI3KCA, and mScarlet (p53EPS) in neural progenitors and radial glia leads to visible fluorescent brain lesions in live zebrafish. The authors performed RNA expression analysis that uncovered a molecular signature consistent with human mesenchymal glioblastoma and identified gene expression patterns associated with inflammation. Live imaging revealed high levels of immune cell infiltration and associations between microglia/macrophages and tumor cells. To define functional roles for regulators of inflammation on specific immune-related responses during tumorigenesis, transient CRISPR/Cas9 gene targeting was used to disrupt interferon regulator factor proteins and showed Inflammation-associated irf7 and irf8 are required to inhibit p53EPS tumor formation. Further, experiments to deplete the macrophages using clodronate liposomes suggest that macrophages contribute to the suppression of tumor engraftment following transplantation. The authors' conclusions are supported by the data and the experiments are thoroughly controlled throughout. Taken together, these results provide new insights into the regulation of glioblastoma initiation and growth by the surrounding microenvironment and provide a novel in vivo platform for the discovery of new molecular mechanisms and testing of therapeutics.

Strengths/Weaknesses:

The authors convincingly show that co-injection of activated human EGFRviii, PI3KCAH1047R, and mScarlet into TP53 null zebrafish promotes formation of fluorescent brain lesions and glioblastoma-like tumor formation. The authors include histological characterization of the tumors, as well as quantifications of p-ERK and p-AKT staining to highlight increased activation of the MAPK/AKT signaling pathways in their tumor model.

The authors use a transplantation assay to further test the tumorigenic potential of dissociated cells from glial-derived tumors in the context of specific manipulations of the tumour microenvironment.

The authors nicely show high levels of immune cell infiltration and associations between microglia/macrophages and tumor cells. Quantification of the emergence of macrophages over time in relation to tumor initiation and growth is provided and supports the observations of tumor suppressive activity of the phagocytes. The authors also attempt to delineate if other leukocyte populations are involved and observe tumor formation without significant infiltration of neutrophils.

The authors provide evidence for key genetic regulators of the local microenvironment, showing increased p53EPS tumor initiation following Ifr7 gene knock-down and loss of irf7 expression in the TME.

Reviewer #2 (Public Review):

Summary:

In this study, Christin Krause et al mapped the hepatic miRNA-transcriptome of type 2 diabetic obese subjects, identified miR-182-5p and its target genes LRP6 as potential drivers of dysregulated glucose tolerance and fatty acid metabolism in obese T2-diabetics.

Strengths:

This study contains some interesting findings and are valuable for the understanding of key regulatory role of miRNAs in the pathogenesis of T2D.

Weaknesses:

The authors didn't systemically investigate the function of miR-182 in T2DM or NAFLD.

Reviewer #2 (Public Review):

Summary:

This work provides important anatomical features of a new species from the Lower Cambrian, which helps advance our understanding of the evolutionary origins of animal body plans. The authors interpreted that the new species possessed a bilateral body covered with cuticular polygonal reticulation and a ventral mouth. Based on cladistic analyses using maximum likelihood, Bayesian, and parsimony, the new species was placed, along with Saccorhytus, in a sister-group ("Saccorhytida") of the Ecdysozoa. The phylogenetic position of Saccorhytida suggests a new scenario of the evolutionary origin of the crown ecdysozoan body plan.

Strengths:

Although the new species reported in this paper show strange morphologies, the interpretation of anatomical features was based on detailed observations of multiple fossil specimens, thereby convincing at the moment. Morphological data about fossil taxa in the Ediacaran and Early Cambrian are quite important for our understanding of the evolution of body plans (and origins of phyla) in paleontology and evolutionary developmental biology, and this paper represents a valuable contribution to such research fields.

Weaknesses:

The preservations of the specimens, in particular on the putative ventral side, are not good, and the interpretation of the anatomical features need to be tested with additional specimens in future. The monophyly of Cycloneuralia (Nematoida + Scalidophora) was not necessarily well-supported by cladistic analyses (Supplementary Figures 7-9), and the evolutionary scenario (Fig. 4) also need to be tested in future works. On the other hand, the revised version provides important contributions from currently available data, and the above-mentioned problems should be studied in a separate paper in future.

Reviewer #2 (Public Review):

Summary:

eQTLs have emerged as a method for interpreting GWAS signals. However, some GWAS signals are difficult to explain with eQTLs. In this paper, the authors demonstrated that caQTLs can explain these signals. This suggests that for GWAS signals to actually lead to disease phenotypes, they must be accessible in the chromatin. This implies that for GWAS signals to translate into disease phenotypes, they need to be accessible within the chromatin.

However, fundamentally, caQTLs, like GWAS, have the limitation of not being able to determine which genes mediate the influence on disease phenotypes. This limitation is consistent with the constraints observed in this study.

(1) For reproducibility, details are necessary in the method section.

- Details about adding YRI samples in ATAC-seq: For example, how many samples are there, and what is used among public data? There is LCL-derived iPSC and differentiated iPSC (cardiomyocytes) data , not LCL itself. How does this differ from LCL, and what is the rationale for including this data despite the differences?

- caQTL is described as having better power than eQTL despite having fewer samples. How does the number of ATAC peaks used in caQTL compare to the number of gene expressions used in eQTL?

- Details about RNA expression data: In the method section, it states that raw data (ERP001942) was accessed, and in data availability, processed data (E-GEUV-1) was used. These need to be consistent.

How many samples were used (the text states 373, but how was it reduced from the original 465, and the total genotype is said to be 493 samples while ATAC has n=100; what are the 20 others?), and it mentions European samples, but does this exclude YRI?

(2) Experimental results determining which TFs might bind to the representative signals of caQTL are required.

(3) It is stated that caQTL is less tissue-specific compared to eQTL; would caQTL performed with ATAC-seq results from different cell types, yield similar results?

Reviewer #2 (Public Review):

In Bing et al, the authors analyze micro-C data from NC14 fly embryos, focusing on the eve locus, to assess different models of chromatin looping. They conclude that fly TADs are less consistent with conventional cohesin-based loop extrusion models and instead rely more heavily on boundary-boundary pairings in an orientation-dependent manner.

Overall, I found the manuscript to be interesting and thought-provoking. However, this paper reads much more like a perspective than a research article. Considering the journal is aimed at the general audience, I strongly suggest the authors spend some time editing their introduction to the most salient points as well as organizing their results section in a more conventional way with conclusion-based titles. It was very difficult to follow the authors' logic throughout the manuscript as written. It was also not clear as written which experiments were performed as part of this study and which were reanalyzed but published elsewhere. This should be made clearer throughout.

It has been shown several times that Drosophila Hi-C maps do not contain all of the features (frequent corner peaks, stripes, etc.) observed when compared to mammalian cells. Considering these features are thought to be products of extrusion events, it is not an entirely new concept that Drosophila domains form via mechanisms other than extrusion. That being said, the authors' analyses do not distinguish between the formation and the maintenance of domains. It is not clear to this reviewer why a single mechanism should explain the formation of the complex structures observed in static Hi-C heatmaps from a population of cells at a single developmental time point. For example, how can the authors rule out that extrusion initially provides the necessary proximity and possibly the cis preference of contacts required for boundary-boundary pairing whereas the latter may more reflect the structures observed at maintenance? Future work aimed at analyzing micro-C data in cohesin-depleted cells might shed additional light on this.

Additional mechanisms at play include compartment-level interactions driven by chromatin states. Indeed, in mammalian cells, these interactions often manifest as a "plume" on Hi-C maps similar to what the authors attribute to boundary interactions in this manuscript. How do the chromatin states in the neighboring domains of the eve locus impact the model if at all?

How does intrachromosomal homolog pairing impact the models proposed in this manuscript (Abed et al. 2019; Erceg et al., 2019). Several papers recently have shown that somatic homolog pairing is not uniform and shows significant variation across the genome with evidence for both tight pairing regions and loose pairing regions. Might loose pairing interactions have the capacity to alter the cis configuration of the eve locus?

In summary, the transgenic experiments are extensive and elegant and fully support the authors' models. However, in my opinion, they do not completely rule out additional models at play, including extrusion-based mechanisms. Indeed, my major issue is the limited conceptual advance in this manuscript. The authors essentially repeat many of their previous work and analyses. The authors make no attempt to dissect the mechanism of this process by modifying extrusion components directly. Some discussion of Rollins et al., 1999 on the discovery of Nipped-B and its role in enhancer-promoter communication should also be made to reconcile their conclusions in the proposed absence of extrusion events.

Reviewer #2 (Public Review):

Summary:

The authors have analyzed ethnogeographic differences in the comorbidity factors, such as a diabetes and heart disease, for the incidences of stroke and whether it leads to mortality.

Strengths:

The idea is interesting and data are compelling. The results are technically solid.

The authors identify specific genetic loci that increase the risk of a stroke and how they differ by region.

Weaknesses:

The presentation is not focused. It would be better to include p-values and focus presentation on the main effects from the dataset analysis.

Reviewer #2 (Public Review):

Summary:

This manuscript aims to tackle the antimicrobial resistance through the development of vaccines. Specifically, the authors test the potential of the RSP protein as a vaccine candidate. The RSP protein contains bacterial Ig-like domains that are typically carried in IncHl1 plasmids like R27. The extracellular location of the RSP protein and its role in the conjugation process makes it a good candidate for a vaccine. The authors then use Salmonella carrying an IncHl plasmid to test the efficacy of the RSP protein as a vaccine antigen in providing protection against infection of antibiotic-resistant bacteria carrying the IncHl plasmid. The authors found no differences in total IgG or IgA levels, nor in pro-inflammatory cytokines between immunized and non-immunized mice. They however found differences in specific IgG and IgA, attenuated disease symptoms, and restricted systemic infection.

The manuscript also evaluates the potential use of nanobodies specifically targeting the RSP protein by expressing it in E. coli and evaluating their interference in the conjugation of IncHl plasmids. The authors found that E. coli strains expressing RSP-specific nanobodies bind to Salmonella cells carrying the R27 plasmid thereby reducing the conjugation efficacy of Salmonella.

Strengths:

- The main strength of this manuscript is that it targets the mechanism of transmission of resistance genes carried by any bacterial species, thus making it broad.

- The experimental setup is sound and with proper replication.

Weaknesses:

- The two main experiments, evaluating the potential of the RSP protein and the effects of nanobodies on conjugation, seem as parts of two different and unrelated strategies.

- The survival rates shown in Figure 1A and Figure 3A for Salmonella pHCM1 and non-immunized mice challenged with Salmonella, respectively, are substantially different. In the same figures, the challenge of immunized mice and Salmonella pHCM1 and mice challenged with Salmonella pHCM1 with and without ampicillin are virtually the same. While this is not the only measure of the effect of immunization, the inconsistencies in the resulting survival curves should be addressed by the authors more thoroughly as they can confound the effects found in other parameters, including total and specific IgG and IgA, and pro-inflammatory cytokines.

- Overall the results are inconsistent and provide only partial evidence of the effectiveness of the RSP protein as a vaccine target.

- The conjugative experiments use very long conjugation times, making it harder to asses if the resulting transconjugants are the direct result of conjugation or just the growth of transconjugants obtained at earlier points in time. While this could be assessed from the obtained results, it is not a direct or precise measure.

- While the potential outcomes of these experiments could be applied to any bacterial species carrying this type of plasmids, it is unclear why the authors use Salmonella strains to evaluate it. The introduction does a great job of explaining the importance of these plasmids but falls short in introducing their relevance in Salmonella.

Reviewer #2 (Public Review):

Summary:

The study by Huang and colleagues focuses on GLP-1 producing entero-endocrine (EEC) L-cells and their regulation of GLP-1 production by a mechano-gated ion channel Piezo1. The study describes Piezo1 expression by L-cells and uses an exciting intersectional mouse model (villin to target epithelium and Gcg to target GLP-1-producing cells and others like glucagon-producing pancreatic endocrine cells), which allows L-cell specific Piezo1 knockout. Using this model, they find an impairment of glucose tolerance, increased body weight, reduced GLP-1 content, and changes to the CaMKKbeta-CaMKIV-mTORC1 signaling pathway using a normal diet and then high-fat diet. Piezo1 chemical agonist and intestinal bead implantation reversed these changes and improved the disrupted phenotype. Using primary sorted L-cells and cell model STC-1, they found that stretch and Piezo1 activation increased GLP-1 and altered the molecular changes described above.

Strengths:

This is an interesting study testing a novel hypothesis that may have important mechanistic and translational implications. The authors generated an important intersectional genetics mouse model that allowed them to target Piezo1 L-cells specifically, and the surprising result of impaired metabolism is intriguing.

Weaknesses:

However, there are several critical limitations that require resolution before making the conclusions that the authors make.

(1) A potential explanation for the data, and one that is consistent with existing literature [see for example, PMC5334365, PMC4593481], is that epithelial Piezo1, which is broadly expressed by the GI epithelium, impacts epithelial cell density and survival, and as such, if Piezo1 is involved in L-cell physiology, it may be through regulation of cell density. Thus, it is critical to determine L-cell densities and epithelial integrity in controls and Piezo1 knockouts systematically across the length of the gut, since the authors do not make it clear which gut region contributes to the phenotype they see. Current immunohistochemistry data are not convincing.

(2) Calcium signaling in L-cells is implicated in their typical role of being gut chemo-sensors, and Piezo1 is a calcium channel, so it is not clear whether any calcium-related signaling mechanism would phenocopy these results.

(3) Intestinal bead implantation, while intriguing, does not have clear mechanisms - and is likely to provide a point of intestinal obstruction and dysmotility.

(4) Previous studies, some that are very important, but not cited, contradict the presented results (e.g., epithelial Piezo1 role in insulin secretion) and require reconciliation.

Overall, this study makes an interesting observation but the data are not currently strong enough to support the conclusions.

Reviewer #3 (Public Review):

Summary<br /> This study investigates the roles of faculty hiring and attrition in influencing gender representation in U.S. academia. It uses a comprehensive dataset covering tenured and tenure-track faculty across various fields from 2011 to 2020. The study employs a counterfactual model to assess the impact of hypothetical gender-neutral attrition and projects future gender representation under different policy scenarios. The analysis reveals that hiring has a more significant impact on women's representation than attrition in most fields and highlights the need for sustained changes in hiring practices to achieve gender parity.

The revisions made by the authors have improved the paper.

Strengths<br /> Overall, the manuscript offers significant contributions to understanding gender diversity in academia through its rigorous data analysis and innovative methodology.

The methodology is robust, employing extensive data covering a wide range of academic fields and institutions.

Weaknesses<br /> The primary weakness of the study lies in its focus on U.S. academia, which may limit the generalizability of its findings to other cultural and academic contexts. Additionally, the counterfactual model's reliance on specific assumptions about gender-neutral attrition could affect the accuracy of its projections.

Additionally, the study assumes that whoever disappeared from the dataset is attrition in academia. While in reality, those attritions could be researchers who moved to another country or another institution that is not indexed by AA.

Reviewer #2 (Public Review):

This is an excellent and timely study from the Rao lab investigating the interactions of enteric glia with the intestinal epithelium. Two early studies in the late 1990s and early 2000s had previously suggested that enteric glia play a pivotal role in control of the intestinal epithelial barrier, as their ablation using mouse models resulted in severe and fatal intestinal inflammation. However, it was later identified that these inflammatory effects could have been an indirect product of the transgenic mouse models used, rather than due to the depletion of enteric glia. In previous studies from this lab, the authors had identified expression of PLP1 in enteric glia, and its use in CRE driver lines to label and ablate enteric glia.

