Reviewer #2 (Public Review):

Vangl2, a core planar cell polarity protein involved in Wnt/PCP signaling, cell proliferation, differentiation, homeostasis, and cell migration. Vangl2 malfunctioning has been linked to various human ailments, including autoimmune and neoplastic disorders. Interestingly, it was shown that Vangl2 interacts with the autophagy regulator p62, and autophagic degradation limits the activity of inflammatory mediators, such as p65/NF-κB. However, the possible role of Vangl2 in inflammation has not been investigated. In this manuscript, Lu et al. describe that Vangl2 expression is upregulated in human sepsis-associated PBMCs and that Vangl2 mitigates experimental sepsis in mice by negatively regulating p65/NF-κB signaling in myeloid cells. Their mechanistic studies further revealed that Vangl2 recruits the E3 ubiquitin ligase PDLIM2 to promote K63-linked poly-ubiquitination of p65. Vangl2 also facilitated the recognition of ubiquitinated p65 by the cargo receptor NDP52. These molecular processes caused selective autophagic degradation of p65. Indeed, abrogation of PDLIM2 or NDP52 functions rescued p65 from autophagic degradation, leading to extended p65/NF-κB activity in myeloid cells. Overall, the manuscript presents convincing evidence for novel Vangl2-mediated control of inflammatory p65/NF-kB activity. The proposed pathway may expand interventional opportunities restraining aberrant p65/NF-kB activity in human ailments.

IKK is known to mediate p65 phosphorylation, which instructs NF-kB transcriptional activity. In this manuscript, Vangl2 deficiency led to an increased accumulation of phosphorylated p65 and IKK also at 30 minutes post-LPS stimulation; however, autophagic degradation of p-p65 may not have been initiated at this early time point. Therefore, this set of data put forward the exciting possibility that Vangl2 could also be regulating the immediate early phase of inflammatory response involving the IKK-p65 axis - a proposition that may be tested in future studies.

Reviewer #2 (Public Review):

The authors screened 21 E2 enzymes for their role in HTTExon1Q72-mCherry (HTT) aggregation in the Drosophila eye. They identified UBE2D, whose knockdown leads to increased HTT aggregation that can be rescued by ectopic expression of the human homolog. The protein levels of UBE2D decrease with aging and knockdown of UBED2 leads to an accumulation of ubiquitinated proteins and a shortened lifespan that can be rescued by ectopic expression of the human homolog. Knockdown of UBE2D leads to proteomic changes with up- and down-regulated proteins that include both components of the proteostasis network.

Comments on revised version:

The authors have not addressed a single critical point experimentally. Their explanations are not resolving my concerns and hence the following critical points remain:

• The readout of HTT aggregation (with methods that are not suitable) as proxy for the role of UBE2D in proteostasis is not convincing.

• UBE2D knockdown increases the number of HTT foci (Fig. 1A), but the quantification is less convincing as depicted in Fig. 1B and other E2 enzymes show a stronger effect (e.g. Ubc6 that is only studied in Figs. 1 + 2 without an explanation and Ubc84D). It does not help or add anything to this study that the authors refer to a previous publication. This review assesses this manuscript.

• The quantification of the HTT fluorescence cannot be used as proxy for HTT aggregation. The authors should assess HTT aggregation by e.g. SDD-AGE, FRAP, filter retardation etc. The quantification of the higher MW species of HTT in the SDS-PAGE is not ideal either as this simply reflects material that is stuck in the wells that could not enter the gel. Aggregation and hence high MW size could be one reason, but it can also be HTT trapped in cell debris etc. This point is critical and I disagree with the response of the authors.

• Does UBE2D ubiquitinate HTT? And thus, is HTT accumulation a suitable readout for the functional assessment of the E2 enzyme UBE2D? The authors state that UBE2D does not ubiquitinate HTT. Thus, HTT accumulation is an indirect consequence of perturbed proteostasis. There are certainly better readouts for the role of UBE2D once they have identified substrates.

• The proteomic analyses could help to identify potential substrates for UBE2D. I think its is a missed chance to not follow up on the proteomic analysis to identify substrates and define the role of UBE2D in maintainig proteostasis.

• Are there mutants available for UBE2D or conditional mutants? One caveat of RNAi are: first not complete knockdown and second, variable knockdown efficiencies that increases variability. So mutants are available and yet the authors refuse to use those.

• The analysis of the E3 enzymes does not add anything to this manuscript and the author's response that this manuscript is a follow-up study on a previous publication of the lab is certainly not a valid argument.

• The manuscript remains at this stage rather descriptive.

Reviewer #2 (Public Review):

Summary:

The authors in this study previously reported that BYL719, an inhibitor of PI3Kα, suppressed heterotopic ossification in mice model of a human genetic disease, fibrodysplasia ossificans progressive, which is caused by the activation of mutant ACVR1/R206H by Activin A. The aim of this study is to identify the mechanism of BYL719 for the inhibition of heterotopic ossification. They found that BYL719 suppressed heterotopic ossification in two ways: one is to inhibit the specification of precursor cells for chondrogenic and osteogenic differentiation and the other is to suppress the activation of inflammatory cells.

Strengths:

This study is based on authors' previous reports and the experimental procedures including the animal model are established. In addition, to confirm the role of PI3Kα, authors used the conditional knock-out mice of the subunit of PI3Kα. They clearly demonstrated the evidence indicating that the targets of PI3Kα are not members of TGFBR by a newly established experimental method.

Weaknesses:

Overall, the presented data were closely related to those previously published by authors' group or others and there were very few new findings. The molecular mechanisms through which BYL719 inhibits HO remain unclear, even in the revised manuscript.

Heterotopic ossification in the mice model was not stable and inappropriate for the scientific evaluation.

The method for chondrogenic differentiation was not appropriate, and the scientific evidence of successful differentiation was lacking.

The design of the gene expression profile comparison was not appropriate and failed to obtain the data for the main aim of this study.

The experiments of inflammatory cells were performed in cell lines without ACVR1/R206H mutation, and therefore the obtained data were not precisely related to the inflammation in FOP.

Reviewer #2 (Public Review):

Summary:

This paper reports the structures of two human biotin-dependent carboxylases. The authors used endogenously purified proteins and solved the structures in high resolutions. Based on the structures, they defined the binding site for acyl-CoA and biotin and reported the potential conformational changes in biotin position.

Strengths:

The authors effectively utilized the biotin of the two proteins and obtained homogeneous proteins from human cells. They determined the high-resolution structures of the two enzymes in apo and substrate-bound states.

Comments and questions to the manuscripts:

(1) I'm quite impressed with the protein purification and structure determination, but I think some functional characterization of the purified proteins should be included in the manuscript. The activity of enzymes should be the foundation of all structures and other speculations based on structures.

(2) In Figure 1B, the structure of MCC is shown as two layers of beta units and two layers of alpha units, while there is only one layer of alpha units resolved in the density maps. I suggest the authors show the structures resolved based on the density maps and show the complete structure with the docked layer in the supplementary figure.

(3) In the introduction, I suggest the author provide more information about the previous studies about the structure and reaction mechanisms of BDCs, what is the knowledge gap, and what problem you will resolve with a higher resolution structure. For example, you mentioned in line 52 that G437 and A438 are catalytic residues, are these residues reported as catalytic residues or this is based on your structures? Has the catalytic mechanism been reported before? Has the role of biotin in catalytic reactions revealed in previous studies?

(4) In the discussion, the authors indicate that the movement of biotin could be related to the recognition of acyl-CoA in BDCs, however, they didn't observe a change in the propionyl-CoA bound MCC structure, which is contradictory to their speculation. What could be the explanation for the exception in the MCC structure?

(5) In the discussion, the authors indicate that the selectivity of PCC to different acyl-CoA is determined by the recognition of the acyl chain. However, there are no figures or descriptions about the recognition of the acyl chain by PCC and MCC. It will be more informative if they can show more details about substrate recognition in Figures 3 and 4.

(6) How are the solved structures compared with the latest Alphafold3 prediction?

Reviewer #2 (Public Review):

This manuscript by Xue et al. describes the effects of a long noncoding RNA, lncDACH1, on the localization of Nav channel expression, the magnitude of INa, and arrhythmia susceptibility in the mouse heart. Because lncDACH1 was previously reported to bind and disrupt membrane expression of dystrophin, which in turn is required for proper Nav1.5 localization, much of the findings are inferred through the lens of dystrophin alterations.

The results report that cardiomyocyte-specific transgenic overexpression of lncDACH1 reduces INa in isolated cardiomyocytes; measurements in the whole heart show a corresponding reduction in conduction velocity and enhanced susceptibility to arrhythmia. The effect on INa was confirmed in isolated WT mouse cardiomyocytes infected with a lncDACH1 adenoviral construct. Importantly, reducing lncDACH1 expression via either a cardiomyocyte-specific knockout or using shRNA had the opposite effect: INa was increased in isolated cells, as was conduction velocity in the heart. Experiments were also conducted with a fragment of lnDACH1 identified by its conservation with other mammalian species. Overexpression of this fragment resulted in reduced INa and greater proarrhythmic behavior. Alteration of expression was confirmed by qPCR.

The mechanism by which lnDACH1 exerts its effects on INa was explored by measuring protein levels from cell fractions and immunofluorescence localization in cells. In general, overexpression was reported to reduce Nav1.5 and dystrophin levels and knockout or knockdown increased them.

The strengths of this manuscript include convincing evidence of a link between lncDACH1 and Na channel function. The identification of a lncDACH1 segment conserved among mammalian species is compelling. The observation that lncDACH1 is increased in a heart failure model and provides a plausible hypothesis for disease mechanism.

Reviewer #2 (Public Review):

In this manuscript, Cai et al use a combination of mouse transgenic lines to re-examine the question of the embryonic origin of telencephalic oligodendrocytes (OLs). Their tools include a novel Flp mouse for labelling mature oligodendrocytes and a number of pre-existing lines (some previously generated by the last author in Josh Huang's lab) that allowed combinatorial or subtractive labelling of oligodendrocytes with different origins. The conclusion is that cortically-derived OLs are the predominant OL population in the motor and somatosensory cortex and underlying corpus callosum, while the LGE/CGE generates OLs for the piriform cortex and anterior commissure rather than the cerebral cortex. Small numbers of MGE-derived OLs persist long-term in the motor, somatosensory and piriform cortex.

Strengths:<br /> The strength and novelty of the manuscript lie in the elegant tools generated and used. These have enabled the resolution of the issue regarding the contribution of different telencephalic progenitor zones to the cortical oligodendrocyte population.