In the current paper, the authors carefully examine the role of enteric glia by first identifying that PLP1-creERT2 is the most useful driver to direct enteric glial ablation, in terms of the number of glial cells targeted, their proximity to the intestinal epithelium, and the relevance for human studies (GFAP expression is rather limited in human samples in comparison). They examined gene expression changes in different regions of the intestine using bulk RNA-seq following ablation of enteric glia by driving expression of diphtheria toxin A (PLP1-creERT2;Rosa26-DTA). Alterations in gene expression were observed in different regions of the gut, with specific effects in different regions. Interestingly, while there were gene expression changes in the epithelium, there were limited changes to the proportions of different epithelial cell types identified using immunohistochemistry in control vs glial-ablated mice. The authors then focused on the investigation of Paneth cells in the ileum, identifying changes in the ultrastructural morphology and lysozyme activity. In addition, they identified alterations in gut microbiome diversity. As Paneth cells secrete antimicrobial peptides, the authors conclude that the changes in gut microbiome are due to enteric glia-mediated impacts on Paneth cell activity.

Overall, the study is excellent and delves into the different possible mechanisms of action, including the investigation of changes in enteric cholinergic neurons innervating the intestinal crypts. The use of different CRE drivers to target enteric glial cells has led to varying results in the past, and the authors should be commended on how they address this in the Discussion.

Reviewer #2 (Public Review):

Summary:

The authors show convincing data that increasing NK cell function/frequency can reduce the development and progression of metastatic disease after primary tumor resection.

Strengths:

The inclusion of a first-in-human trial highlighting some partial responses of metastatic patients treated with in vitro expanded NK cells is tantalising. It is difficult to perform trials in preventing further metastasis since the timelines are very protracted. However, more data like these that highlight the role of NK cells in improving local cDC1/T cells anti-tumor immunity will encourage deeper thinking around therapeutic approaches to target endogenous NK cells to achieve the same.

Weaknesses:

As always, more patient data would help increase confidence in the human relevance of the approach.

Reviewer #2 (Public Review):

Summary:

In "Assessment of the Epigenomic Landscape in Human Myometrium at Term Pregnancy" the authors generate a number of genome-wide data sets to investigate epigenomic and transcriptomic regulation of the myometrium at term pregnancy. These data provide a useful resource for further evaluation of gene regulatory mechanisms in the myometrium and include the first Hi-C data published for this tissue. There is a comprehensive comparison to previously published histone modification data and integration with RNA-seq to highlight potential enhancer-gene regulatory relationships. The authors further investigate putative enhancers upstream of the PLCL2 gene and identify a candidate region that may be regulated by the PGR (progesterone receptor) signaling.

Strengths:

The strengths of this study are in the multi-omics nature of the design as several genome-wide data sets are generated from the same patient samples. Extending this type of approach in the future to a larger number of samples will allow for additional investigation into gene regulation as the correlation between epigenomic features and gene expression across a larger number of samples can reveal regulatory relationships.

Weaknesses:

One of the most interesting aspects of this study is the generation of the first Hi-C data for the human pregnant myometrium, however, there is a minimal description in the results section of the Hi-C data analysis and the only data shown are the number of loops identified and one such loop that includes the PLCL2 promoter shown in Figure 3A. The manuscript would benefit from a more extensive analysis of the Hi-C data, for example, the analysis of TADs (topological associating domains) would be interesting to add and could be used to evaluate to what extent H3K27ac domains and putative regulated genes fall within the same TAD.

The authors present some convincing evidence on the transcriptional regulation of the PLCL2 gene using Perturb-Seq to identify putative upstream enhancer regions and PGR over-expression showing PGR can act as an activator. These two experiments on their own are interesting, however, they are not as mechanistically integrated as they could be to clarify the molecular mechanisms. Deletion of the putative enhancer upstream of PLCL2 followed by over-expression of PGR would clarify the mechanistic relationship between the proposed enhancer, PGR, and PLCL2 expression. Does PGR act through the proposed enhancer? In addition, reporter assays using this proposed enhancer region with and without increased expression of PGR and mutation of any PRE sequences would also provide mechanistic insight. Although CRISPRa and Perturb-Seq can be used to identify potential regulatory regions, the best approach to verify the requirement for a particular enhancer in regulating a specific gene is a deletion approach.

Reviewer #2 (Public Review):

This is a well-presented large analysis from the UK Biobank of nearly 250,000 female adults. The authors examined the associations of breast cancer diagnosis with incident myocardial infarction and heart failure by different onset age groups. Based on results from a series of statistical analyses, the authors concluded that younger onset age of breast cancer was associated with myocardial infarction and heart failure, highlighting the necessity of careful monitoring of cardiovascular status in women diagnosed with breast cancer, especially those younger ones.

Comments to consider:

(1) It's thoughtful for the authors to have included and adjusted for menopausal status, breast cancer surgery, and hormone replacement therapy in their sensitivity analysis. It would be informative if the authors presented the number and percentages of menopause and cancer treatments.

(2) The analytical baseline used for follow-up should be pointed out in the methods section. It's confusing whether the analytic baseline was defined as the study baseline or the time at breast cancer diagnosis.

(3) Did the older onset age group have a longer follow-up duration? Could the authors provide information on the length of follow-up by age of onset in Supplementary Table S4? It would give the readers more information regarding different age groups.

Reviewer #2 (Public Review):

Summary:

Meiotic recombination initiates with the formation of DNA double-strand break (DSB) formation, catalyzed by the conserved topoisomerase-like enzyme Spo11. Spo11 requires accessory factors that are poorly conserved across eukaryotes. Previous genetic studies have identified several proteins required for DSB formation in C. elegans to varying degrees; however, how these proteins interact with each other to recruit the DSB-forming machinery to chromosome axes remains unclear.

In this study, Raices et al. characterized the biochemical and genetic interactions among proteins that are known to promote DSB formation during C. elegans meiosis. The authors examined pairwise interactions using yeast two-hybrid (Y2H) and co-immunoprecipitation and revealed an interaction between a chromatin-associated protein HIM-17 and a transcription factor XND-1. They further confirmed the previously known interaction between DSB-1 and SPO-11 and showed that DSB-1 also interacts with a nematode-specific HIM-5, which is essential for DSB formation on the X chromosome. They also assessed genetic interactions among these proteins, categorizing them into four epistasis groups by comparing phenotypes in double vs. single mutants. Combining these results, the authors proposed a model of how these proteins interact with chromatin loops and are recruited to chromosome axes, offering insights into the process in C. elegans compared to other organisms.

Weaknesses:

This work relies heavily on Y2H, which is notorious for having high rates of false positives and false negatives. Although the interactions between HIM-17 and XND-1 and between DSB-1 and HIM-5 were validated by co-IP, the significance of these interactions was not tested, and cataloging Y2H interactions does not yield much more insight. Moreover, most experiments lack rigor, which raises serious concerns about whether the data convincingly supports the conclusions of this paper. For instance, the XND-1 antibody appears to detect a band in the control IP; however, there was no mention of the specificity of this antibody. Additionally, epistasis analysis of various genetic mutants is based on the quantification of DAPI bodies in diakinesis oocytes, but the comparisons were made without statistical analyses. For cytological data, a single representative nucleus was shown without quantification and rigorous analysis. The rationale for some experiments is also questionable (e.g. the rescue by dsb-2 mutants by him-5 transgenes in Figure 2), making the interpretation of the data unclear. Overall, while this paper claims to present "the first comprehensive model of DSB regulation in a metazoan", cataloging Y2H and genetic interactions did not yield any new insights into DSB formation without rigorous testing of their significance in vivo. The model proposed in Figure 4 is also highly speculative.

Reviewer #2 (Public Review):

The paper by Yan et al. aims to provide evidence for horizontal transmission of the intracellular bacterial symbiont Wolbachia from parasitoid wasps to their whitefly hosts. In my opinion, the paper in its current form consists of major flaws.

Weaknesses:

The dogma in the field is that although horizontal transmission events of Wolbachia occur, in most systems they are so rare that the chances of observing them in the lab are very slim.<br /> For the idea of bacteria moving from a parasitoid to its host, the authors have rightfully cited the paper by Hughes, et al. (2001), which presents the main arguments against the possibility of documenting such transmissions. Thus, if the authors want to provide data that contradict the large volume of evidence showing the opposite, they should present a very strong case.

In my opinion, the paper fails to provide such concrete evidence. Moreover, it seems the work presented does not meet the basic scientific standards.

My main reservations are:

- I think the distribution pattern of bacteria stained by the probes in the FISH pictures presented in Figure 4 looks very much like Portiera, the primary symbiont found in the bacterium of all whitefly species. In order to make a strong case, the authors need to include Portiera probes along with the Wolbachia ones.

- If I understand the methods correctly, the phylogeny presented in Figure 2a is supposed to be based on a wide search for Wolbachia wsp gene done on the NCBI dataset (p. 348). However, when I checked the origin of some of the sequences used in the tree to show the similarity of Wolbachia between Bemisia tabaci and its parasitoids, I found that most of them were deposited by the authors themselves in the course of the current study (I could not find this mentioned in the text), or originated in a couple of papers that in my opinion should not have been published to begin with.

- The authors fail to discuss or even acknowledge a number of published studies that specifically show no horizontal transmission, such as the one claimed to be detected in the study presented.

Reviewer #2 (Public Review):

Summary

Le Roy et al quantify wing morphology and wing kinematics across eight hoverfly species that differ in body mass; the aim is to identify how weight support during hovering is ensured. Wing shape and relative wing size vary significantly with body mass, but wing kinematics are reported to be size-invariant. On the basis of these results, it is concluded that weight support is achieved solely through size-specific variations in wing morphology and that these changes enabled hoverflies to decrease in size throughout their phylogenetic history. Adjusting wing morphology may be preferable compared to the alternative strategy of altering wing kinematics, because kinematics may be under strong evolutionary and ecological constraints, dictated by the highly specialised flight and ecology of the hoverflies.

Strengths

The study deploys a vast array of challenging techniques, including flight experiments, morphometrics, phylogenetic analysis, and numerical simulations; it so illustrates both the power and beauty of an integrative approach to animal biomechanics. The question is well motivated, the methods appropriately designed, and the discussion elegantly and convincingly places the results in broad biomechanical, ecological, evolutionary, and comparative contexts.

Weaknesses

(1) In assessing evolutionary allometry, it is key to identify the variation expected from changes in size alone. The null hypothesis for wing morphology is well-defined (isometry), but the equivalent predictions for kinematic parameters remain unclear. Explicit and well-justified null hypotheses for the expected size-specific variation in angular velocity, angle-of-attack, stroke amplitude, and wingbeat frequency would substantially strengthen the paper, and clarify its evolutionary implications.

(2) By relating the aerodynamic output force to wing morphology and kinematics, it is concluded that smaller hoverflies will find it more challenging to support their body mass - a scaling argument that provides the framework for this work. This hypothesis appears to stand in direct contrast to classic scaling theory, where the gravitational force is thought to present a bigger challenge for larger animals, due to their disadvantageous surface-to-volume ratios. The same problem ought to occur in hoverflies, for wing kinematics must ultimately be the result of the energy injected by the flight engine: muscle. Much like in terrestrial animals, equivalent weight support in flying animals thus requires a positive allometry of muscle force output. In other words, if a large hoverfly is able to generate the wing kinematics that suffice to support body weight, an isometrically smaller hoverfly should be, too (but not vice versa). Clarifying the relation between the scaling of muscle force input, wing kinematics, and weight support would resolve the conflict between these two contrasting hypotheses, and considerably strengthen the biomechanical motivation and interpretation.

(3) The main conclusion - that evolutionary miniaturization is enabled by changes in wing morphology - is only weakly supported by the evidence. First, although wing morphology deviates from the null hypothesis of isometry, the difference is small, and hoverflies about an order of magnitude lighter than the smallest species included in the study exist. Including morphological data on these species, likely accessible through museum collections, would substantially enhance the confidence that size-specific variation in wing morphology occurs not only within medium-sized but also in the smallest hoverflies, and has thus indeed played a key role in evolutionary miniaturization. Second, although wing kinematics do not vary significantly with size, clear trends are visible; indeed, the numerical simulations revealed that weight support is only achieved if variations in wing beat frequency across species are included. A more critical discussion of both observations may render the main conclusions less clear-cut, but would provide a more balanced representation of the experimental and computational results.

In many ways, this work provides a blueprint for work in evolutionary biomechanics; the breadth of both the methods and the discussion reflects outstanding scholarship. It also illustrates a key difficulty for the field: comparative data is challenging and time-consuming to procure, and behavioural parameters are characteristically noisy. Major methodological advances are needed to obtain data across large numbers of species that vary drastically in size with reasonable effort, so that statistically robust conclusions are possible.

Reviewer #2 (Public Review):

Summary:

Protein kinases have been very successfully targeted with small molecules for several decades, with many compounds (including clinical drugs) bringing about conformational changes that are also relevant to broader interactions with the cellular signaling networks that they control. The authors set out to develop a targeted biosensor approach to evaluate distinct kinase conformations in cells for multiple kinases in the context of incoming signals, other proteins and small molecule binding, with a broad goal of using the KinCon assay to confirm (and perhaps predict) how drug binding or signal perception changes conformations and outputs in the presence of cellular complexes; this work will likely impact on the field with cellular reporters of kinase conformations a useful addition to the toolbox.

Strengths:

The KinCon reporter platform has previously been validated for well-known kinases; in this study, the team evaluate how to employ a full-length kinase (often containing a known pathological mutation). The sensitive detection method is based on a Renilla luciferase (RLuc)protein fragment complementation assay, where individual RLuc fragments are present at the N and the C terminus of the kinase. This report, which is both technical and practical in nature, co-expresses the kinase with known interactors (at low levels) in a high throughput format and then performs pharmacological evaluation with known small molecule kinase modulators. This is explained nicely in Figure 1, as are the signaling pathways that are being evaluated. Data demonstrate that V600E BRAF iexposed to vemurafenib is converted to the inactive conformation, as expected. In contrast, the more closed STRAD𝛼 and LKB1 KinCon conformations appear to represent the more active state of the complexed kinase, and a W308C mutation (evaluated alongside others) reverses this effect. The authors then evaluated necroptotic signaling in the context of RIPK1/3 under conditions where RIPK1 and RIPK3 are active, confirming that the reporters highlight the active states of both kinases. Exposure to compounds that are known to engage with the RIPK1 arm of the pathway induce bioluminescence changes consistent with the opening (inactivation) of the kinase. Finally, the authors move to an important drug target for which clinical drugs have arrived relatively recently; the CDK4/6 complexes. These are of additional importance because kinase-independent functions also exist for CDK6, and the effects of drugs in cells usually relies on a downstream marker, rather than demonstration of direct protein complex engagement. The data presented are interpreted as the formation of complexes with the CDK inhibitor p16INK4a; reducing the affinity of the interaction through mutations drives an inactive conformation, whilst the application of CDK4/6 inhibitors does not, implying binding to the active conformation.

Weaknesses:

(1) The work is very solid, and uses examples from the literature and also extends into new experimental space. An obvious weakness is mentioned by the authors for the CKDK data, in that measurements with Cyclin D (the activating subunit) are not characterised, although Cyclin D might be assumed to be present?<br /> (2) The work with the trimeric LKB1 complex involves pseudokinase, STRADalpha, whose conformation is also examined as a function of LKB1 status; since STRAD is an activator of LKB1, a future goal should be the evaluation of the complex in the presence of STRAD inhibitory/activating small molecules.

Reviewer #2 (Public Review):

Summary:

Schmid et al. provide details of their new data management tool Poseidon which is intended to standardise archaeogenetic genotype data and combine it with the associated standardised metadata, including bibliographic references, in a way that conforms to FAIR principles. Poseidon also includes tools to perform standard analyses of genotype files, and the authors pitch it as the potential first port of call for researchers who are planning on using archaeogenetic data in their research. In fact, Poseidon is already up and running and being used by researchers working in ancient human population genetics. To some extent, it is already on its way to becoming a fundamental resource.

Strengths:

A similar ancient genomics resource (The Ancient Allen Database) exists, but Poseidon is several steps ahead in terms of integration and standardisation of metadata, its intrinsic analytical tools, its flexibility, and its ambitions towards being independent and entirely community-driven. It is clear that a lot of thought has gone into each aspect of what is a large and dynamic package of tools and overall it is systematic and well thought through.

Weaknesses:

The main weakness of the plans for Poseidon, which admirably the authors openly acknowledge, is in how to guarantee it is maintained and updated over the long term while also shifting to a fully independent model. The software is currently hosted by the MPI, although the authors do set out plans to move it to a more independent venue. However, the core team comprising the authors is funded by the MPI, and so the MPI is also the main funder of Poseidon. The authors do state their ambition to move towards a community-driven independent model, but the details of how this would happen are a bit vague. The authors imagine that authors of archaeogenetic papers would upload data themselves, thereby making all authors of archaeogenetics papers the voluntary community who would take on the responsibility of maintaining Poseidon. Archaeogeneticists generally are committed enough to their field that there is a good chance such a model would work but it feels haphazard to rely on goodwill alone. Given there needs to be a core team involved in maintaining Poseidon beyond just updating the database, from the paper as it stands it is difficult to see how Poseidon might be weaned off MPI funding/primary involvement and what the alternative is. However, the same anxieties always surround these sorts of resources when they are first introduced. The main aim of the paper is to introduce and explain the resource rather than make explicit plans for its future and so this is a minor weakness of the paper overall.

Reviewer #2 (Public Review):

Summary:

Zhang et al. analyzed the functional role of hepatocyte RIPK1 during metabolic stress, particularly its scaffold function rather than kinase function. They show that Ripk1 knockout sensitizes the liver to cell death and inflammation in response to short-term fasting, a condition that would not induce obvious abnormality in wild-type mice.

Strengths:

The findings are based on a knockout mouse model and supported by bulk RNA-seq and scRNA-seq. The work consolidates the complex role of RIPK1 in metabolic stress.

Weaknesses:

However, the findings are not novel enough because the pro-survival role of RIPK1 scaffold is well-established and several similar pieces of research already exist. Moreover, the mechanism is not very clear and needs additional experiments.

Reviewer 2 (Public Review):

Accumulating data suggests that the presence of immune cell infiltrates in the meninges of the multiple sclerosis brain contributes to the tissue damage in the underlying cortical grey matter by the release of inflammatory and cytotoxic factors that diffuse into the brain parenchyma. However, little is known about the identity and direct and indirect effects of these mediators at a molecular level. This study addresses the vital link between an adaptive immune response in the CSF space and the molecular mechanisms of tissue damage that drive clinical progression. In this short report the authors use a spatial transcriptomics approach using Visium Gene Expression technology from 10x Genomics, to identify gene expression signatures in the meninges and the underlying brain parenchyma, and their interrelationship, in the PLP-induced EAE model of MS in the SJL mouse. MRI imaging using a high field strength (11.7T) scanner was used to identify areas of meningeal infiltration for further study. They report, as might be expected, the upregulation of genes associated with the complement cascade, immune cell infiltration, antigen presentation, and astrocyte activation. Pathway analysis revealed the presence of TNF, JAK-STAT and NFkB signaling, amongst others, close to sites of meningeal inflammation in the EAE animals, although the spatial resolution is insufficient to indicate whether this is in the meninges, grey matter, or both.

UMAP clustering illuminated a major distinct cluster of upregulated genes in the meninges and smaller clusters associated with the grey matter parenchyma underlying the infiltrates. The meningeal cluster contained genes associated with immune cell functions and interactions, cytokine production, and action. The parenchymal clusters included genes and pathways related to glial activation, but also adaptive/B-cell mediated immunity and antigen presentation. This again suggests a technical inability to resolve fully between the compartments as immune cells do not penetrate the pial surface in this model or in MS. Finally, a trajectory analysis based on distance from the meningeal gene cluster successfully demonstrated descending and ascending gradients of gene expression, in particular a decline in pathway enrichment for immune processes with distance from the meninges.

Comments on revised version:

The authors have addressed all of my comments regarding the lack of spatial resolution between the grey matter and the overlying meninges and also concerning the difficulties in extrapolating from this mouse model to MS itself.<br /> I am however very concerned about the lack of the correct control group. Immunization of rodents with complete freunds adjuvant (albeit with pertussis toxin) gives rise to widespread microglial activation, some immune cell infiltration and also structural changes to axons, particularly at nodes of Ranvier (https://doi.org/10.1097/NEN.0b013e3181f3a5b1). This will inevitably make it difficult to interpret the transcriptomics results, depending on whether these changes are reversible or not and the time frame of the reversal. In the C57Bl6 EAE models adjuvant induced microglial activation becomes chronic, whereas the axonal changes do reverse by 10 weeks. Whether this is the same in SJL EAE model using CFA alone is not clear.

Reviewer #2 (Public Review):

Summary of goals:

Untranslated regions are key cis-regulatory elements that control mRNA stability, translation, and translocation. Through interactions with small RNAs and RNA binding proteins, UTRs form complex transcriptional circuitry that allows cells to fine-tune gene expression. Functional annotation of UTR variants has been very limited, and improvements could offer insights into disease relevant regulatory mechanisms. The goals were to advance our understanding of the determinants of UTR regulatory elements and characterize the effects of a set of "disease-relevant" UTR variants.

Strengths:

The use of a massively parallel reporter assay allowed for analysis of a substantial set (6,555 pairs) of 5' and 3' UTR fragments compiled from known disease associated variants. Two cell types were used.

The findings confirm previous work about the importance of AREs, which helps show validity and adds some detailed comparisons of specific AU-rich motif effects in these two cell types.

Using a Lasso regression, TA-dinucleotide content is identified as a strong regulator of RNA stability in a context dependent manner based on GC content and presence of RNA binding protein binding motifs. The findings have potential importance, drawing attention to a UTR feature that is not well characterized.

The use of complementary datasets, including from half-life analyses of RNAs and from random sequence library MRPA's, is a useful addition and supports several important findings. The finding the TA dinucleotides have explanatory power separate from (and in some cases interacting with) GC content is valuable.

The functional enrichment analysis suggests some new ideas about how UTRs may contribute to regulation of certain classes of genes.

Weaknesses:

It is difficult to understand how the calculations for half-life were performed. The sequencing approach measures the relative frequency of each sequence at each time point (less stable sequences become relatively less frequent after time 0, whereas more stable sequences become relatively more frequent after time 0). Since there is no discussion of whether the abundance of the transfected RNA population is referenced to some external standard (e.g., housekeeping RNAs), it is not clear how absolute (rather than relative) half-lives were determined.

Fig. S1A and B are used to assess reproducibility. They show that read counts at a given time point correlate well across replicate experiments. However, this is not a good way to assess reproducibility or accuracy of the measurements of t1/2 are. (The major source of variability in read counts in these plots - especially at early time points - is likely the starting abundance of each RNA sequence, not stability.) This creates concerns about how well the method is measuring t1/2. Also creating concern is the observation that many RNAs are associated with half-lives that are much longer than the time points analyzed in the study. For example, based upon Figure S1 and Table S1 correctly, the median t1/2 for the 5' UTR library in HEK cells appears to be >700 minutes. Given that RNA was collected at 30, 75, and 120 minutes, accurate measurements of RNAs with such long half lives would seem to be very difficult.

There is no direct comparison of t1/2 between the two cell types studied for the full set of sequences studied. This would be helpful in understanding whether the regulatory effects of UTRs are generally similar across cell lines (as has been shown in some previous studies) or whether there are fundamental differences. The distribution of t1/2's is clearly quite different in the two cell lines, but it is important to know if this reflects generally slow RNA turnover in HEK cells or whether there are a large number of sequence-specific effects on stability between cell lines. A related issue is that it is not clear whether the relatively small number of significant variant effects detected in HEK cells versus SH-SY5Y cells is attributable to real biological differences between cell types or to technical issues (many fewer read counts and much longer half lives in HEK cells).

The general assertion is made in many places that TA dinucleotides are the most prominent destabilizing element in UTRs (e.g., in the title, the abstract, Fig. 4 legend, and on p. 12). This appears to be true for only one of the two cell lines tested based on Fig. 3.

Appraisal and impact:

The work adds to existing studies that previously identified sequence features, including AREs and other RNA binding protein motifs, that regulate stability and puts a new emphasis on the role of "TA" (better "UA") dinucleotides. It is not clear how potential problems with the RNA stability measurements discussed above might influence the overall conclusions, which may limit the impact unless these can be addressed.

It is difficult to understand whether the importance of TA dinucleotides is best explained by their occurrence in a related set of longer RBP binding motifs (see Fig 5J, these motifs may be encompassed by the "WWWWWW cluster") or whether some other explanation applies. Further discussion of this would be helpful. Does the LASSO method tend to collapse a more diverse set of longer motifs that are each relatively rare compared to the dinucleotide? It remains unclear whether TA dinucleotides are associated with less stability independent of the presence of the known larger WWWWWWW motif. As noted above, the importance of TA dinucleotides in the HEK experiments appears to be less than is implied in the text.

The inclusion of more than a single cell type is an acknowledgement of the importance of evaluating cell type-specific effects. The work suggests a number of cell type-specific differences, but due to technical issues (especially with the HEK data, as outlined above) and the use of only two cell lines, it is difficult to understand cell type effects from the work.

The inclusion of both 3' and 5' UTR sequences distinguishes this work from most prior studies in the field. Contrasting the effects of these regions on stability is of interest, although the role of these UTRs (especially the 5' UTR) in translational regulation is not assessed here.

Reviewer #2 (Public Review):

Summary:

This work by Bimbard et al., introduces a new implant for Neuropixels probes. While Neuropixels probes have critically improved and extended our ability to record the activity of a large number of neurons with high temporal resolution, the use of these expensive devices in chronic experiments has so far been hampered by the difficulty of safely implanting them and, importantly, to explant and reuse them after conclusion of the experiment. The authors present a newly designed two-part implant, consisting of a docking and a payload module, that allows for secure implantation and straightforward recovery of the probes. The implant is lightweight, making it amenable for use in mice and rats, and customizable. The authors provide schematics and files for printing of the implants, which can be easily modified and adapted to custom experiments by researchers with little to no design experience. Importantly, the authors demonstrate the successful use of this implant across multiple use cases, in head-fixed and freely moving experiments, in mice and rats, with different versions of Neuropixels probes, and across 8 different labs. Taken together, the presented implants promise to make chronic Neuropixel recordings and long-term studies of neuronal activity significantly easier and attainable for both current and future Neuropixels users.

Strengths:

- The implants have been successfully tested across 8 different laboratories, in mice and rats, in head-fixed and freely moving conditions, and have been adapted in multiple ways for a number of distinct experiments.

- Implants are easily customizable and the authors provide a straightforward approach for customization across multiple design dimensions even for researchers not experienced in design.

- The authors provide clear and straightforward descriptions of the construction, implantation, and explant of the described implants.

- The split of the implant into a docking and payload module makes reuse even in different experiments (using different docking modules) easy.

- The authors demonstrate that implants can be re-used multiple times and still allow for high-quality recordings.

- The authors show that the chronic implantations allow for the tracking of individual neurons across days and weeks (using additional software tracking solutions), which is critical for a large number of experiments requiring the description of neuronal activity, e.g. throughout learning processes.

- The authors show that implanted animals can even perform complex behavioral tasks, with no apparent reduction in their performance.

Weaknesses:

- While implanted animals can still perform complex behavioral tasks, the authors describe that the implants may reduce the animals' mobility, as measured by prolonged reaction times. However, the presented data does not allow us to judge whether this effect is specifically due to the presented implant or whether any implant or just tethering of the animals per se would have the same effects.

- While the authors make certain comparisons to other, previously published approaches for chronic implantation and re-use of Neuropixels probes, it is hard to make conclusive comparisons and judge the advantages of the current implant. For example, while the authors emphasize that the lower weight of their implant allows them to perform recordings in mice (and is surely advantageous), the previously described, heavier implants they mention (Steinmetz et al., 2021; van Daal et al., 2021), have also been used in mice. Whether the weight difference makes a difference in practice therefore remains somewhat unclear.

- The non-permanent integration of the headstages into the implant, while allowing for the use of the same headstage for multiple animals in parallel, requires repeated connections and does not provide strong protection for the implant. This may especially be an issue for the use in rats, requiring additional protective components as in the presented rat experiments.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors follow up on their previous work showing that in the absence of the Sir2 deacetylase the MCM replicative helicase at the rDNA spacer region is repositioned to a region of low nucleosome occupancy. Here they show that the repositioned displaced MCMs have increased firing propensity relative to non-displaced MCMs. In addition, they show that activation of the repositioned MCMs and low nucleosome occupancy in the adjacent region depend on the chromatin remodeling activity of Fun30.

Strengths:

The paper provides new information on the role of a conserved chromatin remodeling protein in the regulation of origin firing and in addition provides evidence that not all loaded MCMs fire and that origin firing is regulated at a step downstream of MCM loading.

Weaknesses:

The relationship between the author's results and prior work on the role of Sir2 (and Fob1) in regulation of rDNA recombination and copy number maintenance is not explored, making it difficult to place the results in a broader context. Sir2 has previously been shown to be recruited by Fob1, which is also required for DSB formation and recombination-mediated changes in rDNA copy number. Are the changes that the authors observe specifically in fun30 sir2 cells related to this pathway? Is Fob1 required for the reduced rDNA copy number in fun30 sir2 double mutant cells?

Reviewer #2 (Public Review):

In the present study, Boffi et al. investigate the manner in which the dorsal cortex of the of the inferior colliculus (DCIC), an auditory midbrain area, encodes sound location azimuth in awake, passively listening mice. By employing volumetric calcium imaging (scanned temporal focusing or s-TeFo), complemented with high-density electrode electrophysiological recordings (neuropixels probes), they show that sound-evoked responses are exquisitely noisy, with only a small portion of neurons (units) exhibiting spatial sensitivity. Nevertheless, a naïve Bayesian classifier was able to predict the presented azimuth based on the responses from small populations of these spatially sensitive units. A portion of the spatial information was provided by correlated trial-to-trial response variability between individual units (noise correlations). The study presents a novel characterization of spatial auditory coding in a non-canonical structure, representing a noteworthy contribution specifically to the auditory field and generally to systems neuroscience, due to its implementation of state-of-the-art techniques in an experimentally challenging brain region. However, nuances in the calcium imaging dataset and the naïve Bayesian classifier warrant caution when interpreting some of the results.

Strengths:<br /> The primary strength of the study lies in its methodological achievements, which allowed the authors to collect a comprehensive and novel dataset. While the DCIC is a dorsal structure, it extends up to a millimetre in depth, making it optically challenging to access in its entirety. It is also more highly myelinated and vascularised compared to e.g., the cerebral cortex, compounding the problem. The authors successfully overcame these challenges and present an impressive volumetric calcium imaging dataset. Furthermore, they corroborated this dataset with electrophysiological recordings, which produced overlapping results. This methodological combination ameliorates the natural concerns that arise from inferring neuronal activity from calcium signals alone, which are in essence an indirect measurement thereof.