Comments on latest version:

The revised manuscript by Cai et al has addressed all the issues raised. I have some minor comments:

Figure 2: The y axis in figure 2L should be the same as the y axis in 2M to make the contribution to Mo and SS more clear.

Figure 3: Although this is clear in the figure, A an B should be labelled as classical model and new model to help the reader understand immediately what the two figures show.

Suppl Fig 2: It is not clear what 1-7 represent. It should be made clear in the legend which areas have been pooled into the different bins. The X axis should be labelled.

Reviewer #2 (Public Review):

Summary:

This work addresses a puzzling finding in the viral forecasting literature: high-frequency viral variants evince signatures of neutral dynamics, despite strong evidence for adaptive antigenic evolution. The authors explicitly model interactions between the dynamics of viral adaptations and of the environment of host immune memory, making a solid theoretical and simulation-based case for the essential role of host-pathogen eco-evolutionary dynamics. While the work does not directly address improved data-driven viral forecasting, it makes a valuable conceptual contribution to the key dynamical ingredients (and perhaps intrinsic limitations) of such efforts.

Strengths:

This paper follows up on previous work from these authors and others concerning the problem of predicting future viral variant frequency from variant trajectory (or phylogenetic tree) data, and a model of evolving fitness. This is a problem of high impact: if such predictions are reliable, they empower vaccine design and immunization strategies. A key feature of this previous work is a "traveling fitness wave" picture, in which absolute fitnesses of genotypes degrade at a fixed rate due to an advancing external field, or "degradation of the environment". The authors have contributed to these modeling efforts, as well as to work that critically evaluates fitness prediction (references 11 and 12). A key point of that prior work was the finding that fitness metrics performed no better than a baseline neutral model estimate (Hamming distance to a consensus nucleotide sequence). Indeed, the apparent good performance of their well-adopted "local branching index" (LBI) was found to be an artifact of its tendency to function as a proxy for the neutral predictor. A commendable strength of this line of work is the scrutiny and critique the authors apply to their own previous projects. The current manuscript follows with a theory and simulation treatment of model elaborations that may explain previous difficulties, as well as point to the intrinsic hardness of the viral forecasting inference problem.

This work abandons the mathematical expedience of traveling fitness waves in favor of explicitly coupled eco-evolutionary dynamics. The authors develop a multi-compartment susceptible/infected model of the host population, with variant cross-immunity parameters, immune waning, and infectious contact among compartments, alongside the viral growth dynamics. Studying the invasion of adaptive variants in this setting, they discover dynamics that differ qualitatively from the fitness wave setting: instead of a succession of adaptive fixations, invading variants have a characteristic "expiring fitness": as the immune memories of the host population reconfigure in response to an adaptive variant, the fitness advantage transitions to quasi-neutral behavior. Although their minimal model is not designed for inference, the authors have shown how an elaboration of host immunity dynamics can reproduce a transition to neutral dynamics. This is a valuable contribution that clarifies previously puzzling findings and may facilitate future elaborations for fitness inference methods.

The authors provide open access to their modeling and simulation code, facilitating future applications of their ideas or critiques of their conclusions.

Weaknesses:

The current modeling work does not make direct contact with data. I was hoping to see a more direct application of the model to a data-driven prediction problem. In the end, although the results are compelling as is, this disconnect leaves me wondering if the proposed model captures the phenomena in detail, beyond the qualitative phenomenology of expiring fitness. I would imagine that some data is available about cross-immunity between strains of influenza and sarscov2, so hopefully some validation of these mechanisms would be possible.

After developing the SIR model, the authors introduce an effective "expiring fitness" model that avoids the oscillatory behavior of the SIR model. I hoped this could be motivated more directly, perhaps as a limit of the SIR model with many immune groups. As is, the expiring fitness model seems to lose the eco-evolutionary interpretability of the SIR model, retreating to a more phenomenological approach. In particular, it's not clear how the fitness decay parameter nu and the initial fitness advantage s_0 relate to the key ecological parameters: the strain cross-immunity and immune group interaction matrices.

Reviewer #2 (Public Review):

Summary:

In this study, Bisen et al. characterized the state-dependency of insulin-producing cells in the brain of *Drosophila melanogaster*. They successfully established that IPC activity is modulated by the nutritional state and age of the animal. Interestingly, they demonstrate that IPCs respond to the ingestion of glucose, rather than to perfusion with it, an observation reminiscent of the incretin effect in mammals. The study is well conducted and presented and the experimental data convincingly support the claims made.

Strengths:

The study makes great use of the tools available in *Drosophila* research, demonstrating the effect that starvation and subsequent refeeding have on the physiological activity of IPCs as well as on the behavior of flies to then establish causal links by making use of optogenetic tools.

It is particularly nice to see how the authors put their findings in context to published research and use for example TDC2 neuron activation or DH44 activity to establish baselines to relate their data to.

Weaknesses:

I find the inability of SD to rescue the IPC starvation effect in Figure 1G&H surprising, given that the fully fed flies were raised and kept on that exact diet. Did the authors try to refeed flies with SD for longer than 24 hours? I understand that at some point the age effect would also kick in and counteract potential IPC activity rescue. I think the manuscript would benefit if the authors could indicate the exact age of the SD refed flies and expand a bit on the discussion of that point.

The incretin-like effect is exciting and it will be interesting in the future to find out what might be the signal mediating this effect. It is interesting that IPCs in explants seem to be responsive to glucose. I think it would help if the authors could briefly discuss possible sources for the different findings between these in fact very different preparations. Could the the absence of the inhibitory DH44 feedback in the *ex-vivo* recordings for example play a role?

The incretin-like effect the authors observed seems to start only after 5h which seems longer than in mammals where, as far as I know, insulin peaks around 1h. Do the authors have ideas on how this timescale relates to ingestion and glucose dynamics in flies?

The authors mention "a decrease in the FV of IPC-activated starved flies even before the first optogenetic stimulation (Figure 2I),". Could this be addressed by running an experiment in darkness, only using the IR illumination of their behavioral assay?

The authors show an inhibitory effect of DH44 neuron activation on IPC activity. They further demonstrate that DH44PI neurons are not the ones driving this and thus conclude that "...IPCs are inhibited by DH44Ns outside the PI.". As the authors mentioned the broad expression of the DH44-Gal4 line, can they be sure that the cells labeled outside the PI are actually DH44+? If so they should state this more clearly, if not they should adapt the discussion accordingly.

Reviewer #2 (Public Review):

Summary:

In this study the authors have used pull-down experiments in a cell line overexpressing tagged SERPINE1 mRNA binding protein 1 (SERBP1) followed by mass spectrometry-based proteomics, to establish its interactome. Extensive analyses are performed to connect the data to published resources. The authors attempt to connect SERBP1 to stress granules and Alzheimer's disease-associated tau pathology. Based on the interactome, the authors propose a cross-talk between SERBP1 and PARP1 functions.

Strengths:

The main strength of this study lies in the proteomics data analysis, and its effort to connect the data to published studies.

Weaknesses:

While the authors propose a feedback regulatory model for SERBP1 and PARP1 functions, strong evidence for PARylation modulating SERBP1 functions is lacking. PARP inhibition decreasing the amount of PARylated proteins associated with SERBP1 and likely all other PARylated proteins is expected. This study is also incomplete in its attempt to establish a connection to Alzheimer's disease related tauopathy. A single AD case is not sufficient, and frozen autopsy tissue shows unexplained punctate staining likely due to poor preservation of cellular structures for immunohistochemistry. There is a lack of essential demographic data, source of the tissue, brain regions shown, and whether there was an IRB protocol for the human brain tissue. The presence of phase-separated transient stress granules in an autopsy brain is unlikely, even if G3BP1 staining is present. Normally, stress granule proteins move to the cytoplasm under cellular stress, whereas SERBP1 becomes nuclear. The co-localization of abundant cytoplasmic G3BP1 and SERBP1 under normal conditions does not indicate an association with stress granules.

Reviewer #2 (Public Review):

Summary:

In their manuscript titled "Microbiota from Young Mice Counteracts Susceptibility to Age-Related Gout through Modulating Butyric Acid Levels in Aged Mice," the authors report that fecal transplantation from young mice into old mice alleviates susceptibility to gout. The gut microbiota in young mice is found to inhibit activation of the NLRP3 inflammasome pathway and reduce uric acid levels in the blood in the gout model.

Strengths:

They focused on the butanoate metabolism pathway based on the results of metabolomics analysis after fecal transplantation and identified butyrate as the key factor in mitigating gout susceptibility. In general, this is a well-performed study.

Weaknesses:

The discussion on the current results and previous studies regarding the effect of butyrate on gout symptoms is insufficient. The authors need to provide a more thorough discussion of other possible mechanisms and relevant literature.

Reviewer #2 (Public Review):

Summary:

The authors have collected a significant amount of data from the literature on the flow regimes associated with microorganisms whose propulsion is achieved through the action of cilia or flagella, with particular interest in the competition between sessile and motile lifestyles. They then use several distinct hydrodynamic models for the cilia-driven flows to quantify the nutrient uptake and clearance rate, reported as a function of the Peclet number. Among the interesting conclusions the authors draw concerns the question of whether, for certain ciliates, there is a clear difference in nutrient uptake rates in the sessile versus motile forms. The authors show that this is not the case, thereby suggesting that the evolutionary pressure associated with such a difference is not present. The analysis also includes numerical calculations of the uptake rate for spherical swimmers in the regime of large Peclet numbers, where the authors note an enhancement due to advection-generated thinning of the solutal boundary layer around the organism.

Strengths:

In addressing the whole range of organism sizes and Peclet numbers the authors have achieved an important broad perspective on the problem of nutrient uptake of ciliates, with implications for understanding evolutionary driving forces toward particular lifestyles (e.g. sessile versus motile).

Weaknesses:

The authors appear to be unaware of rather similar calculations that were done some years ago in the context of Volvox, in which the issue of the boundary layer size and nutrient uptake enhancement were clearly recognized [M.B. Short, et al., Flows Driven by Flagella of Multicellular Organisms Enhance Long-Range Molecular Transport, PNAS 103, 8315-8319 (2006)]. This reference also introduced the model of a fixed shear stress at the surface of the sphere as a representation of the action of the cilia, which may be more realistic than the squirmer-type boundary condition, although the two lead to similar large-Pe scalings.