Another strength of the study is its interdisciplinary relevance. For the auditory field, it represents a significant contribution to the question of how auditory space is represented in the mammalian brain. "Space" per se is not mapped onto the basilar membrane of the cochlea and must be computed entirely within the brain. For azimuth, this requires the comparison between miniscule differences between the timing and intensity of sounds arriving at each ear. It is now generally thought that azimuth is initially encoded in two, opposing hemispheric channels, but the extent to which this initial arrangement is maintained throughout the auditory system remains an open question. The authors observe only a slight contralateral bias in their data, suggesting that sound source azimuth in the DCIC is encoded in a more nuanced manner compared to earlier processing stages of the auditory hindbrain. This is interesting, because it is also known to be an auditory structure to receive more descending inputs from the cortex.

Systems neuroscience continues to strive for the perfection of imaging novel, less accessible brain regions. Volumetric calcium imaging is a promising emerging technique, allowing the simultaneous measurement of large populations of neurons in three dimensions. But this necessitates corroboration with other methods, such as electrophysiological recordings, which the authors achieve. The dataset moreover highlights the distinctive characteristics of neuronal auditory representations in the brain. Its signals can be exceptionally sparse and noisy, which provide an additional layer of complexity in the processing and analysis of such datasets. This will be undoubtedly useful for future studies of other less accessible structures with sparse responsiveness.

Weaknesses:<br /> Although the primary finding that small populations of neurons carry enough spatial information for a naïve Bayesian classifier to reasonably decode the presented stimulus is not called into question, certain idiosyncrasies, in particular the calcium imaging dataset and model, complicate specific interpretations of the model output, and the readership is urged to interpret these aspects of the study's conclusions with caution.

I remain in favour of volumetric calcium imaging as a suitable technique for the study, but the presently constrained spatial resolution is insufficient to unequivocally identify regions of interest as cell bodies (and are instead referred to as "units" akin to those of electrophysiological recordings). It remains possible that the imaging set is inadvertently influenced by non-somatic structures (including neuropil), which could report neuronal activity differently than cell bodies. Due to the lack of a comprehensive ground-truth comparison in this regard (which to my knowledge is impossible to achieve with current technology), it is difficult to imagine how many informative such units might have been missed because their signals were influenced by spurious, non-somatic signals, which could have subsequently misled the models. The authors reference the original Nature Methods article (Prevedel et al., 2016) throughout the manuscript, presumably in order to avoid having to repeat previously published experimental metrics. But the DCIC is neither the cortex nor hippocampus (for which the method was originally developed) and may not have the same light scattering properties (not to mention neuronal noise levels). Although the corroborative electrophysiology data largely eleviates these concerns for this particular study, the readership should be cognisant of such caveats, in particular those who are interested in implementing the technique for their own research.

A related technical limitation of the calcium imaging dataset is the relatively low number of trials (14) given the inherently high level of noise (both neuronal and imaging). Volumetric calcium imaging, while offering a uniquely expansive field of view, requires relatively high average excitation laser power (in this case nearly 200 mW), a level of exposure the authors may have wanted to minimise by maintaining a low the number of repetitions, but I yield to them to explain. Calcium imaging is also inherently slow, requiring relatively long inter-stimulus intervals (in this case 5 s). This unfortunately renders any model designed to predict a stimulus (in this case sound azimuth) from particularly noisy population neuronal data like these as highly prone to overfitting, to which the authors correctly admit after a model trained on the entire raw dataset failed to perform significantly above chance level. This prompted them to feed the model only with data from neurons with the highest spatial sensitivity. This ultimately produced reasonable performance (and was implemented throughout the rest of the study), but it remains possible that if the model was fed with more repetitions of imaging data, its performance would have been more stable across the number of units used to train it. (All models trained with imaging data eventually failed to converge.) However, I also see these limitations as an opportunity to improve the technology further, which I reiterate will be generally important for volume imaging of other sparse or noisy calcium signals in the brain.

Transitioning to the naïve Bayesian classifier itself, I first openly ask the authors to justify their choice of this specific model. There are countless types of classifiers for these data, each with their own pros and cons. Did they actually try other models (such as support vector machines), which ultimately failed? If so, these negative results (even if mentioned en passant) would be extremely valuable to the community, in my view. I ask this specifically because different methods assume correspondingly different statistical properties of the input data, and to my knowledge naïve Bayesian classifiers assume that predictors (neuronal responses) are assumed to be independent within a class (azimuth). As the authors show that noise correlations are informative in predicting azimuth, I wonder why they chose a model that doesn't take advantage of these statistical regularities. It could be because of technical considerations (they mention computing efficiency), but I am left generally uncertain about the specific logic that was used to guide the authors through their analytical journey.

That aside, there remain other peculiarities in model performance that warrant further investigation. For example, what spurious features (or lack of informative features) in these additional units prevented the models of imaging data from converging? In an orthogonal question, did the most spatially sensitive units share any detectable tuning features? A different model trained with electrophysiology data in contrast did not collapse in the range of top-ranked units plotted. Did this model collapse at some point after adding enough units, and how well did that correlate with the model for the imaging data? How well did the form (and diversity) of the spatial tuning functions as recorded with electrophysiology resemble their calcium imaging counterparts? These fundamental questions could be addressed with more basic, but transparent analyses of the data (e.g., the diversity of spatial tuning functions of their recorded units across the population). Even if the model extracts features that are not obvious to the human eye in traditional visualisations, I would still find this interesting.

Finally, the readership is encouraged to interpret certain statements by the authors in the current version conservatively. How the brain ultimately extracts spatial neuronal data for perception is anyone's guess, but it is important to remember that this study only shows that a naïve Bayesian classifier could decode this information, and it remains entirely unclear whether the brain does this as well. For example, the model is able to achieve a prediction error that corresponds to the psychophysical threshold in mice performing a discrimination task (~30 {degree sign}). Although this is an interesting coincidental observation, it does not mean that the two metrics are necessarily related. The authors correctly do not explicitly claim this, but the manner in which the prose flows may lead a non-expert into drawing that conclusion. Moreover, the concept of redundancy (of spatial information carried by units throughout the DCIC) is difficult for me to disentangle. One interpretation of this formulation could be that there are non-overlapping populations of neurons distributed across the DCIC that each could predict azimuth independently of each other, which is unlikely what the authors meant. If the authors meant generally that multiple neurons in the DCIC carry sufficient spatial information, then a single neuron would have been able to predict sound source azimuth, which was not the case. I have the feeling that they actually mean "complimentary", but I leave it to the authors to clarify my confusion, should they wish.

In summary, the present study represents a significant body of work that contributes substantially to the field of spatial auditory coding and systems neuroscience. However, limitations of the imaging dataset and model as applied in the study muddles concrete conclusions about how the DCIC precisely encodes sound source azimuth and even more so to sound localisation in a behaving animal. Nevertheless, it presents a novel and unique dataset, which, regardless of secondary interpretation, corroborates the general notion that auditory space is encoded in an extraordinarily complex manner in the mammalian brain.

Reviewer #2 (Public Review):

Summary:

The authors inquire in particular whether the receptor Gpr156, which is necessary for hair cells to reverse their polarities in the zebrafish lateral line and mammalian otolith organs downstream of the differential expression of the transcription factor Emx2, also controls the mechanosensitive properties of hair cells and ultimately an animal's behavior. This study thoroughly addresses the issue by analyzing the morphology, electrophysiological responses, and afferent connections of hair cells found in different regions of the mammalian utricle and the Ca2+ responses of lateral line neuromasts in both wild-type animals and gpr156 mutants. Although many features of hair cell function are preserved in the mutants-such as development of the mechanosensory organs and the Emx2-dependent, polarity-specific afferent wiring and synaptic pairing-there are a few key changes. In the zebrafish neuromast, the magnitude of responses of all hair cells to water flow resembles that of the wild-type hair cells that respond to flow arriving from the tail. These responses are larger than those observed in hair cells that are sensitive to flow arriving from the head and resemble effects previously observed in Emx2 mutants. The authors note that this behavior suggests that the Emx2-GPR156 signaling axis also impinges on hair cell mechanotransduction. Although mutant mice exhibit normal posture and balance, they display defects in swimming behavior. Moreover, their vestibulo-ocular reflexes are perturbed. The authors note that the gpr156 mutant is a good model to study the role of opposing hair cell polarity in the vestibular system, for the wiring patterns follow the expression patterns of Emx2, even though hair cells are all of the same polarity. This paper excels at describing the effects of gpr156 perturbation in mouse and zebrafish models and will be of interest to those studying the vestibular system, hair cell polarity, and the role of inner-ear organs in animal behavior.

Strengths:

The study is exceptional in including, not only morphological and immunohistochemical indices of cellular identity but also electrophysiological properties. The mutant hair cells of murine maculæ display essentially normal mechanoelectrical transduction and adaptation-with two or even three kinetic components-as well as normal voltage-activated ionic currents.

Reviewer #2 (Public Review):

Summary:

Napoli et al. provide a compelling study showing the importance of cytosolic S100A8/9 in maintaining calcium levels at LFA-1 nanoclusters at the cell membrane, thus allowing the successful crawling and adherence of neutrophils under shear stress. The authors show that cytosolic S100A8/9 is responsible for retaining stable and high concentrations of calcium specifically at LFA-1 nanoclusters upon binding to ICAM-1, and imply that this process aids in facilitating actin polymerisation involved in cell shape and adherence. The authors show early on that S100A8/9 deficient neutrophils fail to extravasate successfully into the tissue, thus suggesting that targeting cytosolic S100A8/9 could be useful in settings of autoimmunity/acute inflammation where neutrophil-induced collateral damage is unwanted.

Strengths:

Using multiple complementary methods from imaging to western blotting and flow cytometry, including extracellular supplementation of S100A8/9 in vivo, the authors conclusively prove a defect in intracellular S100A8/9, rather than extracellular S100A8/9 was responsible for the loss in neutrophil adherence, and pinpointed that S100A8/9 aided in calcium stabilisation and retention at the plasma membrane.

Weaknesses:

(1) Extravasation is shown to be a major defect of Mrp14-/- neutrophils, but the Giemsa staining in Figure 1H seems to be quite unspecific to me, as neutrophils were determined by nuclear shape and granularity. It would have perhaps been more clear to use immunofluorescence staining for neutrophils instead as seen in Supplementary Figure 1A (staining for Ly6G or other markers instead of S100A9).

(2) The representative image for Mrp14-/- neutrophils used in Figure 4K to demonstrate Ripley's K function seems to be very different from that shown above in Figures 4C and 4F.

(3) Although the authors have done well to draw a path linking cytosolic S100A8/9 to actin polymerisation and subsequently the arrest and adherence of neutrophils in vitro, the authors can be more explicit with the analysis - for example, is the F-actin co-localized with the LFA-1 nanoclusters? Does S100A8/9 localise to the membrane with LFA-1 upon stimulation? Lastly, I think it would have been very useful to close the loop on the extravasation observation with some in vitro evidence to show that neutrophils fail to extravasate under shear stress.

Reviewer #2 (Public Review):

Summary:

How dynamics of gene expression accompany cell fate and cellular morphological changes is important for our understanding of molecular mechanisms that govern development and diseases. The phenomenon is particularly prominent during spermatogenesis, the process which spermatogonia stem cells develop into sperm through a series of steps of cell division, differentiation, meiosis, and cellular morphogenesis. The intricacy of various aspects of cellular processes and gene expression during spermatogenesis remains to be fully understood. In this study, the authors found that testis-specific actin-related proteins (which usually participate in modifying cells' cytoskeletal systems) ACTL7A and ACTL7B were expressed and localized in the nuclei of mouse spermatocytes and spermatids. Based on this observation, the authors analyzed protein sequence conservations of ACTL7B across dozens of species and identified a putative nuclear localization sequence (NLS) that is often responsible for the nuclear import of proteins that carry them. Using molecular biology experiments in a heterologous cell system, the authors verified the potential role of this internal NLS and found it indeed could facilitate the nuclear localization of marker proteins when expressed in cells. Using gene-deleted mouse models they generated previously, the authors showed that deletion of Actl7b caused changes in gene expression and mis-localization of nucleosomal histone H3 and chromatin regulator histone deacetylase HDAC1 and 2, supporting their proposed roles of ACTL7B in regulating gene expression. The authors further used alpha-Fold 2 to model the potential protein complexes that could be formed between the ARPs (ACTL7A and ACTL7B) and known chromatin modifiers, such as INO80 and SWI/SNF complexes and found that consistent with previous findings, it is likely that ACTL7A and ACTL7B interact with the chromatin-modifying complexes through binding to their alpha-helical HSA domain cooperatively. These results suggest that ACTL7B possesses novel functions in regulating chromatin structure and thus gene expression beyond conventional roles of cytoskeleton regulation, providing alternative pathways for understanding how gene expression is regulated during spermatogenesis and the etiology of relevant infertility diseases.

Strengths:

The authors provided sufficient background to the study and discussions of the results. Based on their previous research, this study utilized numerous methods, including protein complex structural modeling method alpha-fold 2 Multimers, to further investigate the functional roles of ACTL7B. The results presented here are in general of good quality. The identification of a potential internal NLS in ACTL7B is mostly convincing, in line with the phenotypes presented in the gene deletion model.

Weaknesses:

While the study offered an interesting new look at the functions of ARP proteins during spermatogenesis, some of the study is mainly theoretical speculations, including the protein complex formation. Some of the results may need further experimental verifications, for example, differentially expressed genes that were found in potentially spermatogenic cells at different developmental stages, in order to support the conclusions and avoid undermining the significance of the study.

Reviewer #2 (Public Review):

Summary:

The authors present the Perceptual Error Adaptation (PEA) model, a computational approach offering a unified explanation for behavioral results that are inconsistent with standard state-space models. Beginning with the conventional state-space framework, the paper introduces two innovative concepts. Firstly, errors are calculated based on the perceived hand position, determined through Bayesian integration of visual, proprioceptive, and predictive cues. Secondly, the model accounts for the eccentricity of vision, proposing that the uncertainty of cursor position increases with distance from the fixation point. This elegantly simple model, with minimal free parameters, effectively explains the observed plateau in motor adaptation under the implicit motor adaptation paradigm using the error-clamp method. Furthermore, the authors experimentally manipulate visual cursor uncertainty, a method established in visuomotor studies, to provide causal evidence. Their results show that the adaptation rate correlates with perturbation sizes and visual noise, uniquely explained by the PEA model and not by previous models. Therefore, the study convincingly demonstrates that implicit motor adaptation is a process of Bayesian cue integration

Strengths:

In the past decade, numerous perplexing results in visuomotor rotation tasks have questioned their underlying mechanisms. Prior models have individually addressed aspects like aiming strategies, motor adaptation plateaus, and sensory recalibration effects. However, a unified model encapsulating these phenomena with a simple computational principle was lacking. This paper addresses this gap with a robust Bayesian integration-based model. Its strength lies in two fundamental assumptions: motor adaptation's influence by visual eccentricity, a well-established vision science concept, and sensory estimation through Bayesian integration. By merging these well-founded principles, the authors elucidate previously incongruent and diverse results with an error-based update model. The incorporation of cursor feedback noise manipulation provides causal evidence for their model. The use of eye-tracking in their experimental design, and the analysis of adaptation studies based on estimated eccentricity, are particularly elegant. This paper makes a significant contribution to visuomotor learning research.