The findings reported in Figure 4, that the uptake rate is robust to variations in cilia coverage and absorption fraction, are similar in spirit to an observation made recently in the context of the somatic cell neighbourhood areas in Vovox [Day, et al., eLife 11, e72707 (2022)]. There, it was found that while there is a broad distribution of those areas, and hence of the coarse-grained tangential flagellar force acting on the fluid, the propulsion speed is rather insensitive to those variations.

Reviewer #2 (Public Review):

Summary:

Translation of CGG repeats leads to the accumulation of poly G, which is associated with neurological disorders. This is a valuable paper in which the authors sought out proteins that modulate RAN translation. They determined which proteins in Hela cells bound to CGG repeats and affected levels of polyG encoded in the 5'UTR of the FMR1 mRNA. They then showed that siRNA depletion of ribosomal protein RPS26 results in less production of FMR1polyG than in control. There are data supporting the claim that RPS26 depletion modulates RAN translation in this RNA, although for some results, the Western results are not strong. The data to support increased aggregation by polyG expression upon S26 KD are incomplete.

Strengths:

The authors have proteomics data that show the enrichment of a set of proteins on FMR1 RNA but not a related RNA.

Weaknesses:

-It is insinuated that RPS26 binds the RNA to enhance CGG-containing protein expression. However, RPS26 reduction was also shown previously to affect ribosome levels, and reduced ribosome levels can result in ribosomes translating very different RNA pools.

-A significant claim is that RPS26 KD alleviates the effects of FMR polyG expression, but those data aren't presented well.

Reviewer #3 (Public Review):

Summary:

This article is a direct follow-up to the paper published last year in eLife by the same group. In the previous article, the authors discovered a zinc finger protein, Kipferl, capable of guiding the HP1 protein Rhino towards certain genomic regions enriched in GRGGN motifs and packaged in heterochromatin marked by H3K9me3. Unlike other HP1 proteins, Rhino recruitment activates the transcription of heterochromatic regions, which are then converted into piRNA source loci. The molecular mechanism by which Kipferl interacts specifically with Rhino (via its chromodomain) and not with other HP1 proteins remained enigmatic.

In this latest article, the authors go a step further by elucidating the molecular mechanisms important for the specific interaction of Rhino and not other HP1 proteins with Kipferl. A phylogenetic study carried out between the HP1 proteins of 5 Drosophila species led them to study the importance of an AA Glycine at position 31 located in the Rhino chromodomain, an AA different from the AA (aspartic acid) found at the same position in the other HP1 proteins. The authors then demonstrate, through a series of structure predictions, biochemical and genetic experiments, that this specific AA in the Rhino-specific chromodomain explains the difference in the chromatin binding pattern between Rhino and the other Drosophila HP1 proteins. Importantly, the G31D conversion of the Rhino protein prevents interaction between Rhino and Kipferl, phenocopying a Kipfer mutant.

Strengths:

The strength of this study is to test at the molecular and genetic level whether the difference in the AA sequence- encovered by phylogenetic analysis of HP1 proteins including Rhino combined with structure prediction- can explain the difference in chromatin binding patterns between HP1 proteins and Rhino.<br /> To do so they have created a Rhino mutant by introducing a point mutation into the endogenous rhino gene, reverting the Glycine in position 31 to the aspartic acid found in all other HP1 proteins. Even if the Rhino G31D mutant retains its ability to interact with H3K9me3 (predictive and biochemistry approaches that I'm less familiar with) it does not localize correctly on the chromatin preventing certain regions such as locus 80F from being converted into piRNA source loci. However other regions such as satellite regions attract the Rhino mutant protein converting them into super piRNA source loci, phenocopying the effects observed in a Kipferl mutant. Why Rhino when not bound to Kipferl concentrates in satellite regions is a question that remains unanswered.

Weaknesses:

In this new version of the manuscript, the authors have answered all the questions and weaknesses raised previously.

Reviewer #2 (Public Review):

Summary:

Jellinger, Suthard, et al. investigated the transcriptome of positive and negative valence engram cells in the ventral hippocampus, revealing anti- and pro-inflammatory signatures of these respective valences. The authors further reactivated the negative valence engram ensembles to assay the effects of chronic negative memory reactivation in young and old mice. This chronic re-activation resulted in differences in aspects of working memory, fear memory, and caused morphological changes in glia. Such reactivation-associated changes are putatively linked to GABA changes and behavioral rumination.

Strengths:

Much the content of of this manuscript is of benefit to the community, such as the discovery of differential engram transcriptomes dependent on memory valence. The chronic activation of neurons, and the resultant effects on glial cells and behavior, also provide the community with important data. Laudable points of this manuscript include the comprehensiveness of behavioral experiments, as well as the cross-disciplinary approach.

Weaknesses:

Weaknesses noted in the previous version of the manuscript have been accounted for.

Reviewer #3 (Public Review):

Summary:

In their article Jack Lindsey and Ashok Litwin-Kumar describe a new model for systems memory consolidation. Their idea is that a short-term memory acts not as a teacher for a long-term memory - as is common in most complementary learning systems -, but as a selection module that determines which memories are eligible for long term storage. The criterion for the consolidation of a given memory is a sufficient strength of recall in the short term memory.

The authors provide an in-depth analysis of the suggested mechanism. They demonstrate that it allows substantially higher SNRs than previous synaptic consolidation models, provide an extensive mathematical treatment of the suggested mechanism, show that the required recall strength can be computed in a biologically plausible way for three different learning paradigms, and illustrate how the mechanism can explain spaced training effects.

Strengths:

The suggested consolidation mechanism is novel and provides a very interesting alternative to the classical view of complementary learning systems. The analysis is thorough and convincing.

Weaknesses:

The main weakness of the paper is the equation of recall strength with the synaptic changes brought about by the presentation of a stimulus. In most models of learning, synaptic changes are driven by an error signal and hence cease once the task has been learned. The suggested consolidation mechanism would stop at that point, although recall is still fine. The authors should discuss other notions of recall strength that would allow memory consolidation to continue after the initial learning phase. Aside from that, I have only a few technical comments that I'm sure the authors can address with a reasonable amount of work.

Reviewer #2 (Public Review):

The paper from Liu et al shows a mechanism by which axons can change direction during development. They use the sLNv neurons as a model. They find that the appearance of a new group of neurons (DNs) during post-embryonic proliferation secretes netrins and repels horizontally towards the midline, the axonal tip of the LNvs. The experiments are well done and the results are conclusive.

Reviewer #2 (Public Review):

Summary:

Here the effect of overall transcription blockade, and then specifically depletion of YAP/TAZ transcription factors was tested on cytoskeletal responses, starting from a previous paper showing YAP/TAZ-mediated effects on the cytoskeleton and cell behaviors. Here, primary endothelial cells were assessed on substrates of different stiffness and parameters such as migration, cell spreading, and focal adhesion number/length were tested upon transcriptional manipulation. Zebrafish subjected to similar manipulations were also assessed during the phase of intersegmental vessel elongation. The conclusion was that there is a feedback loop of 4 hours that is important for the effects of mechanical changes to be translated into transcriptional changes that then permanently affect the cytoskeleton.

The idea is intriguing and a previous paper contains data supporting the overall model. The fish washout data is quite interesting and supports the kinetics conclusions. New transcriptional profiling in this version supports that cytoskeletal genes are differentially regulated with YAP/TAZ manipulations.

Major strengths: The combination of in vitro and in vivo assessment provides evidence for timing in physiologically relevant contexts, and rigorous quantification of outputs is provided. The idea of defining temporal aspects of the system is quite interesting. New RNA profiling supports the model.

Weaknesses:

Actinomycin D blocks most transcription so exposure for hours likely leads to secondary and tertiary effects and perhaps effects on viability.

Reviewer #2 (Public Review):

The manuscript by Okholm and colleagues identified an interesting new instance of ceRNA involving a circular RNA. The data are clearly presented and support the conclusions. Quantification of the copy number of circRNA and quantification of the protein were performed, and this is important to support the ceRNA mechanism.

This is the second rebuttal and the authors further improved the manuscript. The data are of interest to the large spectrum of readers of the journal.

Comments on revision:

The authors explain that they have compared primer efficiencies of two linear Laccase version amplicons and their divergent primers targeting circHIPK3 using amplification standard curves (not shown). They claim that all amplicons were found to be directly comparable, ensuring that their estimation of cirRNA:lineal ratio estimation by RT-qPCR was accurate. I agree that this is not a technically trivial experiment. However, for this measurement to be valid, it is not enough to compare the efficiencies of primers using cDNA/DNA standard curves in the context of the qPCR reaction alone. Instead, one should perform the full RT-qPCR tandem reactions in the context of standard curves of the specific RNAs (for example, obtained by in vitro synthesis). RNA absolute amounts in these standard curves should be known in order to compare the different RNA species (linear or circular).

I do not have major concerns about this issue.

Reviewer #2 (Public Review):

The authors of this study have investigated how oscillations may promote fear learning using a network model. They distinguished three types of rhythmic activities and implemented an STDP rule to the network aiming to understand the mechanisms underlying fear learning in the BLA. My comments are the following.

(1) Gamma oscillations are generated locally; thus, it is appropriate to model in any cortical structure. However, the generation of theta rhythms is based on the interplay of many brain areas therefore local circuits may not be sufficient to model these oscillations. Moreover, to generate the classical theta, a laminal structure arrangement is needed (where neurons form layers like in the hippocampus and cortex)(Buzsaki, 2002), which is clearly not present in the BLA. To date, I am not aware of any study which has demonstrated that theta is generated in the BLA. All studies that recorded theta in the BLA performed the recordings referenced to a ground electrode far away from the BLA, an approach that can easily pick up volume conducted theta rhythm generated e.g., in the hippocampus or other layered cortical structure. To clarify whether theta rhythm can be generated locally, one should have conducted recordings referenced to a local channel (see Lalla et al., 2017 eNeuro). In summary, at present, there is no evidence that theta can be generated locally within the BLA. Though, there can be BLA neurons, firing of which shows theta rhythmicity, e.g., driven by hippocampal afferents at theta rhythm, this does not mean that theta rhythm per se can be generated within the BLA as the structure of the BLA does not support generation of rhythmic current dipoles. This questions the rationale of using theta as a proxy for BLA network function which does not necessarily reflect the population activity of local principal neurons in contrast to that seen in the hippocampus.

(2) The authors distinguished low and high theta. This may be misleading, as the low theta they refer to is basically a respiratory-driven rhythm typically present during an attentive state (Karalis and Sirota, 2022; Bagur et al., 2021, etc.). Thus, it would be more appropriate to use breathing-driven oscillations instead of low theta. Again, this rhythm is not generated by the BLA circuits, but by volume conducted into this region. Yet, the firing of BLA neurons can still be entrained by this oscillation. I think it is important to emphasize the difference.

(3) The authors implemented three interneuron types in their model, ignoring a large fraction of GABAergic cells present in the BLA (Vereczki et al., 2021). Recently, the microcircuit organization of the BLA has been more thoroughly uncovered, including connectivity details for PV interneurons, firing features of neurochemically identified interneurons (instead of mRNA expression-based identification, Sosulina et al., 2010), synaptic properties between distinct interneuron types as well as principal cells and interneurons using paired recordings. These recent findings would be vital to incorporate into the model instead of using results obtained in the hippocampus and neocortex. I am not sure that a realistic model can be achieved by excluding many interneuron types.

(4) The authors set the reversal potential of GABA-A receptor-mediated currents to -80 mV. What was the rationale for choosing this value? The reversal potential of IPSCs has been found to be -54 mV in fast-spiking (i.e., parvalbumin) interneurons and around -72 mV in principal cells (Martina et al., 2001, Veres et al., 2017).

(5) Proposing neuropeptide VIP as a key factor for learning is interesting. Though, it is not clear why this peptide is more important in fear learning in comparison to SST and CCK, which are also abundant in the BLA and can effectively regulate the circuit operation in cortical areas.

Reviewer #2 (Public Review):

The authors of this study have investigated how oscillations may promote fear learning using a network model. They distinguished three types of rhythmic activities and implemented an STDP rule to the network aiming to understand the mechanisms underlying fear learning in the BLA.

After the revision, the fundamental question, namely, whether the BLA networks can or cannot intrinsically generate any theta rhythms, is still unanswered. The author added this sentence to the revised version: "A recent experimental paper, (Antonoudiou et al., 2022), suggests that the BLA can intrinsically generate theta oscillations (3-12 Hz) detectable by LFP recordings under certain conditions, such as reduced inhibitory tone." In the cited paper, the authors studied gamma oscillations, and when they applied 10 uM Gabazine to the BLA slices observed rhythmic oscillations at theta frequencies. 10 uM Gabazine does not reduce the GABA-A receptor-mediated inhibition but eliminates it, resulting in rhythmic populations burst driven solely by excitatory cells. Thus, the results by Antonoudiou et al., 2022 contrast with, and do not support, the present study, which claims that rhythmic oscillations in the BLA depend on the function of interneurons. Thus, there is still no convincing evidence that BLA circuits can intrinsically generate theta oscillations in intact brain or acute slices. If one extrapolates from the hippocampal studies, then this is not surprising, as the hippocampal theta depends on extra-hippocampal inputs, including, but not limited to the entorhinal afferents and medial septal projections (see Buzsaki, 2002). Similarly, respiratory related 4 Hz oscillations are also driven by extrinsic inputs. Therefore, at present, it is unclear which kind of physiologically relevant theta rhythm in the BLA networks has been modelled.

Reviewer #2 (Public Review):

Summary:

Molecular dynamics (MD) data is deposited in public, non-specialist repositories. This work starts from the premise that these data are a valuable resource as they could be used by other researchers to extract additional insights from these simulations; it could also potentially be used as training data for ML/AI approaches. The problem is that mining these data is difficult because they are not easy to find and work with. The primary goal of the authors was to discover and index these difficult-to-find MD datasets, which they call the "dark matter of the MD universe" (in contrast to data sets held in specialist databases).

The authors developed a search strategy that avoided the use of ill-defined metadata but instead relied on the knowledge of the restricted set of file formats used in MD simulations as a true marker for the data they were looking for. Detection of MD data marked a data set as relevant with a follow-up indexing strategy of all associated content. This "explore-and-expand" strategy allowed the authors for the first time to provide a realistic census of the MD data in non-specialist repositories.

As a proof of principle, they analyzed a subset of the data (primarily related to simulations with the popular Gromacs MD package) to summarize the types of simulated systems (primarily biomolecular systems) and commonly used simulation settings.

Based on their experience they propose best practices for metadata provision to make MD data FAIR (findable, accessible, interoperable, reusable).

A prototype search engine that works on the indexed datasets is made publicly available. All data and code are made freely available as open source/open data.

Strengths:

- The novel search strategy is based on relevant data to identify full datasets instead of relying on metadata and thus is likely to have many true positives and few false positives.

- The paper provides a first glimpse at the potential hidden treasures of MD simulations and force field parametrizations of molecules.

- Analysis of parameter settings of MD simulations from how researchers *actually* run simulations can provide valuable feedback to MD code developers for how to document/educate users. This approach is much better than analyzing what authors write in the Methods sections.

- The authors make a prototype search engine available.

- The guidelines for FAIR MD data are based on experience gained from trying to make sense of the data.

Weaknesses:

- So far the work is a proof-of-concept that focuses on MD data produced by Gromacs (which was prevalent under all indexed and identified packages).

As discussed in the manuscript, some types of biomolecules are likely underrepresented because different communities have different preferences for force fields/MD codes (for example: carbohydrates with AMBER/GLYCAM using AMBER MD instead of Gromacs).

- Materials sciences seem to be severely under-represented - commonly used codes in this area such as LAMMPS are not even detected, and only very few examples could be identified. As it is, the paper primarily provides an insight into the *biomolecular* MD simulation world.

The authors succeed in providing a first realistic view on what MD data is available in public repositories. In particular, their explore-expand approach has the potential to be customized for all kinds of specialist simulation data, whereby specific artifacts are<br /> used as fiducial markers instead of metadata. The more detailed analysis is limited to Gromacs simulations and primarily biomolecular simulations (even though MD is also widely used in other fields such as the materials sciences). This restricted view may simply be correlated with the user community of Gromacs and hopefully, follow-up studies from this work will shed more light on this shortcoming.

The study quantified the number of trajectories currently held in structured databases as ~10k vs ~30k in generalist repositories. To go beyond the proof-of-principle analysis it would be interesting to analyze the data in specialist repositories in the same way as the one in the generalist ones, especially as there are now efforts underway to create a database for MD simulations (Grant 'Molecular dynamics simulation for biology and chemistry research' to establish MDDB' DOI 10.3030/101094651). One should note that structured databases do not invalidate the approach pioneered in this work; if anything they are orthogonal to each other and both will likely play an important role in growing the usefulness of MD simulations in the future.

Reviewer #2 (Public Review):

Summary:

The paper sought to determine the number of myosin 10 molecules per cell and localized to filopodia, where they are known to be involved in formation, transport within, and dynamics of these important actin-based protrusions. The authors used a novel method to determine the number of molecules per cell. First, they expressed HALO tagged Myo10 in U20S cells and generated cell lysates of a certain number of cells and detected Myo10 after SDS-PAGE, with fluorescence and a stained free method. They used a purified HALO tagged standard protein to generate a standard curve which allowed for determining Myo10 concentration in cell lysates and thus an estimate of the number of Myo10 molecules per cell. They also examined the fluorescence intensity in fixed cell images to determine the average fluorescence intensity per Myo10 molecule, which allowed the number of Myo10 molecules per region of the cell to be determined. They found a relatively small fraction of Myo10 (6%) localizes to filopodia. There are hundreds of Myo10 in each filopodia, which suggests some filopodia have more Myo10 than actin binding sites. Thus, there may be crowding of Myo10 at the tips, which could impact transport, the morphology at the tips, and dynamics of the protrusions themselves. Overall, the study forms the basis for a novel technique to estimate the number of molecules per cell and their localization to actin-based structures. The implications are broad also for being able to understand the role of myosins in actin protrusions, which is important for cancer metastasis and wound healing.

Strengths:

The paper addresses an important fundamental biological question about how many molecular motors are localized to a specific cellular compartment and how that may relate to other aspects of the compartment such as the actin cytoskeleton and the membrane. The paper demonstrates a method of estimating the number of myosin molecules per cell using the fluorescently labeled HALO tag and SDS-PAGE analysis. There are several important conclusions from this work in that it estimates the number of Myo10 molecules localized to different regions of the filopodia and the minimum number required for filopodia formation. The authors also establish a correlation between number of Myo10 molecules filopodia localized and the number of filopodia in the cell. There is only a small % of Myo10 that tip localized relative to the total amount in the cell, suggesting Myo10 have to be activated to enter the filopodia compartment. The localization of Myo10 is log-normal, which suggests a clustering of Myo10 is a feature of this motor.

One of the main critiques of the manuscript was that the results were derived from experiments with overexpressed Myo10 and therefore are hard to extrapolate to physiological conditions. The authors counter this critique with the argument that their results provide insight into a system in which Myo10 is a limiting factor for controlling filopodia formation. They demonstrate that U20S cells do not express detectable levels of Myo10 (supplementary Figure 1E) and thus introducing Myo10 expression demonstrates how triggering Myo10 expression impacts filopodia. An example is given how melanoma cells often heavily upregulation Myo10.

In addition, the revised manuscript addresses the concerns about the method to quantitate the number of Myo10 molecules per cell and therefore puncta in the cell. The authors have now made a good faith effort to correct for incomplete labeling of the HALO tag (Figure 2A-C, supplementary Figure 2D-E). The authors also address the concerns about variability in transfection efficiency (Figure 1D-E).

A very interesting addition to the revised manuscript was the quantitation of the number of Myo10 molecules present during an initiation event when a newly formed filopodia just starts to elongate from the plasma membrane. They conclude that 100s of Myo10 molecules are present during an initiation event. They also examined other live cell imaging events in which growth occurs from a stable filopodia tip and correlated with elongation rates.

Weaknesses:

The authors acknowledge that a limitation of the study is that all of the experiments were performed with overexpressed Myo10. They address this limitation in the discussion but also provide important comparisons for how their work relates to physiological conditions, such as melanoma cells that only express large amounts of Myo10 when they are metastatic. Also, the speculation about how fascin can outcompete Myo10 should include a mechanism for how the physiological levels of fascin can complete with the overabundance of Myo10 (page 10, lines 401-408).

Reviewer #2 (Public Review):

Summary:

Most polymerases and nucleases use two or three divalent metal ions in their catalytic functions. The family of His-Me nucleases, however, use only one divalent metal ion, along with a conserved histidine, to catalyze DNA hydrolysis. The mechanism has been studied previously but, according to the authors, it remained unclear. By use of a time resolved X-ray crystallography, this work convincingly demonstrated that only one M2+ ion is involved in the catalysis of the His-Me I-PpoI 19 nuclease, and proposed concerted functions of the metal and the histidine.