The authors discussed in the revised version that the proposed model can capture the general implicit motor learning process in addition to the visuomotor rotation task. In the discussion, they emphasize two main principles: the automatic tracking of effector position and the combination of movement cues using Bayesian integration. These principles are suggested as key to understanding and modeling various motor adaptations and skill learning. The proposed model could potentially become a basis for creating new computational models for skill acquisition, especially where current models fall short.

Weaknesses:

The proposed model is described as elegant. In this paper, the authors test the model within a limited example condition, demonstrating its relevance to the sensorimotor adaptation mechanisms of the human brain. However, the scope of the model's applicability remains unclear. It has shown the capacity to explain prior data, thereby surpassing previous models that rely on elementary mathematics. To solidify its credibility in the field, the authors must gather more supporting evidence.

Reviewer #2 (Public Review):

This study compares the activity of neural populations in the primary and non-primary auditory cortex of ferrets while the animals actively behaved or passively listened to a sound discrimination task. Using a variety of methods, the authors convincingly show differential effects of task engagement on population neural activity in primary vs non-primary auditory cortex; notably that in the primary auditory cortex, task-engagement (1) improves discriminability for both task-relevant and non-task relevant dimensions, and (2) improves the alignment between covariability and sound discrimination axes; whereas in the non-primary auditory cortex, task-engagement (1) improves discriminability for only task-relevant dimensions, and (2) does not affect the alignment between covariability and sound discrimination axes. They additionally show that task-engagement changes in gain can account for the selectivity noted in the discriminability of non-primary auditory neurons. They also admirably attempt to isolate task-engagement from arousal fluctuations, by using fluctuations in pupil size as a proxy for physiological arousal. This is a well-carried out study with thoughtful analyses which in large part achieves its aims to evaluate how task-engagement changes neural activity across multiple auditory regions . As with all work, there are several caveats or areas for future study/analysis. First, the sounds used here (tones, and narrow-band noise) are relatively simple sounds; previous work suggests that exactly what activity is observed within each region (e.g., sensory only, decision-related, etc) may depend in part upon what stimuli are used. Therefore, while the current study adds importance to the literature, future work may consider the use of more varied stimuli. Second, the animals here were engaged in a behavioral task; but apart from an initial calculation of behavioral d', the task performance (and its effect on neural activity) is largely unaddressed.

Reviewer #3 (Public Review):

Summary:<br /> The study conducted by Pisanski et al investigates the role of the lateral parafacial area (pFL) in controlling active expiration. Stereotactic injections of bicuculline were utilized to map various pFL sites and their impact on respiration. The results indicate that injections at more rostral pFL locations induce the most robust changes in tidal volume, minute ventilation, and combined respiratory responses. The study indicates that the rostro-caudal organization of the pFL and its influence on breathing is not simple and uniform.

Strengths:<br /> The data provide novel insights into the importance of rostral locations in controlling active expiration. The authors use innovative analytic methods to characterize the respiratory effects of bicuculline injections into various areas of the pFL.

Weaknesses:<br /> Bicuculline injections increase the excitability of neurons. Aside of blocking GABA receptors, bicuculline also inhibits calcium-activated potassium currents and potentiates NMDA currents, thus insights into the role of GABAergic inhibition are limited.<br /> Increasing the excitability of neurons provides little insights into the activity pattern and function of the activated neurons. Without recording from the activated neurons, it is impossible to know whether an effect on active expiration or any other respiratory phase is caused by bicuculline acting on rhythmogenic neurons or tonic neurons that modulate respiration. While this approach is inappropriate to study the functional extent of the conditional "oscillator" for active expiration, it still provides valuable insights into this region's complex role in controlling breathing .

Reviewer #2 (Public Review):

Summary:

The authors conducted a time-course of whole-body transcriptional analysis of a pest aphid, Rhopalosiphum padi, and identified four major clusters of the genes that show diurnal rhythmicity in transcription. In addition, they conducted the analysis of aphid feeding behaviour and showed that aphids salivate longer from the end of the day toward the beginning of the night while their phloem feeding time does not change throughout the day. The genes up-regulated at night time were enriched with the genes involved in metabolic activities, collaborating with the results showing a higher number of honeydew excretion at night. The authors identified the list of candidate salivary genes that show diurnal rhythmicity in the transcription and silenced a salivary gene C002 and the candidate salivary gene E8696. Silencing of these genes reduced aphid fecundity and survival rate on the host plant but not on the artificial diet.

Strengths:

The time-course transcription study and its analysis will be of interest to researchers studying diurnal rhythms in insect biology. Also, the analysis of aphid feeding behaviour at different times of day is interesting. This study provides variable resources for those who study insect biology.

Weaknesses:

It is not clear to me which data was used to define the putative salivary effectors for R. padi, but the candidate salivary gene list made by Thorpe et al consists of the aphid genes encoding secreted proteins that are up-regulated in the head samples compared to the body samples. Although some proteins were confirmed to be secreted into the aphid saliva, many genes in the list are not confirmed to be expressed in the aphid salivary glands, and their products are not confirmed to be secreted into the saliva and the plant. Is E8696 expressed in the aphid salivary glands and secreted into its host plant? Without the data confirming the expression of the gene in the salivary glands and its secretion into the saliva and into the host plant, we cannot call the protein a salivary protein. Furthermore, without the observation that E8696 has some effect on plant biology, we cannot call it an aphid effector. Therefore, I cannot agree with the parts of the manuscript that refer to E8686 as an aphid salivary effector.

It is interesting to know that some candidate salivary gene expression showed a diurnal rhythm. However, without the knowledge of the functions of the salivary effectors, especially their targets, it is not possible to conclude that the rhythmical expression is important for the aphid performance. In addition, I wonder whether the increase in gene expression is directly correlated with the increase of protein secretion into the saliva and the plant.

Finally, the authors examined aphid survival, fecundity, and feeding behaviour. Those are important for overall aphid performance, but they do not "shape" aphid colonization. Aphid colonisation is shaped by the mechanisms by which aphids find and select their host plant and start to feed on it. Therefore, I do not agree with the title of this manuscript and some parts of the discussion.

I would like the authors to develop how the knowledge of the diurnal rhythm of aphid feeding can contribute to optimise pest management. I see that there are some differences in aphid metabolism and feeding behaviour between day and night, but I would like to hear how such knowledge can optimise pest management strategies.

Reviewer #2 (Public Review):

Weng et al. perform a comprehensive study of gene expression changes in young and old animals, in wild-type and daf-2 insulin receptor mutants, in the whole animal and specifically in the nervous system. Using this data, they identify gene families that are correlated with neuronal ageing, as well as a distinct set of genes that are upregulated in neurons of aged daf-2 mutants. This is particularly interesting as daf-2 mutants show both extended lifespan and healthier neurons in aged animals, reflected by better learning/memory in older animals compared with wild-type controls. Indeed, knockdown of several of these upregulated genes resulted in poorer learning and memory. In addition, the authors showed that several genes upregulated during ageing in wild-type neurons also contribute to learning and memory; specifically, knockdown of these genes in young animals resulted in improved memory. This indicates that (at least in this small number of cases), genes that show increased transcript levels with age in the nervous system somehow suppress memory, potentially by having damaging effects on neuronal health.

Finally, from a resource perspective, the neuronal transcriptome provided here will be very useful for C. elegans researchers as it adds to other existing datasets by providing the transcriptome of older animals (animals at day 8 of adulthood) and demonstrating the benefits of performing tissue-specific RNAseq instead of whole-animal sequencing.

The work presented here is of high quality and the authors present convincing evidence supporting their conclusions.

Reviewer #2 (Public Review):

Summary:

The study explores how single striatal projection neurons (SPNs) utilize dendritic nonlinearities to solve complex integration tasks. It introduces a calcium-based synaptic learning rule that incorporates local calcium dynamics and dopaminergic signals, along with metaplasticity to ensure stability for synaptic weights. Results show SPNs can solve the nonlinear feature binding problem and enhance computational efficiency through inhibitory plasticity in dendrites, emphasizing the significant computational potential of individual neurons. In summary, the study provides a more biologically plausible solution to single-neuron learning and gives further mechanical insights into complex computations at the single-neuron level.

Strengths:

The paper introduces a novel learning rule for training a single multicompartmental neuron model to perform nonlinear feature binding tasks (NFBP), highlighting two main strengths: the learning rule is local, calcium-based, and requires only sparse reward signals, making it highly biologically plausible, and it applies to detailed neuron models that effectively preserve dendritic nonlinearities, contrasting with many previous studies that use simplified models.

Weaknesses:

I am concerned that the manuscript was submitted too hastily, as evidenced by the quality and logic of the writing and the presentation of the figures. These issues may compromise the integrity of the work. I would recommend a substantial revision of the manuscript to improve the clarity of the writing, incorporate more experiments, and better define the goals of the study.

Major Points:

(1) Quality of Scientific Writing: The current draft does not meet the expected standards. Key issues include:

i. Mathematical and Implementation Details: The manuscript lacks comprehensive mathematical descriptions and implementation details for the plasticity models (LTP/LTD/Meta) and the SPN model. Given the complexity of the biophysically detailed multicompartment model and the associated learning rules, the inclusion of only nine abstract equations (Eq. 1-9) in the Methods section is insufficient. I was surprised to find no supplementary material providing these crucial details. What parameters were used for the SPN model? What are the mathematical specifics for the extra-synaptic NMDA receptors utilized in this study? For instance, Eq. 3 references [Ca2+]-does this refer to calcium ions influenced by extra-synaptic NMDARs, or does it apply to other standard NMDARs? I also suggest the authors provide pseudocodes for the entire learning process to further clarify the learning rules.

ii. Figure quality. The authors seem not to carefully typeset the images, resulting in overcrowding and varying font sizes in the figures. Some of the fonts are too small and hard to read. The text in many of the diagrams is confusing. For example, in Panel A of Figure 3, two flattened images are combined, leading to small, distorted font sizes. In Panels C and D of Figure 7, the inconsistent use of terminology such as "kernels" further complicates the clarity of the presentation. I recommend that the authors thoroughly review all figures and accompanying text to ensure they meet the expected standards of clarity and quality.

iii. Writing clarity. The manuscript often includes excessive and irrelevant details, particularly in the mathematical discussions. On page 24, within the "Metaplasticity" section, the authors introduce the biological background to support the proposed metaplasticity equation (Eq. 5). However, much of this biological detail is hypothesized rather than experimentally verified. For instance, the claim that "a pause in dopamine triggers a shift towards higher calcium concentrations while a peak in dopamine pushes the LTP kernel in the opposite direction" lacks cited experimental evidence. If evidence exists, it should be clearly referenced; otherwise, these assertions should be presented as theoretical hypotheses. Generally, Eq. 5 and related discussions should be described more concisely, with only a loose connection to dopamine effects until more experimental findings are available.

(2) Goals of the Study: The authors need to clearly define the primary objective of their research. Is it to showcase the computational advantages of the local learning rule, or to elucidate biological functions?

i. Computational Advantage: If the intent is to demonstrate computational advantages, the current experimental results appear inadequate. The learning rule introduced in this work can only solve for four features, whereas previous research (e.g., Bicknell and Hausser, 2021) has shown capability with over 100 features. It is crucial for the authors to extend their demonstrations to prove that their learning rule can handle more than just three features. Furthermore, the requirement to fine-tune the midpoint of the synapse function indicates that the rule modifies the "activation function" of the synapses, as opposed to merely adjusting synaptic weights. In machine learning, modifying weights directly is typically more efficient than altering activation functions during learning tasks. This might account for why the current learning rule is restricted to a limited number of tasks. The authors should critically evaluate whether the proposed local learning rule, including meta-plasticity, actually offers any computational advantage. This evaluation is essential to understand the practical implications and effectiveness of the proposed learning rule.

ii. Biological Significance: If the goal is to interpret biological functions, the authors should dig deeper into the model behaviors to uncover their biological significance. This exploration should aim to link the observed computational features of the model more directly with biological mechanisms and outcomes.

Reviewer #2 (Public Review):

The authors investigate the properties of the transcriptional co-activator Taiman in regulating tissue growth. In previously published work they had shown that cells that overexpress Tiaman in the pupal wing can cause the death of thoracic cells adjacent to the wing tip to die and thus allow the wing to invade the thorax. This was mediated by the secretion of Spz ligands. Here, they investigate the properties of cells that are homozygous for a hypomorphic allele of taiman (tai). They show that homozygous mutant clones are much smaller than their wild-type twin spots and that cells in the clones are dying by apoptosis which is inferred from elevated levels of anti-Dcp1 staining (Figure 1).

By generating clones during eye development, the authors screen for dominant modifiers that increase the representation of homozygous tai tissue in the adult eye (Figure 2). They find that reducing the levels of hid, the entire rpr/hid/grim locus and Apc (and/or Apc2) each increase the representation of tai clones. They then show that the survival of tissue to the adult stage correlates with the size of lones in the third-instar larval wing disc (Figure 3). The rest of the study derives from the modification of the phenotype by Apc and investigates the interaction between Wnt signaling and tai clone survival.

The authors then investigate interactions between tai and the wingless (wg) pathway. First, they show that increasing tai expression increases the expression of a wg reporter (nkd-lacZ) while reducing tai levels decreases its expression (Figure 4) indicating that wg signaling is likely reduced when tai levels are decreased. This finding is strengthened by examining wg-lacZ expression since the expression of this reporter is normally restricted to the D/V boundary in the wing disc by feedback inhibition via Wg signaling. Expression of the reporter is increased when tai expression is reduced and decreased when tai expression is increased (Figure 5).

The authors then look at Wg protein away from the DV boundary. They find increased levels when tai expression is increased and decreased levels when tai is decreased. They conclude that tai activity increased Wg protein in cells (Figure 6). They suggest that this could be the result of the regulation of expression of Dally-like protein (Dlp). Consistent with this idea, increasing tai expression increases Dlp levels, and decreasing tai decreases Dlp levels (Figure 7). They then show that increasing Dlp levels when tai is reduced increases Wg levels which presumably means that Dlp is epistatic to tai. Puzzlingly, increasing both tai and Dlp decreases Wg.

The authors then examine the effect of reducing Dlp in the cells that secrete Wg. They find that increasing tai results in the diffusion of Wg further from its source while reducing tai reduces its spread (Figure 8). They then show that in clones with reduced tai, there is increased cytoplasmic Dlp (Figure 9). They therefore propose that tai clones fail to survive because they do not secrete enough Dlp which results in reduced capture of the Wg for those cells and hence decreased Wg signaling.

Evaluation

While the authors present good evidence in support of most of their conclusions, there are alternative explanations in many cases that have not been excluded.

From the results in Figure 1 (and Figure 3), the authors conclude that "The data indicate the existence of an extracellular competition mechanism that allows normal tai[wt] cells to kill tai[k15101] neighbors" (line 127). However, the experiments have been done with a single allele, and these experiments do not exclude the possibility that there is another mutation on the same chromosome arm that is responsible for the observed phenotype. Since the authors have a UAS-tai stock, they could strengthen their results using a MARCM experiment where they could test whether the expression of UAS-tai rescues the elimination of tai mutant clones. Alternatively, they could use a second (independent) allele to demonstrate that the phenotype can be attributed to a reduction in tai activity.