Strengths:

This work performs mechanistic studies, including the number and roles of metal ion, pH dependence, and activation mechanism, all by structural analyses, coupled with some kinetics and mutagenesis. Overall, it is a highly rigorous work. This approach was first developed in Science (2016) for a DNA polymerase, in which Yang Cao was the first author. It has subsequently been applied to just 5 to 10 enzymes by different labs, mainly to clarify two versus three metal ion mechanisms. The present study is the first one to demonstrate a single metal ion mechanism by this approach.

Furthermore, on the basis of the quantitative correlation between the fraction of metal ion binding and the formation of product, as well as the pH dependence, and the data from site-specific mutants, the authors concluded that the functions of Mg2+ and His are a concerted process. A detailed mechanism is proposed in Figure 6.

Even though there are no major surprises in the results and conclusions, the time-resolved structural approach and the overall quality of the results represent a significant step forward for the Me-His family of nucleases. In addition, since the mechanism is unique among different classes of nucleases and polymerases, the work should be of interest to readers in DNA enzymology, or even mechanistic enzymology in general.

Weaknesses:

Two relatively minor issues are raised here for consideration:<br /> p. 4, last para, lines 1-2: "we next visualized the entire reaction process by soaking I-PpoI crystals in buffer....". This is a little over-stated. The structures being observed are not reaction intermediates. They are mixtures of substrates and products in the enzyme-bound state. The progress of the reaction is limited by the progress of the soaking of the metal ion. Crystallography has just been used as a tool to monitor the reaction (and provide structural information about the product). It would be more accurate to say that "we next monitored the reaction progress by soaking....".

p. 5, the beginning of the section. The authors on one hand emphasized the quantitative correlation between Mg ion density and the product density. On the other hand, they raised the uncertainty in the quantitation of Mg2+ density versus Na+ density, thus they repeated the study with Mn2+ which has distinct anomalous signals. This is a very good approach. However, there is still no metal ion density shown in the key Figure 2A. It will be clearer to show the progress of metal ion density in a figure (in addition to just plots), whether it is Mg or Mn.

Reviewer #2 (Public Review):

Summary:

The authors demonstrate that a low parenteral glucose regimen can lead to improved bacterial clearance and survival from Staph epi sepsis in newborn pigs without inducing hypoglycemia, as compared to a high glucose regimen. Using RNA-seq, metabolomic, and proteomic data, the authors conclude that this is primarily mediated by altered hepatic metabolism.

Strengths:

Well-defined controls for every time point, with multiple time points and biological replicates.

The authors used different experimental strategies to arrive at the same conclusion, which lends credibility to their findings.

The authors have published the negative findings associated with their study, including the inability to reverse sepsis-related mortality after switching from SE-high to SE-low at 3h or 6h and after administration of hIAIP.

Weaknesses:

(1) The authors mention, and it is well-known, that Staph epi is primarily involved in late-onset sepsis. The model of S. epi sepsis used in this study clearly replicates early-onset sepsis, but S. epi is extremely rare in this time period. How do the authors justify the clinical relevance of this model?

(2) The authors find that the neutrophil subset of the leukocyte population is diminished significantly in the SE-low and SE-high populations. However, they conclude on page 10 that "modulations of hepatic, but not circulating immune cell metabolism, by reduced glucose supply..." and this is possible because the authors have looked at the entire leukocyte transcriptome. I am curious about why the authors did not sequence the neutrophil-specific transcriptome.

(3) The authors use high (30g/k/d) and low (7.2g/k/d) glucose regimens. These translate into a GIR of 21 and 5 mg/k/min respectively. A normal GIR for a preterm infant is usually 5-8, and sometimes up to 10. Do the authors have a "safe GIR" or a threshold they think we cannot cross? Maybe a point where the metabolism switch takes place? They do not comment on this, especially as GIR and glucose levels are continuous variables and not categorical.

(4) In Figures 2B and C the authors show that SE-high and SE-low animals have differences in the oxphos, TCA, and glycolytic pathways. The authors themselves comment in the Supplementary Table S1B, E-F that these same metabolic pathways are also different in the Con-Low and Con-high animals, it is just the inflammatory pathways that are not different in the non-infected animals. How can they then justify that it is these metabolic pathways specifically which lead to altered inflammatory pathways, and not just the presence of infection along with some other unfound mechanism?

(5) The authors mention in Figure 1F that SE-low animals had lower bacterial burdens than SE-high animals, but then go on to infer that the inflammatory cytokine differences are attributed to a rewiring of the immune response. However, they have not normalized the cytokine levels to the bacterial loads, as the differences in the cytokines might be attributed purely to a difference in bacterial proliferation/clearing.

(6) The authors mention that switching from SE-high to SE-low at 3 or 6 h time points does not reduce mortality. Have the authors considered the reverse? Does hyperglycemia after euglycemia initially, worsen mortality? That would really conclude that there is some metabolic reprogramming happening at the very onset of sepsis and it is a lost battle after that.

Reviewer #2 (Public Review):

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria and plays a critical role in bacterial virulence. The LPS export mechanism is a potential target for new antibiotics. Inhibiting this process can render bacteria more susceptible to the host immune system or other antibacterial agents. Given the rise of antibiotic-resistant bacteria, novel targets are urgently needed. The seven LPS transport (Lpt) proteins, A-G, move LPS from the inner to the outer membrane. This study investigated the conformational changes in the LptB2FG-LptC complex using site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy, revealing how ATP binding and hydrolysis affect the LptF β-jellyroll domain and lateral gates. The findings highlight the role of LptC in regulating LPS entry, ensuring efficient and unidirectional transport across the periplasm.

The β-jellyrolls are not fully resolved in the vanadate-trapped structure of LptB2FG and LptB2FGC. Therefore, the current study provides valuable information on the functional dynamics of these periplasmic domains, their interactions, and their roles in the unidirectional transport of LPS. Additionally, the dynamic perspective of the lateral gates in LptFG in the presence and absence of LptC is another strength of this study. Moreover, at least in detergent samples, more comprehensive intermediates of the ATP turnover cycle are studied than in the available structures, providing crucial missing mechanistic details.

Other major strengths of the study include high-quality DEER distance measurements in both detergent and proteoliposomes, the latter providing valuable dynamics information in the lipid environment. However, lipid composition is not mentioned. The proteoliposome study is crucial since the previous structural study (Li, Orlando & Liao 2019) was done in rather small-diameter nanodiscs, which might affect the overall dynamics of the complex. It would have been beneficial if the investigators had reconstituted the complex in lipid nanodiscs with the same composition as proteoliposomes. The mixed lipid/detergent micelles provide an alternative. It seems the ATPase activity of the protein complex is much lower in detergent compared with lipid nanodiscs (Li, Orlando & Liao 2019). In the current study, ATPase activity in proteoliposomes is not provided. Also, the reviewer assumes cysteine-less (CL) constructs of the complex components were utilized. The ATPase assay on CL complex is not presented.

Additionally, from previous structural studies and the mass spectrometry data presented here, LPS co-purifies and is already bound to the complex, thus the Apo state may represent the LPS-bound state without nucleotides.

The selection of sites to probe lateral gate 2, which forms the main LPS entry site, may pose an issue. Although the authors provide justification based on the available structures, one site (position 325 in LptF) is located on a flexible loop, and position 52 in LptG is on the neighboring transmembrane helix, separated by a potentially flexible loop from the gating TM1. These labeling sites could exhibit significant local dynamics, resulting in a broader distribution of distances and potentially masking the gating-related conformational changes.

Reviewer #2 (Public Review):

Summary:

Using an innovative task design and analysis approach, the authors set out to show that the activity patterns in the hippocampus related to the development of social relationships with multiple partners in a virtual game. While I found the paper highly interesting (and would be thrilled if the claims made in the paper turned out to be true), I found many of the analyses presented either unconvincing or slightly unconnected to the claims that they were supposed to support. I very much hope the authors can alleviate these concerns in a revision of the paper.

Strengths & Weaknesses:

(1) The innovative task design and analyses, and the two independent samples of participants are clear strengths of the paper.

(2) The RSA analysis is not what I expected after I read the abstract and tile of the result section "The hippocampus represents abstract dimensions of affiliation and power". To me, the title suggests that the hippocampus has voxel patterns, which could be read out by a downstream area to infer the affiliation and power value, independent of the exact identity of the character in the current trial. The presented RSA analysis however presents something entirely different - namely that the affiliation trials and power trials elicit different activity patterns in the area indicated in Figure 3. What is the meaning of this analysis? It is not clear to me what is being "decoded" here and alternative explanations have not been considered. How do affiliation and power trials differ in terms of the length of sentences, complexity of the statements, and reaction time? Can the subsequent decision be decoded from these areas? I hope in the revision the authors can test these ideas - and also explain how the current RSA analysis relates to a representation of the "dimensions of affiliation and power".

(3) Overall, I found that the paper was missing some more fundamental and simpler RSA analyses that would provide a necessary backdrop for the more complicated analyses that followed. Can you decode character identity from the regions in question? If you trained a simple decoder for power and affiliation values (using the LLE, but without consideration of the sequential position as used in the spline analysis), could you predict left-out trials? Are affiliation and power represented in a way that is consistent across participants - i.e. could you train a model that predicts affiliation and power from N-1 subjects and then predict the Nth subject? Even if the answer to these questions is "no", I believe that they are important to report for the reader to get a full understanding of the nature of the neural representations in these areas. If the claim is that the hippocampus represents an "abstract" relationship space, then I think it is important to show that these representations hold across relationships. Otherwise, the claim needs to be adjusted to say that it is a representation of a relationship-specific trajectory, but not an abstract social space.

(4) To determine that the location of a specific character can be decoded from the hippocampal activity patterns, the authors use a sequential analysis in a low-dimensional space (using local linear embedding). In essence, each trial is decoded by finding the pair of two temporally sequential trials that is closest to this pattern, and then interpolating the power/affiliation values linearly between these two points. The obvious problem with this analysis is that fMRI pattern will have temporal autocorrelation and the power and affiliation values have temporal autocorrelation. Successful decoding could just reflect this smoothness in both time series. The authors present a series of control analyses, but I found most of them to not be incisive or convincing and I believe that they (and their explanation of their rationale) need to be improved. For example, the circular shifting of the patterns preserves some of the autocorrelation of the time series - but not entirely. In the shifted patterns, the first and last items are considered to be neighboring and used in the evaluation, which alone could explain the poor performance. The simplest way that I can see is to also connect the first and last item in a circular fashion, even when evaluating the veridical ordering. The only really convincing control condition I found was the generation of new sequences for every character by shuffling the sequence of choices and re-creating new artificial trajectories with the same start and endpoint. This analysis performs much better than chance (circular shuffling), suggesting to me that a lot of the observed decoding accuracy is indeed simply caused by the temporal smoothness of both time series.

(5) Overall, I found the analysis of the brain-behavior correlation presented in Figure 5 unconvincing. First, the correlation is mostly driven by one individual with a large network size and a 6.5 cluster. I suspect that the exclusion of this individual would lead to the correlation losing significance. Secondly, the neural measure used for this analysis (determining the number of optimal clusters that maximize the overlap between neural clustering and behavioral clustering) is new, non-validated, and disconnected from all the analyses that had been reported previously. The authors need to forgive me for saying so, but at this point of the paper, would it not be much more obvious to use the decoding accuracy for power and affiliation from the main model used in the paper thus far? Does this correlate? Another obvious candidate would be the decoding accuracy for character identity or the size of the region that encodes affiliation and power. Given the plethora of candidate neural measures, I would appreciate if the authors reported the other neural measures that were tried (and that did not correlate). One way to address this would have been to select the method on the initial sample and then test it on the validation sample - unfortunately, the measure was not pre-registered before the validation sample was collected. It seems that the correlation was only found and reported on the validation sample?

Reviewer #2 (Public Review):

Summary:

Lamination is a layered neuronal arrangement that provides a basic frame to establish functional connectivity in the brain. The formation of a layered structure requires a highly coordinated interaction between migrating neurons and the developing microenvironment. Earlier studies revealed that to reach specific locations, migrating neurons typically follow various morphogen gradients. Here, Hallada et al. showed that cerebellar granule neurons (CGNs) could navigate via adhesive interaction with Junctional Adhesion Molecule C (JAM-C) followed by recruitment and distribution of intercellular partners (Pard3 and debris) at the contact sites. These results show that neuronal migration could be structured by specific interactions with adhesion molecules and spatial re-arrangements of downstream effectors.

Strengths:

The authors concluded that cis/trans binding sites of JAM-C on CGNs are crucial for contact formation with cerebellar glial cells (Bergman glial cells, BGs) and recruitment of Pard3 and drebrin to contact sites. This conclusion was based on the data obtained utilizing several advanced tools and technical approaches, such as cutting-edge microscopy, detailed visualization of cell-cell recognition, and a new correlation analysis.

Weaknesses:

(1) Despite multiple advanced methodologies, the study has weaknesses related primarily to the lack of specific evidence in support of findings and data interpretation issues. For example, it is unclear how JAM-C-mediated adhesion facilitates the entry of CGNs into the cerebellar molecular layer (ML). The authors described that CGN-CGN JAM recognition recruits more Pard3 and drebrin compared to CGN-BG recognition, which could increase the dwelling time of CGNs before moving to ML. However, such a mechanism does not explain what would initiate the entry of CGNs into ML. Perhaps the authors could provide a detailed explanation of this phenomenon in the Discussion (but certainly not in the Abstract). Also, the authors could consider revising the content of the Abstract, emphasizing their findings, and leaving out the speculations.

(2) To allow for comparison, it would be very helpful to indicate specific numerical values for each data point throughout the manuscript. For example, the authors stated that a change in instantaneous migration angle due to JAM-C silencing negatively affects CGNs movement to the ML (Figure 2) and that spatial distribution of negative JAM-Drebrin correlation is altered at CGN-CGN contacts (Figure 7). However, without specific values, it remains unclear what the magnitude of the discussed changes is or whether they were actually significant. It was not certainly straightforward to make specific conclusions based on graphical presentation alone.

Reviewer #2 (Public Review):

I think this is a very promising paper. The combination of EEG and fMRI is unique and original. However, I also have some suggestions that I think could help improve the manuscript.

This manuscript reports the findings of an EEG-fMRI study (n = 50) on the effects of expectations on pain. The combination of EEG with fMRI is extremely original and well-suited to study the transition from expectation to perception. However, I think that the current treatment of the data, as well as the way that the manuscript is currently written, does not fully capitalize on the potential of this unique dataset. Several findings are presented but there is currently no clear message coming out of this manuscript.

First, one positive point is that the experimental manipulation clearly worked. However, it should be noted that the instructions used are not typical of studies on placebo/nocebo. Participants were not told that the stimulations would be of higher/lower intensity. Rather, they were told that objective intensities were held constant, but that EEG recordings could be used to predict whether they would perceive the stimulus as more or less intense. I think that this is an interesting way to manipulate expectations, but there could have been more justification in the introduction for why the authors have chosen this unusual procedure.

Also, the introduction mentions that little is known about potential cerebral differences between expectations of high vs. low pain expectations. I think the fear conditioning literature could be cited here. Activations in ACC, SMA, Ins, parahippocampal gyrus, PAG, etc. are often associated with upcoming threat, whereas activations vmPFC/default mode network are associated with safety.

The fact that the authors didn't observe a clearer distinction between high and low expectations here could be related to their specific instructions that imply that the stimulus is the same and that it is the subjective perception that is expected to change. In any case, this is a relatively minor issue that is easy to address.

Towards the end of the introduction, the authors present the aims of the study in mainly exploratory terms:<br /> (1) What are the differences between anticipation and perception?<br /> (2) What regions display a difference between high and low expectations (high > low or low < high) vs. an effect of expectation regardless of the direction (high and low different than neutral)?<br /> I think these are good questions, but the authors should provide more justification, or framework, for these questions. More specifically, what will they be able to conclude based on their observations?

For instance (note that this is just an example to illustrate my point. I encourage the authors to come up with their own framework/predictions) :

(1) Possibility #1: A certain region encodes expectations in a directed fashion (high > low) and that same region also responds to perception in the same direction (high > low). This region would therefore modulate pain by assimilating perception towards expectations.<br /> (2) Possibility # 2: different regions are involved in expectation and perception. Perhaps this could mean that certain regions influence pain processing through descending facilitation for instance...

Regarding analyses, I think that examining the transition from expectations to perception is a strong angle of the manuscript given the EGG-fMRI nature of the study. However, I feel that more could have been done here. One problem is that the sequence of analyses starts by identifying an fMRI signal of interest and then attempts to find its EEG correlates. The problem is that the low temporal resolution of fMRI makes it difficult to differentiate expectation from perception, which doesn't make this analysis a good starting point in my opinion. Why not start by identifying an EEG signal that differentiates perception vs expectation, and then look for its fMRI correlates?

Finally, I found the hypotheses on "valenced" vs. "absolute" effects a little bit more difficult to follow. This is because "neutral" is not really neutral: it falls in between low and high. If I follow correctly, participants know that the temperature is always the same. Therefore, if they are told that the machine cannot predict whether their perception is going to be low or high, then it must be because it is likely to be in between. Ratings of expectation and pain ratings confirm that. The neutral condition is not "devoid" of expectations as the authors suggest. Therefore, it would make sense to look at regions with the following pattern low > neutral > high, or vice-versa, low < neutral < high. Low & high being different than neutral is more difficult to interpret. I don't think that you can say that it reflects "absolute" expectations because neutral is also the expectation of a medium temperature. Perhaps it reflects "certainty/uncertainty" or something like that, but it is not clear that it reflects "expectations".

Reviewer #2 (Public Review):

Background and Summary:

This study addresses the intriguing question of whether and how tumors can develop in the freshwater polyp hydra and how they influence the fitness of the animals. Hydra is notable for its significant morphogenetic plasticity and nearly unlimited capacity for regeneration. While its growth through asexual reproduction (budding) and the associated processes of pattern formation have been extensively studied at the cellular level, the occurrence of tumors was only recently described in two strains of Hydra oligactis (Domazet-Lošo et al, 2014). In that research, an arrest in the differentiation of female germ cells led to an accumulation of germline cells that failed to develop into eggs. In hydra, fertile egg cells typically incorporate nurse cells, which originate from large interstitial stem cells (ISCs) restricted to the germline, through apoptosis. However, this increase in apoptosis activity is absent in "germline tumors," and germline ISCs instead form slowly growing patches that do not compromise tissue integrity. Despite the upregulation of certain genes associated with mammalian neoplasms (such as tpt1 and p23) in this tissue, determining whether this differentiation arrest and the resulting egg patches truly constitute neoplasms remains a challenge.

The authors have recently published two papers on the ecological and evolutionary aspects of hydra tumor formation (Boutry et al 2022, 2023), which is also the focus of this manuscript. They transplanted tissues derived from animals with germline tumors to wildtype animals and analyzed their growth patterns, specifically the number of tentacles in the host tissue. They observed that such tissues induced the growth of additional tentacles compared to tissues without germline tumors. The authors conclude that this growth pattern (increased number of tentacles) is correlated with "reducing the burden on the host by (over-)compensating for the reproductive costs of tumors" and claim that "transmissible tumors in hydra have evolved strategies to manipulate the phenotype of their host". While it might be stimulating to add a fresh view from other disciplines (here, ecological and evolutionary aspects), the authors completely ignore the current knowledge of the underlying cell biology of the processes they analyze.

Strengths:

The study focuses on intriguing questions. Whether and how tumors can develop in the freshwater polyp hydra, and how they influence the fitness of the animals?

Weaknesses:

Concept of germline tumors.<br /> The conceptual foundation of their experiments on germline tumors was the study of Domazet-Lošo et al (2014) introducing the concept of germline tumors in hydra (see above). While this is an intriguing hypothesis, there has been little advancement in comprehending the molecular mechanisms underlying tumor formation in hydra beyond this initial investigation. Germline tumors in hydra do not fully meet the typical criteria for neoplasms observed in mammalian tissues. More importantly, a similar phenotype was already reported by the work of Paul Brien and described as "crise gametique" (Brien, 1966, Biologie de la reproduction animale - Blastogenèse, Gamétogenèse, Sexualisation, ed. Masson & Cie, Paris). This phenomenon of gametic crisis is unique to Hydra oligactis, a stenotherm, cold-adapted cosmopolitan species. In this species, gametogenesis severely impacts the vitality of the polyps, often leading to complete exhaustion and death (Tardent, 1974). Animals can only be rescued during the initial phase of the cold-induced sexual period (see also the research of Littlefield (1984, 1985, 1986, 1991). The observed arrest in differentiation arrest in germline tumors might represent an epigenetically established consequence of surviving gametogenesis. Regrettably, this important work was not mentioned by the authors or by Domazet-Lošo et al. (2014), highlighting a notable gap in the recognition of basic research in this area that might challenge the hydra tumor hypothesis.