By screening for dominant modifiers of a phenotype one would not expect to identify all interacting genes - only those that are haploinsufficient in this situation. The authors have screened a total of 21 chromosomes for modification and have not really explained which alleles are nulls and which are hypomorphs. The nature of each of the alleles screened needs to be explained better. Also, the absence of a dominant modification does not necessarily exclude a function of that gene or pathway in the process. This is especially relevant for the Spz/Toll pathway which the authors have previously implicated in the ability of tai-overexpressing cells to kill wild-type cells. The most important discovery from this screen is the modification by the Apc alleles. This part of the paper would be strengthened by testing for modification by other components of the Wingless pathway. The authors show modification by Apc[MI01007] and the double mutant Apc[Q8] Apc2[N175A]. Without showing the Apc[Q8] and Apc2[N175A] alleles separately, it is hard to know if the effect of the double mutant is due to Apc, Apc2,` or the combination.

RNAi of tai seems to block the formation of the Wg gradient. If so, one might expect a reduction in wing size. Indeed, this could explain why the wings of tai/Df flies are smaller. The authors mention briefly that the posterior compartment size is reduced when tai-RNAi is expressed in that compartment. However, this observation merits more emphasis since it could explain why tai/Df flies are smaller (Are their wings smaller?).

In Figure 7, the authors show the effect of manipulating Tai levels alone or in combination with increasing Dlp levels. However, they do not include images of Wg protein distribution upon increasing Dlp levels alone.

In Figure 8, there is more Wg protein both at the DV boundary and spreading when tai is overexpressed in the source cells using bbg-Gal4. However, in an earlier experiment (Figure 5C) they show that the wg-lacZ reporter is downregulated at the DV boundary when tai is overexpressed using en-Gal4. They therefore conclude that wg is not transcriptionally upregulated but is, instead secreted at higher levels when tai is expressed in the source cells. Wg protein is reduced in the DV stripe with tai is overexpressed using the en-Gal4 driver (Figure 6B') and is increased at the same location when tai is overexpressed with the bbg-Gal4 driver. (Figure 8) I don't know how to reconcile these observations.

In Figure 9, the tai-low clones have elevated levels of Dlp. How can this be reconciled with the tai-RNAi knockdown shown in Figure 7C' where reducing tai levels causes a strong reduction in Dlp levels?

Reviewer #2 (Public Review):

Summary:

This manuscript uses cell lines representative of germ line cells, somatic cells and pluripotent cells to address the question of how the endocrine disrupting compound BPS affects these various cells with respect to gene expression and DNA methylation. They find a relationship between the presence of estrogen receptor gene expression and the number of DNA methylation and gene expression changes. Notably, PGCLCs do not express estrogen receptors and although they do have fewer changes, changes are nevertheless detected, suggesting a nonconical pathway for BPS-induced perturbations. Additionally, there was a significant increase in the occurrence of BPS-induced epimutations near EREs in somatic and pluripotent cell types compared to germ cells. Epimutations in the somatic and pluripotent cell types were predominantly in enhancer regions whereas that in the germ cell type was predominantly in gene promoters.

Strengths:

The strengths of the paper include the use of various cell types to address sensitivity of the lineages to BPS as well as the observed relationship between the presence of estrogen receptors and changes in gene expression and DNA methylation.

Weaknesses:

The weakness includes the fact that exposures are more complicated in a whole organism than in an isolated cell line.

Reviewer #2 (Public Review):

Summary:

Scx is a well-established marker for tenocytes, but the expression in myogenic-lineage cells was unexplored. In this study, the authors performed lineage-trace and scRNA-seq analyses and demonstrated that Scx is expressed in activated SCs. Further, the authors showed that Scx is essential for muscle regeneration using conditional KO mice and identified the target genes of Scx in myogenic cells, which differ from those of tendons.

Strengths:

Sometimes, lineage-trace experiments cause mis-expression and do not reflect the endogenous expression of the target gene. In this study, the authors carefully analyzed the unexpected expression of Scx in myogenic cells using some mouse lines and scRNA-seq data.

Weaknesses:

Scx protein expression has not been verified.

Reviewer #2 (Public Review):

This article describes a novel mechanism of host defense in the gut of Drosophila larvae. Pathogenic bacteria trigger the activation of a valve that blocks them in the anterior midgut where they are subjected to the action of antimicrobial peptides. In contrast, beneficial symbiotic bacteria do not activate the contraction of this sphincter, and can access the posterior midgut, a compartment more favorable to bacterial growth.

Strengths:

The authors decipher the underlying mechanism of sphincter contraction, revealing that ROS production by Duox activates the release of DH31 by enteroendocrine cells that stimulate visceral muscle contractions. The use of mutations affecting the Imd pathway or lacking antimicrobial peptides reveals their contribution to pathogen elimination in the anterior midgut.

Weaknesses:

- The mechanism allowing the discrimination between commensal and pathogenic bacteria remains unclear.

- The use of only two pathogens and one symbiotic species may not be sufficient to draw a conclusion on the difference in treatment between pathogenic and symbiotic species.

- We can also wonder how the process of sphincter contraction is affected by the procedure used in this study, where larvae are starved. Does the sphincter contraction occur in continuous feeding conditions? Since larvae are continuously feeding, is this process physiologically relevant?

Reviewer #2 (Public Review):

Sharp wave ripples are transient oscillations occurring in the hippocampus that are thought to play an important role in organising temporal sequences during the reactivation of neuronal activity. This study addresses the mechanism by which these temporal sequences are generated in the CA3 region focusing on two different subtypes of pyramidal neurons, thorny and athorny. Using high-quality electrophysiological recordings from up to 8 pyramidal neurons at a time the authors measure the connectivity rates between these pyramidal cell subtypes in a large dataset of 348 cells. This is a significant achievement and provides important data. The most striking finding is how similar connection characteristics are between cell types. There are no differences in synaptic strength or failure rates and some small differences in connectivity rates and short-term plasticity. Using model simulations, the authors explore the implications of the differences in connectivity rates for the temporal specificity of pyramidal cell firing within sharp-wave ripple events. The simulations show that the experimentally observed connectivity rates may contribute to the previously observed temporal sequence of pyramidal cell firing during sharp wave ripples.

The conclusions drawn from the simulations are not experimentally tested so remain theoretical. In the simple network model, the authors include basket cell and anti-SWR interneurons but the connectivity of these cell types is not measured experimentally and variations in interneuron parameters may also influence temporal specificity of firing. In addition, the influence of short-term plasticity measured in their experiments is not tested in the model. Interestingly, the experimental data reveal a large variability in many of the measured parameters. This may strongly influence the firing of pyramidal cells during SWRs but it is not represented within the model which uses the averaged data.

Reviewer #2 (Public Review):

Summary:

This is an interesting manuscript that describes a series of molecular dynamics studies on the peptide transporter PepT2 (SLC15A2). They examine, in particular, the effect on the transport cycle of protonation of various charged amino acids within the protein. They then validate their conclusions by mutating two of the residues that they predict to be critical for transport in cell-based transport assays. The study suggests a series of protonation steps that are necessary for transport to occur in Petp2. Comparison with bacterial proteins from the same family show that while the overall architecture of the proteins and likely mechanism are similar, the residues involved in the mechanism may differ.

Strengths:

This is an interesting and rigorous study that uses various state of the art molecular dynamics techniques to dissect the transport cycle of PepT2 with nearly 1ms of sampling. It gives insight into the transport mechanism, investigating how protonation of selected residues can alter the energetic barriers between various states of the transport cycle. The authors have, in general, been very careful in their interpretation of the data.

Weaknesses:

Interestingly, they suggest that there is an additional protonation event that may take place as the protein goes from occluded to inward-facing (clear from Figure 8) but as the authors comment they have not identified this residue(s).

Reviewer #2 (Public Review):

Summary:

This study presents a significant finding that enhances our understanding of spermatogenesis. TMC7 belongs to a family of transmembrane channel-like proteins (TMC1-8), primarily known for their role in the ear. Mutations to TMC1/2 are linked to deafness in humans and mice and were originally characterized as auditory mechanosensitive ion channels. However, the function of the other TMC family members remains poorly characterized. In this study, the authors begin to elucidate the function of TMC7 in acrosome biogenesis during spermatogenesis. Through analysis of transcriptomics datasets, they elevated levels of TMC7 in round spermatids in both mouse and human testis. They then generate Tmc7-/- mice and find that male mice exhibit smaller testes and complete infertility. Examination of different developmental stages reveals spermatogenesis defects, including with reduced sperm count, elongated spermatids and large vacuoles. Additionally, abnormal acrosome morphology are observed beginning at the early-stage Golgi phase, indicating TMC7's involvement in proacrosomal vesicle trafficking and fusion. They observed localization of TMC7 in the cis-Golgi and suggest that its presence is required for maintaining Golgi integrity, with Tmc7-/- leading to reduced intracellular Ca2+, elevated pH and increased ROS levels, likely resulting in spermatid apoptosis. Overall, the work delineates a new function of TMC7 in spermatogenesis and the authors propose that that its ion channel and/or scramblase activity is likely important for Golgi homeostasis. This work is of significant interest to the community and is of high quality.

Strengths:

The biggest strength of the paper is the phenotypic characterization of the TMC7-/- mouse model, which has clear acrosome biogenesis/spermatogenesis defects. This is the main claim of the paper and it is supported with the data that are presented.

Weaknesses:

It isn't clear whether TMC7 functions as an ion channel from the current data presented in this paper, but the authors are careful in their interpretation and present this merely as a hypothesis supporting this idea.

Reviewer #2 (Public Review):

Summary:

This is an interesting paper that delves into the post-translational modifications of the yeast Srs2 helicase and proteins with which it interacts in coping with DNA damage. The authors use mutants in some interaction domains with RPA and Srs2 to argue for a model in which there is a balance between RPA binding to ssDNA and Srs2's removal of RPA. The idea that a checkpoint is being regulated is based on observing Rad53 and Rad9 phosphorylation (so there are the attributes of a checkpoint), but evidence of cell cycle arrest is lacking. The only apparent delay in the cell cycle is the re-entry into the second S phase (but it could be an exit from G2/M); but in any case, the wild-type cells enter the next cell cycle most rapidly. No direct measurement of RPA residence is presented.

Strengths:

Data concern viability assays in the presence of camptothecin and in the post-translational modifications of Srs2 and other proteins.

Weaknesses:

There are a couple of overriding questions about the results, which appear technically excellent. Clearly, there is an Srs2-dependent repair process here, in the presence of camptothecin, but is it a consequence of replication fork stalling or chromosome breakage? Is repair Rad51-dependent, and if so, is Srs2 displacing RPA or removing Rad51 or both? If RPA is removed quickly what takes its place, and will the removal of RPA result in lower DDC1-MEC1 signaling?

Moreover, It is worth noting that in single-strand annealing, which is ostensibly Rad51 independent, a defect in completing repair and assuring viability is Srs2-dependent, but this defect is suppressed by deleting Rad51. Does deleting Rad51 have an effect here?

Neither this paper nor the preceding one makes clear what really is the consequence of having a weaker-binding Rfa1 mutant. Is DSB repair altered? Neither CPT nor MMS are necessarily good substitutes for some true DSB assay.

With camptothecin, in the absence of site-specific damage, it is difficult to test these questions directly. (Perhaps there is a way to assess the total amount of RPA bound, but ongoing replication may obscure such a measurement). It should be possible to assess how CPT treatment in various genetic backgrounds affects the duration of Mec1/Rad53-dependent checkpoint arrest, but more than a FACS profile would be required.

It is also notable that MMS treatment does not seem to yield similar results (Fig. S1).

Reviewer #3 (Public Review):

The authors presented point light displays of human walkers to children (mean = 9 years) with and without ADHD to compare their biological motion perception abilities, and relate them to IQ, social responsiveness scale (SRS) scores and age. They report that children with ADHD were worse at all three biological motion tasks, but that those loading more heavily on local processing related to social interaction skills and global processing to age. The valuable and solid findings are informative for understanding this complex condition, as well as biological motion processing mechanisms in general. However, the correlations present a pattern that needs further examination in future studies because many of the differences between correlations are not significant.

Strengths:

The authors present differences between ADHD and TD children in biological motion processing, and this question has not received as much attention as equivalent processing capabilities in autism. They use a task that appears well controlled. They raise some interesting mechanistic possibilities for differences in local and global motion processing, which are distinctions worth exploring. The group differences will therefore be of interest to those studying ADHD, as well as other developmental conditions, and those examining biological motion processing mechanisms in general.

Weaknesses:

The data are not strong enough to support claims about differences between global and lobal processing wrt social communication skills and age. The mechanistic possibilities for why these abilities may dissociate in such a way are interesting, but the crucial tests of differences between correlations do not present a clear picture. Further empirical work would be needed to test this further. Specifics:

The authors state frequently that it was the local BM task that related to social communication skills (SRS) and not the global tasks. However, the results section shows a correlation between SRS and all three tasks. The only difference is that when looking specifically within the ADHD group, the correlation is only significant for the local task. The supplementary materials demonstrate that tests of differences between correlations present an incomplete picture. Currently they have small samples for correlations, so this is unsurprising.

Theoretical assumptions. The authors make some statements about local vs global biological motion processing that may have been made in previous studies, but would appear controversial and not definitive. E.g., that local BM processing does not improve with age.

Reviewer #3 (Public Review):

It is well established that there is extensive post-transcriptional gene regulation in nervous systems, including the fly brain. For example, dynamic regulation of hundreds of genes during photoreceptor development could only be observed at the level of translated mRNAs, but not the entire transcriptomes. The present study instead addresses the role of differential translational regulation between cell types (or rather classes: neurons and glia, as both are still highly heterogenous groups) in the adult fly brain. By performing bulk RNA-seq and Ribo-seq on the same lysates, the authors are able to compare translation efficiency (TE) of all transcripts between neurons and glia. Many genes display differential TE, but interestingly, they tend to be the genes that already show strong differences at their mRNA level. The most striking observation is the finding that neuronal transcripts in glia display increased ribosome stalling at their 5' UTR, and in particular at the start codons of short "upstream ORFs". This could suggest that glia specifically employ a mechanism to upregulate upstream ORF translation, enabling them to better suppress the expression of the genes that have them. And neuronal genes tend to have longer 5' UTRs, perhaps to facilitate this type of regulation.

However, it is difficult to evaluate the functional significance of these differences because the authors provide only one follow-up experiment to their RNA-seq analysis. Venus expressed with the Rh1 UTR sequences may be displaying differential levels between glia and neurons, but I find this image (Fig. 5C) rather unconvincing to support that conclusion. There are no quantifications of colocalization, or even sample size information provided for this experiment. And if there is indeed a difference, it would still be difficult argue this is because of the 5' stalling phenomenon authors observe with Rh1, because they switched both the 5' and 3' UTRs.

I also find it puzzling that the TE differences between the groups are mostly among the transcripts that are already strongly differentially expressed at the transcriptional level. The authors would like to frame this as a mechanism of 'contrast sharpening'; but it is unclear why that would be needed. Rh1, for instance, is not just differentially expressed between neurons and glia, but it is actually only expressed by a very specific neuronal type (photoreceptors). Thus it's not clear to me why the glia would need this 5' stalling mechanism to fully suppress Rh1 expression, while all the other neurons can apparently do so without it.

Response to authors' revisions:

The authors have addressed most of the technical points in their revised manuscript. However, it is still rather unclear whether this mechanism would have any significant impact on differential gene expression between cell types in vivo. Considering that it's mostly occurring on genes that are already strongly differentially transcribed, that doesn't appear very likely.