"Super-nummary" tentacles in graft experiments.<br /> The authors describe that after grafting tissue from animals with germline tumors to wild-type animals, the number of tentacles in the host tissue increased when the donor tissue had germline tumors. A maximum effect of four additional tentacles was found with donor strain H. oligactis robusta and three additional tentacles with donor strain H.oligactis St Petersburg. In general, H.oligactis wild-type host strains had fewer tentacles than H.oligactis St Petersburg strains. This is consistent with the results of Domazet-Lošo et al (2014) who showed that the number of tentacles increased in the strains with germline tumors. What conclusions can be drawn from these experiments? The authors might want to conclude that transmissible tumors in Hydra have developed strategies to manipulate the phenotype of their host. But there is no evidence for this, as essential controls are missing. It is known that the size of hydra polyps is proportion-regulated, i.e. the number of tentacles varies with the size and number of (epithelial) cells. Such controls are missing in the experiments. There is also a lack of controls from wild-type animals in gametogenesis: it is very likely that grafts with wild-type animals with egg spots of comparable size as the germline tumors (see above) will result in similar numbers of tentacles in host tissue.

Reviewer #2 (Public Review):

Summary:

This MR study by Zhao et al. provides a comprehensive hypothesis-free approach to identifying risk and protective factors causal to Alzheimer's Disease (AD).

Strengths:

The study employs a comprehensive, hypothesis-free approach, which is novel over traditional hypothesis-driven studies. Also, causal associations between risk/protective factors and AD were addressed using genetic instruments and analysis.

Major comments:

(1) The authors used the inverse-variance weighted (IVW) model as the primary method and other MR methods (MR-Egger, weighted mean, etc.) for sensitivity analysis. However, each method has its own assumption, and IVW is only robust when pleiotropy and heterogeneity are not severe. Rather than using IVW imprudently across all associations, it would be more appropriate to choose the best MR method for each association based on heterogeneity/Egger intercept tests. This customized approach, based on tests of MR assumption violations, yields more stable and reliable results. For reference, please follow up on work by Milad et al. (EHJ - "Plasma lipids and risk of aortic valve stenosis: a Mendelian randomization study"). This study selected the best MR model for each association based on pleiotropy and heterogeneity tests. Given the large number of tests in this work, I suggest initially screening significant signals using IVW, as done, and then validating the results using multiple MR methods for those signals. It is common for MR estimates from different methods to vary significantly (with some being statistically significant and others not), and in such cases, the MR estimates from the best-fitted model should be trusted and highlighted.

(2) Lines 157-160 mentioned "But to date, AD has been reported as hypothesis-driven MR study based on a single factor, ignoring the potential role of a huge number of other risk factors. Also, due to the high degree of heterogeneity present in AD subtypes, which have different biological and genetic characteristics. Thus, the previous studies cannot offer a systematic and complete viewpoint.". This statement overlooks a similar study published in Molecular Psychiatry ("A Phenome-wide Association and Mendelian Randomization Study for Alzheimer's Disease: A Prospective Cohort Study of 502,493"), which rigorously assessed the effects of 4171 factors spanning 10 different categories on AD using observational analysis and MR. The authors should revise their statement on the novelty of their study type throughout the manuscript and discuss how their work differs from and potentially strengthens previous studies.

(3) Given the large number of tests, the multiple testing issue is concerning. To mitigate potential false positives, I recommend employing the Bonferroni threshold or FDR. The authors should only interpret exposures that are significant at the Bonferroni threshold.

(4) In the discussion, the authors should interpret or highlight exposures that remain significant after multiple testing corrections.

Reviewer #2 (Public Review):

Summary:

Mitchell & Mohammadkhani et al. used an Orexin-Cre mouse line with a Cre-dependent GCaMP virus to perform lateral hypothalamic (LH) Ca2+ fiber photometry recordings in mice during the approach to food under various metabolic and saliency conditions. They also used a Vgat-Cre mouse line with Cre-dependent ChR2 in various regions of the ventral striatopallidal (VSP) complex in combination with an Orexin promoter-driven reporter virus labeling Orx-LH neurons to assess electrophysiological connectivity of inhibitory/excitatory inputs from VSP to Orx-LH. Overall, authors note that Orx-LH Ca2+ activation occurs during approach to food (but not consumption of food), and that VSP->Orx-LH connectivity is primarily monosynaptic and inhibitory, although this varies across subregions, with some monosynaptic excitatory input as well. While their methods and analyses are technically sound and the manuscript is clearly written and presented, the further knowledge gained over previous work is rather incremental and does not produce a substantial shift in the current existing framework.

Strengths:

Cell type specificity of OX/HT recordings is confirmed by post-hoc immunostaining, both for fiber photometry and electrophysiological connectivity. This is an important strength given the contentious history of cell specificity in various transgenic OX/HT mouse lines.

Clearly implicating metabolic state and food saliency as factors impacting OX/HT activity dynamics is a strength, and linking the influence of ghrelin receptor signaling is relatively novel.

Weaknesses:

In fiber photometry traces, OX/HT activity begins increasing 2-3 seconds prior to the food approach (Figures 1F and 1G), requiring an explanation. One possibility is that mice may be detecting odorant cues indicative of food prior to the physical approach.

Figure 1F - the authors' interpretation that OX/HT activity doesn't actually decrease during consumption, but simply "trends toward baseline" is complicated by the fact that the authors shaded 20s-30s intervals labeled "eating". Mice do not typically consume food for 20-30s nonstop. Mice typically consume for ~1-5 seconds, then they take a break, then they resume.

The authors state in the Discussion "... the reduction in OX/HT cell activity was more closely correlated with the termination of approach behavior" (rather than with eating per se). However, in many cases, mice begin consuming food immediately after approaching it, so it is puzzling that there is an activity reduction following the approach, but not an activity reduction upon consumption. In other words, the cessation of approach and the beginning of consumption are often tightly linked together in rapid sequence.

Figure 2E - the single polysynaptic oIPSC appears to have the same/similar latency as many of the Monosynaptic oIPSCs. Close proximity of consecutive oIPSCs may affect the analysis of amplitude and latency. For example, in representative traces of Figure 2C, it is unlikely to get an accurate measure of the second oIPSC.

The comparison of apparent connectivity differences between VP vs. mNAcSh vs. lNAcSh is limited by appropriate anatomical quantification and demonstration. When using a Vgat-Cre mouse line and targeting the VSP, there is the potential for massive viral spread across the entire Nucleus accumbens/VP/SI/BNST area.

How do the electrophysiological properties of OX/HT neurons (and VSP inputs) change across metabolic/saliency states? For example, under High Fat Diet, chronic Food Restriction, and chronic Ghrelin. This seems to be the fundamental question that the authors are working toward, but it is not resolved with the current data set.

Potential Ephys Pitfall: a high Chloride internal solution means that oEPSCs might actually be GABAergic after all. Low Chloride solution, so Cl reversal potential is closer to RMP (or put more Chloride in pipette so it has more depolarized potential than resting- to reverse current mediated by Chloride ions). However, the internal solution used for oEPSCs was calculated to have a Cl reversal potential at ~ -20mV; thus, the Cl-mediated PSCs would be depolarizing when cells were held at -65mV. Did the authors apply any blockers in the bath to confirm that recorded oEPSCs were glutamatergic?

Reviewer #2 (Public Review):

Summary:

With this work, the authors tried to expand and integrate the concept of realized niche in the context of movement ecology by using fine-scale GPS data of 55 juvenile Golden eagles in the Alps. Authors found that ontogenic changes influence the percentage of area flyable to the eagles as individuals exploit better geographic uplifts that allow them to reduce the cost of transport.

Strengths:

Authors made insightful work linking changes in ontogeny and energy landscapes in large soaring birds. It may not only advance the understanding of how changes in the life cycle affect the exploitability of aerial space but also offer valuable tools for the management and conservation of large soaring species in the changing world.

Weaknesses:

Future research may test the applicability of the present work by including more individuals and/or other species from other study areas.

Reviewer #2 (Public Review):

Summary:

In this work, Lao et al. develop an open-source software (OpenNucleome) for GPU-accelerated molecular dynamics simulation of the human nucleus accounting for chromatin, nucleoli, nuclear speckles, etc. Using this, the authors investigate the steady-state organization and dynamics of many of the nuclear components.

Strengths:

This is a comprehensive open-source tool to study several aspects of the nucleus, including chromatin organization, interactions with lamins and organization, and interactions with nuclear speckles and nucleoli. The model is built carefully, accounting for several important factors and optimizing the parameters iteratively to achieve experimentally known results. Authors have simulated the entire genome at 100kb resolution (which is a very good resolution to simulate and study the entire diploid genome) and predict several static quantities such as the radius of gyration and radial positions of all chromosomes, and time-dependent quantities like the mean-square displacement of important genomic regions.

Weaknesses:

One weakness of the model is that it has several parameters. Some of them are constrained by the experiments. However, the role of every parameter is not clear in the manuscript.

Reviewer #2 (Public Review):

Summary:

It was first reported in 2000 that Smad2/3/4 are sequestered to microtubules in resting cells and TGF-β stimulation releases Smad2/3/4 from microtubules, allowing activation of the Smad signaling pathway. Although the finding was subsequently confirmed in a few papers, the underlying mechanism has not been explored. In the present study, the authors found that Rudhira/breast carcinoma amplified sequence 3 is involved in the release of Smad2/3 from microtubules in response to TGF-β stimulation. Rudhira is also induced by TGF-β and is probably involved in the stabilization of microtubules in the delayed phase after TGF-β stimulation. Therefore, Rudhira has two important functions downstream of TGF-β in the early as well as delayed phase.

Strengths:

This work aimed to address an unsolved question on one of the earliest events after TGF-β stimulation. Based on loss-of-function experiments, the authors identified a novel and potentially important player, Rudhira, in the signal transmission of TGF-β,

Weaknesses:

The authors have identified a key player that triggers Smad2/3 released from microtubules after TGF-β stimulation probably via its association with microtubules. This is an important first step for understanding the regulation of Smad signaling, but underlying mechanisms as well as upstream and downstream events largely remain to be elucidated.