Reviewer #2 (Public Review):

Summary:

In this study, the authors study how the deubiquitinase USP8 regulates endosome maturation in C. elegans and mammalian cells. The authors have isolated USP8 mutant alleles in C. elegans and used multiple in vivo reporter lines to demonstrate the impact of USP8 loss-of-function on endosome morphology and maturation. They show that in USP8 mutant cells, the early endosomes and MVB-like structures are enlarged while the late endosomes and lysosomal compartments are reduced. They elucidate that USP8 interacts with Rabx5, a guanine nucleotide exchange factor (GEF) for Rab5, and show that USP8 likely targets specific lysine residue of Rabx5 to dissociate it from early endosomes. They also find that localization of USP8 to early endosomes are disrupted in Rabx5 mutant cells. They observe that in both Rabx5 and USP8 mutant cells, the Rab7 GEF SAND-1 puncta which likely represents late endosomes are diminished, although that Rabex5 are accumulated in USP8 mutant cells. The authors provide evidence that USP8 regulates endosomal maturation in a similar fashion in mammalian cells. Based on their observations they propose that USP8 dissociates Rabex5 from early endosomes and enhances the recruitment of SAND-1 to promote endosome maturation.

Strengths:

The major highlights of this study include the direct visualization of endosome dynamics in a living multi-cellular organism, C. elegans. The high-quality images provide clear in vivo evidences to support the main conclusions. The authors have generated valuable resources to study mechanisms involved in endosome dynamics regulation in both the worm and mammalian cells, which would benefit many members in the cell biology community. The work identifies a fascinating link between USP8 and the Rab5 guanine nucleotide exchange factor Rabx5, which expands the targets and modes of action of USP8. The findings make a solid contribution toward the understanding of how endosomal trafficking is controlled.

Weaknesses:

- The authors utilized multiple fluorescent protein reporters, including those generated by themselves, to label endosomal vesicles. Although these are routine and powerful tools for studying endosomal trafficking, these results cannot tell that whether the endogenous proteins (Rab5, Rabex5, Rab7, etc.) are affected in the same fashion.<br /> - The authors clearly demonstrated a link between USP8 and Rabx5, and they showed that cells deficient of both factors displayed similar defects in late endosomes/lysosomes. But the authors didn't confirm whether and/or to which extent that USP8 regulates endosome maturation through Rabx5. Additional genetic and molecular evidence might be required to better support their working model.

Reviewer #2 (Public Review):

Summary:

Golluscio et al. addresses one of the mechanisms of IKs (KCNQ1/KCNE1) channel upregulation by a polyunsaturated fatty acid (PUFA). PUFAs are known to upregulate KCNQ1 and KCNQ1/KCNE1 channels by two mechanisms: one shifts the voltage dependence to the negative direction, and the other increases the maximum conductance (Gmax). While the first mechanism is known to affect the voltage sensor equilibrium by charge effect, the second mechanism is less known. By applying the single-channel recordings and mutagenesis on the putative binding sites (most of them related to the selectivity filter), they concluded that the selectivity filter is stabilized to a conductive state by PUFA binding.

Strengths:

The manuscript employed single-channel recordings and directly assessed the behavior of the selectivity filter. The method is straightforward and convincing enough to support the claims.

Weaknesses:

Although the analysis using selectivity filter mutants supports the hypothesis that PUFA binding stabilizes the conducting state of the filter, it may be somewhat speculative how PUFAs bind to the KCNQ1 channel in the presence of KCNE1.

Reviewer #2 (Public Review):

Summary:

The primary goal of this study was to identify the transport pathway that is responsible for the release of dietary citrate from enterocytes into blood across the basolateral membrane.

Strengths:

The transport pathway responsible for the entry of dietary citrate into enterocytes was already known, but the transporter responsible for the second step remained unidentified. The studies presented in this manuscript identify SLC35G1 as the most likely transporter that mediates the release of absorbed citrate from intestinal cells into the serosal side. This fills an important gap in our current knowledge of the transcellular absorption of dietary citrate. The exclusive localization of the transporter in the basolateral membrane of human intestinal cells and the human intestinal cell line Caco-2 and the inhibition of the transporter function by chloride support this conclusion.

Weaknesses:

(i) The substrate specificity experiments have been done with relatively low concentrations of potential competing substrates, considering the relatively low affinity of the transporter for citrate. Given that NaDC1 brings in not only citrate as a divalent anion but also other divalent anions such as succinate, it is possible that SLC35G1 is responsible for the release of not only citrate but also other dicarboxylates. But the substrate specificity studies show that the dicarboxylates tested did not compete with citrate, meaning that SLc35G1 is selective for the citrate (2-), but this conclusion might be flawed because of the low concentration of the competing substrates used in the experiment.

(ii) The authors have used MDCK cells for assessment of the transcellular transfer of citrate via SLC35G1, but it is not clear whether this cell line expresses NaDC1 in the apical membrane as the enterocytes do. Even though the authors expressed SLC35G1 ectopically in MDCK cells and showed that the transporter localizes to the basolateral membrane, the question as to how citrate actually enters the apical membrane for SLC35G1 in the other membrane to work remains unanswered.

(iii) There is one other transporter that has already been identified for the efflux of citrate in some cell types in the literature (SLC62A1, PLoS Genetics; 10.1371/journal.pgen.1008884), but no mention of this transporter has been made in the current manuscript.

Reviewer #2 (Public Review):

Summary:

This work is of great significance in revealing the regulatory mechanisms of pathogenic fungi in toxin production, pathogenicity, and in its prevention and pollution control. Overall, this is generally an excellent manuscript.

Strengths:

The data in this manuscript is robust and the experiments conducted are appropriate.

Weaknesses:

(1) The authors found that SntB played key roles in oxidative stress response of A. flavus by ChIP-seq and RNA sequencing. To confirm the role of SntB in oxidative stress, authors have better to measure the ROS levels in the ΔsntB and WT strains, besides the ΔcatC strain.<br /> (2) Why the authors only studied the function of catC among the 7 genes related to oxidative response listed in Table S14.

Reviewer #2 (Public Review):

In this manuscript, the authors first developed a new small molecular inhibitor that could target specifically the M1 metalloproteases of both important malaria parasite species Plasmodium falciparum and P. vivax. This was done by a chemical modification of a previously developed molecule that targets PfM1 as well as PfM17 and possibly other Plasmodial metalloproteases. After the successful chemical synthesis, the authors showed that the derived inhibitor, named MIPS2673, has a strong antiparasitic activity with IC50 342 nM and it is highly specific for M1. With this in mind, the authors first carried out two large-scale proteomics to confirm the MIPS2673 interaction with PfM1 in the context of the total P. falciparum protein lysate. This was done first by using thermal shift profiling and subsequently limited proteolysis. While the first demonstrated overall interaction, the latter (limited proteolysis) could map more specifically the site of MIPS2673-PfM1 interaction, presumably the active site. Subsequent metabolomics analysis showed that MIPS2673 cytotoxic inhibitory effect leads to the accumulation of short peptides many of which originate from hemoglobin. Based on that the authors argue that the MIPS2673 mode of action (MOA) involves inhibition of hemoglobin digestion that in turn inhibits the parasite growth and development.

Comments on the revised version:

The authors addressed all my comments from the previous round of reviews.

Reviewer #2 (Public Review):

Summary:

Bone resorption by osteoclasts plays an important role in bone modeling and homeostasis. The multinucleated mature osteoclasts have higher bone-resorbing capacity than their mononuclear precursors. The previous work by authors has identified that increased cell-surface level of La protein promotes the fusion of mononuclear osteoclast precursor cells to form fully active multinucleated osteoclasts. In the present study, the authors further provided convincing data obtained from cellular and biochemical experiments to demonstrate that the nuclear-localized La protein where it regulates RNA metabolism was oxidized by redox signaling during osteoclast differentiation and the modified La protein was translocated to the osteoclast surface where it associated with other proteins and phospholipids to trigger cell-cell fusion process. The work provides novel mechanistic insights into osteoclast biology and provides a potential therapeutic target to suppress excessive bone resorption in metabolic bone diseases such as osteoporosis and arthritis.

Strengths:

Increased intracellular ROS induced by osteoclast differentiation cytokine RANKL has been widely studied in enhancing RANKL signaling during osteoclast differentiation. The work provides novel evidence that redox signaling can post-translationally modify proteins to alter the translocation and functions of critical regulators in the late stage of osteoclastogenesis. The results and conclusions are mostly supported by the convincing cellular and biochemical assays,

Weaknesses:

The lack of in vivo studies in animal models of bone diseases such as postmenopausal osteoporosis, inflammatory arthritis, and osteoarthritis reduces the translational potential of this work.

Reviewer #2 (Public Review):

Summary:

The authors studied the sequence determinants of C-terminal tags that govern protein degradation in bacteria. They introduce a new strategy to determine degron sequences: Detox (Degron Enrichment by toxin). This unbiased approach links degron efficiency to cell growth as degrons are C-terminally fused to the toxin VapC, which inhibits protein translation. Selecting for bacterial growth and thus toxin degradation enabled the identification of potent degron derived from a randomized library of pentapeptides. Remarkably, most degrons show sequence similarity to the SsrA-tag, which is fused to incomplete polypeptides at stalled ribosomes by the tmRNA-tagging system. These findings underline the extraordinary efficiency of the SsrA-tag and the ClpXP protease in removing incomplete polypeptides and demonstrate that most proteins are spared from degradation by harboring different C-termini. The introduced method will be highly useful to determine degron sequences in other positions and other bacterial species.

Strengths:

The work introduces an innovative and powerful strategy to identify degron sequences in bacteria. The study is well-controlled and results have been thoroughly analyzed. It will now become important to broaden the technology, making it also accessible for more complex degrons.

Weaknesses:

The approach is efficient in identifying strong degron sequences that are predominantly recognized by the ClpXP protease. The sequence specificity of other proteolytic systems, however, is not efficiently addressed, pointing to a potential limitation of this technology. The GS-rich linker sequence connecting the degron and the toxin might also impact proteolysis and thus outcome.

Reviewer #2 (Public Review):

Summary:

Shimin Wang et al. investigated the role of Sertoli cells in mediating spermatogenesis disorders in non-obstructive azoospermia (NOA) through stage-specific communications. The authors utilized scRNA-seq and scATAC-seq to analyze the molecular and epigenetic profiles of germ cells and Sertoli cells at different stages of spermatogenesis.

Strengths:

By understanding the gene expression patterns and chromatin accessibility changes in Sertoli cells, the authors sought to uncover key regulatory mechanisms underlying male infertility and identify potential targets for therapeutic interventions. They emphasized that the absence of the SC3 subtype would be a major factor contributing to NOA.

Weaknesses:

Although the authors used cutting-edge techniques to support their arguments, it is difficult to find conceptual and scientific advances compared to Zeng S et al.'s paper (Zeng S, Chen L, Liu X, Tang H, Wu H, and Liu C (2023) Single-cell multi-omics analysis reveals dysfunctional Wnt signaling of spermatogonia in non-obstructive azoospermia. Front. Endocrinol. 14:1138386.). Overall, the authors need to improve their manuscript to demonstrate the novelty of their findings in a more logical way.

Reviewer #2 (Public Review):

Summary:

In the manuscript "Temporally controlled nervous system-to-gut signaling bidirectionally regulates longevity in C. elegans", Xu and colleagues examine the role of cholinergic signaling by C. elegans motor neurons in modulating lifespan. The authors show that manipulating motor neuronal activity using genetic techniques can be beneficial or detrimental to lifespan, depending on when motor neuron activity is modulated.

Strengths:

A large body of data showing the effects of knockdown of cholinergic receptors and neurotransmitters on lifespan is presented. This would be of value to the community.

Weaknesses:

However, the studies are incomplete. More rigorous approaches would be needed to support the key conclusions, and substantiate the main findings and pathway components.

Reviewer #2 (Public Review):

Summary:

The authors explored the importance of data quality and representation for ligand-based virtual screening approaches. I believe the results could be of potential benefit to the drug discovery community, especially to those scientists working in the field of machine learning applied to drug research. The in silico design is comprehensive and adequate for the proposed comparisons.

This manuscript by Chong A. et al describes that it is not necessary to resort to the use of sophisticated deep learning algorithms for virtual screening, since based on their results considering conventional ML may perform exceptionally well if fed by the right data and molecular representations.

The article is interesting and well-written. The overview of the field and the warning about dataset composition are very well thought-out and should be of interest to a broad segment of the AI ​​in drug discovery readership. This article further highlights some of the considerations that need to be taken into consideration for the implementation of data-centric AI for computer-aided drug design methods.

Strengths:

This study contributes significantly to the field of machine learning and data curation in drug discovery. The paper is, in general, well-written and structured. However, in my opinion, there are some suggestions regarding certain aspects of the data analyses.

Weaknesses:

The conclusions drawn in the study are based on the analysis of a single dataset, and I am not sure they can be generalized. Therefore, in my opinion, the conclusions are only partially supported by the data. To generalize the conclusions, it is imperative to conduct a benchmark with diverse datasets, for different molecular targets.<br /> The conclusion cannot be immediately extended to molecular descriptors or features different from the ones used in this study<br /> It is advisable to present statistical analyses to ascertain whether the observed differences in metrics hold statistical significance.

Reviewer #2 (Public Review):

Summary

Patrícia Graça et al., examined the role of the putative oxidoreductase MftG in regeneration of redox cofactors from the mycofactocin family in Mycolicibacerium smegmatis. The authors show that the mftG is often co-encoded with genes from the mycofactocin synthesis pathway in M. smegmatis genomes. Using a mftG deletion mutant, the authors show that mftG is critical for growth when ethanol is the only available carbon source, and this phenotype can be complemented in trans. The authors demonstrate the ethanol associated growth defect is not due to ethanol induced cell death, but is likely a result of carbon starvation, which was supported by multiple lines of evidence (imaging, transcriptomics, ATP/ADP measurement and respirometry using whole cells and cell membranes). The authors next used LC-MS to show that the mftG deletion mutant has much lower oxidised mycofactocin (MFFT-8 vs MMFT-8H2) compared to WT, suggesting an impaired ability to regenerate myofactocin redox cofactors during ethanol metabolism. These striking results were further supported by mycofactocin oxidation assays after over-expression of MftG in the native host, but also with recombinantly produced partially purified MftG from E. coli. The results showed that MftG is able to partially oxidise mycofactocin species, finally respirometry measurements with M. smegmatis membrane preparations from WT and mftG mutant cells show that the activity of MftG is indispensable for coupling of mycofactocin electron transfer to the respiratory chain. Overall, I find this study to be comprehensive and the conclusions of the paper are well supported by multiple complementary lines of evidence that are clearly presented.

Strengths

The major strengths of the paper are that it is clearly written and presented and contains multiple, complementary lines of experimental evidence that support the hypothesis that MftG is involved in the regeneration of mycofactocin cofactors, and assists with coupling of electrons derived from ethanol metabolism to the aerobic respiratory chain. The data appear to support the the authors hypotheses.

Weaknesses

No major weaknesses were identified, only minor weaknesses mostly surrounding presentation of data in some figures.

Reviewer #2 (Public Review):

Summary: Blocking a weak base compound's protonation increased intracellular diffusion and fractional recovery in the cytoplasm, which may improve the intracellular availability and distribution of weakly basic, small molecule drugs and be impactful in future drug development.

Strengths:

1) The intracellular distribution of drugs and the chemical properties that drive their distribution are much needed in the literature. Thus, the idea behind this paper is of relevance.

2) The study used common compounds that were relevant to others.

3) Altering a compound's pKa value and measuring cytosolic diffusion rates certainly is inciteful on how weak base drugs and their relatively high pKa values affect distribution and pharmacokinetics. This particular experiment demonstrated relevance to drug targeting and drug development.

4) The manuscript was fairly well written.

Weaknesses:

1) Small sample sizes. 2 acids and 1 neutral compound vs 6 weak bases (Figure 1).

2) A comparison between the percentage of neutral and weak base drug accumulation in lysosomes would have helped indicate weak base ion trapping. Such a comparison would have strengthened this study.

3) When cytosolic diffusion rates of compounds were measured, were the lysosomes extracted from the image using Imaris to determine a realistic cytosolic value? In real-time, lysosomes move through the cytosol at different rates. Because weak base drugs get trapped, it is likely the movement of a weak base in the lysosome being measured rather than the movement of a weak base itself throughout the cytosol. This was unclear in the methods. Please explain.

4) Because weak base drugs can be protonated in the cytoplasm, the authors need to elaborate on why they thought that inhibiting lysosome accumulation of weak bases would increase cytosolic diffusion rates. Ion trapping is different than "micrometers per second" in the cytosol. Moreover, treating cells with sodium azide de-acidifies lysosomes and acidifies the cytosol; thus, more protons in the cytosol means more protonation of weak base drugs. The diffusion rates were slowed down in the presence of lysosome inhibition (Figure 7), which is more fitting of the story about blocking protonation increases diffusion rates, but in this case, increasing cytosolic protonation via lysosome de-acidification agents decreases diffusion rates. Please elaborate.

A discussion of the likely impact:<br /> The manuscript certainly adds another dimension to the field of intracellular drug distribution, but the manuscript needs to be strengthened in its current form. Additional experiments need to be included, and there are clarifications in the manuscript that need to be addressed. Once these issues are resolved, then the manuscript, if the conclusions are further strengthened, is much needed and would be inciteful to drug development.

Reviewer #2 (Public Review):

Summary:

Transmembrane signaling in plants is crucial for homeostasis. In this study, the authors set out to understand to what extent catalytic activity in the EFR tyrosine kinase is required in order to transmit a signal. This work was driven by mounting data that suggest many eukaryotic kinases do not rely on catalysis for signal transduction, relying instead on conformational switching to relay information. The crucial findings reported here involve the realisation that a kinase-inactive EFR can still activate (ie lead to downstream phosphorylation) of its partner protein BAK1. Using a convincing set of biochemical, mass spectrometric (HD-exchange) and in vivo assays, the team suggests a model in which EFR is likely phosphorylated in the canonical activation segment (where two Ser residues are present), which is sufficient to generate a conformation that can activate BAK1 through dimersation. A model is put forward involving C-helix positioning in BAK1, and the model is extended to other 'non-RD' kinases in Arabidopsis kinases that likely do not require kinase activity for signaling.

Strengths:

The work uses logical and well-controlled approaches throughout, and is clear and convincing in most areas, linking data from IPs, kinase assays (including clear 32P-based biochemistry), HD-MX data (from non-phosphorylated EFR) structural biology, oxidative burst data and infectivity assays. Repetitions and statistical analysis all appear appropriate.

Overall, the work builds a convincing story and the discussion does a clear job of explaining the potential impact of these findings (and perhaps an explanation of why so many Arabidopsis kinases are 'pseudokinases', including XPS1 and XIIa6, where this is shown explicitly).

Impact:

The work is an important new step in the huge amount of follow-up work needed to examine how kinases and pseudokinases 'talk' to each other in (especially) the plant kingdom, where significant genetic expansions have occurred. The broader impact is that we might understand better how to manipulate signaling for the benefit of plants and mankind; as the authors suggest, their study is a natural progression both of their own work and the kingdom-wide study of the Kannan group.

Reviewer #2 (Public Review):

Diaphorina citri is the primary vector of Candidatus Liberibacter asiaticus (CLas), but the mechanism of how D. citri maintains a balance between lipid metabolism and increased fecundity after infection with CLas remains unknown. In their study, Li et al. presented convincing methodology and data to demonstrate that CLas exploits AKH/AKHR-miR-34-JH signaling to enhance D. citri lipid metabolism and fecundity, while simultaneously promoting CLas replication. These findings are both novel and valuable, not only have theoretical implications for expanding our understanding of the interaction between insect vectors and pathogenic microorganisms but also provide new targets for controlling D. citri and HLB in practical implications. The conclusions of this paper are well supported by data.

Reviewer #2 (Public Review):

Summary:

This paper investigates a rare and severe brain disease called Hereditary Diffuse Leukoencephalopathy with Axonal Spheroids (HDLS). The authors aimed to understand how mutations in the gene CSF-1R affect microglia, the resident immune cells in the brain, and which alterations and factors lead to the specific pathophysiology. To model the human brain with the pathophysiology of HDLS, they used the human-specific model system of induced pluripotent stem cell (iPSC)-derived forebrain organoids with integrated iPSC-derived microglia (iMicro) from patients with the HDLS-causing mutation and an isogenic cell line with the corrected genome. They found that iPSC-derived macrophages (iMac) with HDLS mutations showed changes in their response, including increased inflammation and altered metabolism. Additionally, they studied these iMacs in forebrain organoids, where they differentiate into iMicro, and showed transcriptional differences in isolated iMicro when carrying the HDLS mutation. In addition, the authors described the influence of the mutation within iMicro on the transcriptional level of neurons and neural progenitor cells (NPCs) in the organoid. They observed that the one mutation showed implications for impaired development of neurons, possibly contributing to the progression of the disease. Overall, this study provides valuable insights into the mechanisms underlying HDLS and emphasizes the importance of studying diseases like these with a suitable model system. These findings, while promising, represent only an initial step towards understanding HDLS and similar neurodegenerative diseases, and thus, their direct translation into new treatment options remains uncertain.

Strengths:

The strength of the work lies in the successful reprogramming of two HDLS patient-derived induced pluripotent stem cells (iPSCs) with different mutations, which is crucial for the study of HDLS using human forebrain organoid models. The use of corrected isogenic iPSC lines as controls increases the validity of the mutation-specific observations. In addition, the model effectively mimics HDLS, particularly concerning deficits in the frontal lobe, mirroring observations in the human brain. Obtaining iPSCs from patients with different CSF1R mutations is particularly valuable given the limitations of rodent and zebrafish models when studying adult-onset neurodegenerative diseases. The study also highlights significant metabolic changes associated with the CSF1R mutation, particularly in the HD2 mutant line, which is confirmed by the HD1 line. In addition, the work shows transcriptional upregulation of the proinflammatory cytokine IL-1beta in cells carrying the mutation, particularly when they phagocytose apoptotic cells, providing further insight into disease mechanisms.

Weaknesses:

Most of the points have been addressed in the revision, but some points remain (see below) and are well within the scope of the current manuscript in this reviewer's opinion.

(1) The characterization of iMicros is incomplete, with limited protein-level analysis (e.g. validate RNA-seq data via flow cytometry, ELISA etc.).

(2) Additionally, the claim of microglial-like morphology lacks adequate evidence, as the provided image is insufficient for such an assessment (also the newly provided Supp. Fig. 3C is insufficient and looks rather like background). Show single channels for each staining. Show examples for both cell lines.

(3) RNA-seq experiments are still difficult to read. A combination of data from both lines into one big analysis would be advantageous. E.g. showing overlapping GO terms for both lines. What is common, what is different in both lines?

(4) Statistical test information is missing in the legends.

Reviewer #2 (Public Review):

Summary:

This work presents new genetic tools for enhanced Cre-mediated gene deletion and genetic lineage tracing. The authors optimise and generate mouse models that convert temporally controlled CreER or DreER activity to constitutive Cre expression, coupled with the expression of tdT reporter for the visualizing and tracing of gene-deleted cells. This was achieved by inserting a stop cassette into the coding region of Cre, splitting it into N- and C-terminal segments. Removal of the stop cassette by Cre-lox or Dre-rox recombination results in the generation of modified Cre that is shown to exhibit similar activity to native Cre. The authors further demonstrate efficient gene knockout in cells marked by the reporter using these tools, including intersectional genetic targeting of pericentral hepatocytes.

Strengths:

The new models offer several important advantages. They enable tightly controlled and highly effective genetic deletion of even alleles that are difficult to recombine. By coupling Cre expression to reporter expression, these models reliably report Cre-expressing i.e. gene-targeted cells, and circumvent false positives that can complicate analyses in genetic mutants relying on separate reporter alleles. Moreover, the combinatorial use of Dre/Cre permits intersectional genetic targeting, allowing for more precise fate mapping.

Weaknesses:

The scenario where the lines would demonstrate their full potential compared to existing models has not been tested. Mosaic genetics is increasingly recognized as a key methodology for assessing cell-autonomous gene functions. The challenge lies in performing such experiments, as low doses of tamoxifen needed for inducing mosaic gene deletion may not be sufficient to efficiently recombine multiple alleles in individual cells while at the same time accurately reporting gene deletion. Therefore, a demonstration of the efficient deletion of multiple floxed alleles in a mosaic fashion would be a valuable addition.

In addition, a drawback of this line is the constitutive expression of Cre. When combined with the confetti line, the reporter cassette will continue flipping, potentially leading to misleading lineage tracing results. Constitutive expression of Cre is also associated with toxicity, as discussed by the authors in the introduction. These drawbacks should be acknowledged.

Reviewer #2 (Public Review):

In this study, the authors investigate how diverse bacterial species influence Orsay virus transmission and host susceptibility in C. elegans. They find that Ochrobactrum species increase infection rates, while Pseudomonas species decrease infection rates, and they identify regulators of quorum sensing and the gacA two-component system as genetic factors in the effects of Pseudomonas on infection. These findings provide important insights into the species-specific effects that bacteria can have on viral infection in C. elegans, and they may have relevance for the impact of bacterial species on viral infection in other systems. Overall the manuscript has high rigor. However, a few minor concerns are listed below.

(1) The authors state that the amount of bacteria added to each plate was standardized by seeding plates with equivalent volumes of overnight culture. This approach does not account for differences in bacterial growth rate. A more rigorous approach would be to standardize based on OD600 measurements or CFU's. Alternatively, the authors could include bacterial growth curves to demonstrate that each strain/species has reached a similar growth phase (i.e. late log) at the time of plating, as bacterial physiology and virulence is dependent on the stage of growth. At the least, if it is not possible to perform these experiments, it would be useful to include a statement that potential differences in bacterial growth rate may influence their conclusions.

(2) Line 314-315: The claim "We did not observe any potent effect on host susceptibility to infection by Orsay virus from any supernatant (Supp. Fig. 9)" is not fully supported by the data, as the data in Fig S9 only show pals-5p::GFP levels. To confirm that host susceptibility is not affected, the authors would also measure the viral infection rate and/or viral load. Otherwise, the authors should rephrase the conclusion to increase accuracy. For example, "We did not observe any potent effect on pals-5p::GFP activation upon Orsay virus infection when animals were exposed to bacterial culture supernatant".

(3) The Ct values shown in Fig 3B-F should be normalized to a reference gene (i.e. Ct values for snb-1).

Reviewer #3 (Public Review):

The work by Ghasemahmad et al. has the potential to significantly advance our understanding of how neuromodulators provide internal-state signals to the basolateral amygdala (BLA) while an animal listens to social vocalizations.

Ghasemahmad et al. made changes to the manuscript that have significantly improved the work. In particular, the transparency in showing the underlying levels of Ach, DA, and 5HIAA is excellent. My previous concerns have been adequately addressed.

Reviewer #2 (Public Review):

Cerebellar diseases can manifest as various behavioral phenotypes, such as ataxia, dystonia, and tremor. In this study, van der Heijden and colleagues aim to understand whether these differing behavioral phenotypes are associated with disease-specific changes in the firing patterns of cerebellar output neurons in the cerebellar nuclei (CN). The authors effectively demonstrate that across different mouse models of cerebellar disease, there are distinct changes in the firing properties of CN neurons. They take a crucial step further by attempting to replicate disease-specific firing patterns in the cerebellar output neurons of healthy (control) mice using optogenetics. When Purkinje cells are stimulated in a manner that results in similar firing properties in CN neurons, the authors observe a variety of atypical behavioral responses, many of which align with the behavioral phenotypes observed in mouse models of the respective diseases.

Overall, the primary results are quite convincing. Specifically, they show that (1) different mouse models of cerebellar disease exhibit different statistics of firing in CN neurons, and (2) driving CN neurons in a time-varying manner that mimics the statistics measured in disease models results in behavioral phenomena reminiscent of the disease states. These findings suggest that aberrant activity in the CN can originate from various sources (e.g., developmental circuit deficits, abnormal plasticity, insult), but ultimately, these changes are funneled through the CN neurons, whose firing rates are affected, and this, in turn, drives some portion of the aberrant behavior. This is a noteworthy observation that underscores the potential of targeting these output neurons in the treatment of cerebellar disease. Moreover, this manuscript provides valuable insights into the firing patterns associated with the most common cerebellar-dependent disease phenotypes.

However, the applicability of the classifier for identifying mice cerebellar behavioral phenotypes directly from the spiking activity of neurons in the cerebellar nuclei remains this paper's weak point. Cross-validated performance of the model on a single mouse model of tremor is, for instance, only 54%. However, a benefit of this classifier is its overall simplicity; only three parameters are required to achieve average classifier performance of 76%. While more sophisticated models might provide improved classifier performance and enhanced generalization, such models would suffer from a lack of interpretability. This paper, therefore, represents a reasonable starting point for understanding the parameter space of cerebellar nuclei firing and its relationship to behavioral phenotypes during disease.

Reviewer #2 (Public Review):

Reward and punishment learning have long been seen as emerging from separate networks of frontal and subcortical areas, often studied separately. Nevertheless, both systems are complimentary and distributed representations of reward and punishments have been repeatedly observed within multiple areas. This raised the unsolved question of the possible mechanisms by which both systems might interact, which this manuscript went after. The authors skillfully leveraged intracranial recordings in epileptic patients performing a probabilistic learning task combined with model-based information theoretical analyses of gamma activities to reveal that information about reward and punishment was not only distributed across multiple prefrontal and insular regions, but that each system showed specific redundant interactions. The reward subsystem was characterized by redundant interactions between orbitofrontal and ventromedial prefrontal cortex, while the punishment subsystem relied on insular and dorsolateral redundant interactions. Finally, the authors revealed a way by which the two systems might interact, through synergistic interaction between ventromedial and dorsolateral prefrontal cortex.

Here, the authors performed an excellent reanalysis of a unique dataset using innovative approaches, pushing our understanding on the interaction at play between prefrontal and insular cortex regions during learning. Importantly, the description of the methods and results is truly made accessible, making it an excellent resource to the community. The authors also carefully report individual subjects' data, which brings confidence in the reproducibility of their observations.

This manuscript goes beyond what is classically performed using intracranial EEG dataset, by not only reporting where a given information, like reward and punishment prediction errors, is represented but also by characterizing the functional interactions that might underlie such representations. The authors highlight the distributed nature of frontal cortex representations and proposed new ways by which the information specifically flows between nodes. This work is well placed to unify our understanding of the complementarity and specificity of the reward and punishment learning systems.

Reviewer #2 (Public Review):

Summary:

In the current manuscript, Tresenrider et al., present their recent study focusing on screening of small molecules to enhance the conversion from Müller cells (MG) to retina neurons induced by ectopic Ascl1 expression.

Strengths:

To analyze results from multiple treatment conditions in a single experiment, the authors employed a method called sci-Plex to perform scRNA-seq on mixed samples to investigate the effects of different durations of Ascl1 expression and screen for potential small molecules to promote reprogramming. Ultimately, they identified two compounds with intended activities on mouse retina. The findings may aid in future development of a cell replacement strategy for treating retinal degeneration.

Weaknesses:

The mechanistic insights are limited. Certain claims are confusing or superficial at this point.