(1) The process of how Rudhira causes the release of Smad proteins from microtubules remains unclear. The statement that "Rudhira-MT association is essential for the activation and release of Smad2/3 from MTs" (lines 33-34) is not directly supported by experimental data.

(2) The process of how Rudhira is mobilized to microtubules in response to TGF-β remains unclear.

(3) After Rudhira releases Smad proteins from microtubules, Rudhira stabilizes microtubules. The process of how cells return to a resting state and recover their responsiveness to TGF-β remains unclear.

This reviewer is also afraid that some of the biochemical data lack appropriate controls and are not convincing enough.

Reviewer #2 (Public Review):

Summary

The authors aimed to characterise the evolutionary dynamics that occur during the resistance to androgen receptor signalling inhibition, and how this differs in established tumours vs. residual disease, in prostate cancer. By using a barcoding method, they aimed to both characterise the distribution of clones that support therapy resistance in these settings, while also then being able to isolate said clones from the pre-graft population via single-cell cloning to characterise the mechanisms of resistance and dependency on cooperativity.

While, interestingly, the timing of combination therapies has been shown to be critical to avoid cross-resistance, the timing of therapy has not been specifically considered as a factor dictating resistance pathways. Additionally, the role of residual disease and dormant populations in driving relapse is of increasing interest, yet a lot remains to be understood of these populations. The question of whether different clinical manifestations of therapy resistance follow similar evolutionary pathways to resistance is therefore interesting and relevant for the field.

The methods applied are elegant and the body of work is substantial. The proposed divergent evolutionary pathways pose interesting questions, and the findings on cooperativity provide insight. However, whether the model truly reflects minimal residual disease to the extent that the authors suggest may limit the relevance of the findings at this stage. Certain patterns in the DNA barcoding results also call into question whether the results fully support the strong claims of the authors, or whether alternative explanations could exist. While the potential to isolate individual clones in the pre-graft setting is a great strength of the method applied and the isolation of these clones is a huge body of work in itself, the limited number of clones that could be isolated also somewhat limits the validation of the findings.

Strengths

• Very relevant and interesting question, clear clinical relevance, applying elegant methods that hold the potential to provide a novel understanding of multiple aspects of therapy resistance, through from evolutionary patterns to intracellular and cooperative mechanisms of resistance.

• The text is clearly written, logical, and the structure is easy to follow.

Weaknesses

(1) The extent to which the model used truly mimics residual disease

The main conclusions of the paper are built upon results using a model for minimal residual disease. However, the extent to which this truly recapitulates minimal residual disease, particularly with regard to their focus on the timings of therapy, could be discussed further. If in the clinical setting residual disease occurs following the existence of a tumour and its microenvironment, there might be many aspects of the process that are missed when coinciding treatment with engraftment of a xenograft tumour with pre-castration. If any characterisation of the minimal residual disease was possible (such as histologically or through RNA sequencing), this may help demonstrate in what ways this model recapitulates minimal residual disease.

(2) Whether the observed enrichment of pre-resistant clones is truly that

The authors strongly make the case that their barcoding experiments provide evidence for pre-existing resistance in the context of minimal residual disease. However, it seems that the clones enriched in the ARSIR tumours are consistently the most enriched clones in the pregraft. Is it possible that the high selective pressure in the pre-engraftment ARSI condition simply leads to an enrichment of the most populous clones from the pregraft? Whereas in the control setting, the reduced selective pressure at the point of engraftment allows for a wider variety of clones to establish in the tumour? Additionally, is there the possibility that the clones highly enriched in the pregraft are in fact a heterogeneous group of cells bearing the same barcode due to stochastic events in the process of viral transduction? Addressing these questions would greatly improve the study.

(3) The robustness of the subsequent work based on 1-2 pre-resistant clones

While appreciating the volume of work involved in isolating and culturing individual pre-resistant clones, given the previous point, the conclusions would benefit from very robust validations with these single-cell clones. There are only two clones, and the results seem to focus more on one than the other, for which the data is less convincing. For instance, the Enz IC50 data, which in the case for pre-ARSI R2 is restricted to the supplementary, compares the clones A-D. In Figure S8 B, pre-ARSI R2 is compared to clone B, which is, of the four clones shown in the main figure when compared to R1, the one with the lowest Enz IC50. Therefore, while the resistant clones seem to have a significantly higher Enz IC50, comparing both clones to clones A-D may not have achieved this significance. It would also be useful to know how abundant the resistant clones were in the original barcode experiments.

(4) The logic used in the final section requires further explanation

In the final section, the authors suggest that a pre-ARSIR clone is able to cooperate with a pre-Intact clone to aid adaptive ARSI resistance. If this is true, then could it not be that rare, pre-resistant clones support adaptive resistance in established tumours? And, therefore, the mechanism underlying resistance could be through pre-existing resistant clones in both settings. The work would benefit from a discussion to clarify this discrepancy in the interpretation of the findings. This is particularly necessary given the strong wording the authors use regarding their findings, such as that they have provided 'conclusive evidence' for acquired resistance.

Reviewer #2 (Public Review):

Roy et al. investigated the role of non-canonical DNA structures called G-quadruplexes (G4s) in long-range chromatin interactions and gene regulation. Introducing a G4 array into chromatin significantly increased the number of long-range interactions, both within the same chromosome (cis) and between different chromosomes (trans). G4s functioned as enhancer elements, recruiting p300 and boosting gene expression even 5 megabases away. The study reveals that G4s directly influence 3D chromatin organization via facilitating communication between regulatory elements and genes.

Strengths:

The authors' findings are valuable for understanding the role of G4-DNA in 3D genome organization and gene transcription. The authors provide convincing evidence to support their claims.

Reviewer #2 (Public Review):

Sur and colleagues investigate the role of ATP6V0A1 in mitochondrial function in cystinotic proximal tubule cells. They propose that loss of cystinosin downregulates ATP6V0A1 resulting in acidic lysosomal pH loss, and adversely modulates mitochondrial function and lifespan in cystinotic RPTECs. They further investigate the use of a novel therapeutic Astaxanthin (ATX) to upregulate ATP6V0A1 that may improve mitochondrial function in cystinotic proximal tubules.

The new information regarding the specific proximal tubular injuries in cystinosis identifies potential molecular targets for treatment. As such, the authors are advancing the field in an experimental model for potential translational application to humans.

Reviewer #2 (Public Review):

In the manuscript by Weber and colleagues, the authors investigated the role of a DEAD-box helicase DDX6 in regulating mRNA stability upon ribosome slowdown in human cells. The authors knocked out DDX6 KO in HEK293T cells and showed that the half-life of a reporter containing a rare codon repeat is elongated in the absence of DDX6. By analogy to the proposed function of fission yeast Dhh1p (DDX6 homolog) as a sensor for slow ribosomes, the authors demonstrated that recombinant DDX6 interacted with human ribosomes. The interaction with the ribosome was mediated by the FDF motif of DDX6 located in its RecA2 domain, and rescue experiments showed that DDX6 requires the FDF motif as well as its interaction with the CCR4-NOT deadenylase complex and ATPase activity for degrading a reporter mRNA with rare codons. To identify endogenous mRNAs regulated by DDX6, they performed RNA-Seq and ribosome footprint profiling. The authors focused on mRNAs whose stability is increased in DDX6 KO cells with high local ribosome density and validated that such mRNA sequences induced mRNA degradation in a DDX6-dependent manner.

The experiments were well-performed, and the results clearly demonstrated the requirement of DDX6 in mRNA degradation induced by slowed ribosomes.

[Editors' note: The authors have addressed the key points from the previous public reviews in their revised manuscript.]

Reviewer #2 (Public Review):

SUMMARY:

In this manuscript, Ger and colleagues propose two complementary analytical methods aimed at quantifying the model misspecification and irreducible stochasticity in human choice behavior. The first method involves fitting recurrent neural networks (RNNs) and theoretical models to human choices and interpreting the better performance of RNNs as providing evidence of the misspecifications of theoretical models. The second method involves estimating the number of training iterations for which the fitted RNN achieves the best prediction of human choice behavior in a separate, validation data set, following an approach known as "early stopping". This number is then interpreted as a proxy for the amount of explainable variability in behavior, such that fewer iterations (earlier stopping) correspond to a higher amount of irreducible stochasticity in the data. The authors validate the two methods using simulations of choice behavior in a two-stage task, where the simulated behavior is generated by different known models. Finally, the authors use their approach in a real data set of human choices in the two-stage task, concluding that low-IQ subjects exhibit greater levels of stochasticity than high-IQ subjects.

STRENGTHS:

The manuscript explores an extremely important topic to scientists interested in characterizing human decision-making. While it is generally acknowledged that any computational model of behavior will be limited in its ability to describe a particular data set, one should hope to understand whether these limitations arise due to model misspecification or due to irreducible stochasticity in the data. Evidence for the former suggests that better models ought to exist; evidence for the latter suggests they might not.

To address this important topic, the authors elaborate carefully on the rationale of their proposed approach. They describe a variety of simulations -- for which the ground truth models and the amount of behavioral stochasticity are known -- to validate their approaches. This enables the reader to understand the benefits (and limitations) of these approaches when applied to the two-stage task, a task paradigm commonly used in the field. Through a set of convincing analyses, the authors demonstrate that their approach is capable of identifying situations where an alternative, untested computational model can outperform the set of tested models, before applying these techniques to a realistic data set.

WEAKNESSES:

The most significant weakness is that the paper rests on the implicit assumption that the fitted RNNs explain as much variance as possible, an assumption that is likely incorrect and which can result in incorrect conclusions. While in low-dimensional tasks RNNs can predict behavior as well as the data-generating models, this is not always the case, and the paper itself illustrates (in Figure 3) several cases where the fitted RNNs fall short of the ground-truth model. In such cases, we cannot conclude that a subject exhibiting a relatively poor RNN fit necessarily has a relatively high degree of behavioral stochasticity. Instead, it is at least conceivable that this subject's behavior is generated precisely (i.e., with low noise) by an alternative model that is pooly fit by an RNN -- e.g., a model with long-term sequential dependencies, which RNNs are known to have difficulties in capturing.

These situations could lead to incorrect conclusions for both of the proposed methods. First, the model mis-specification analysis might show equal predictive performance for a particular theoretical model and for the RNN. While a scientist might be inclined to conclude that the theoretical model explains the maximum amount of explainable variance and therefore that no better model should exist, the scenario in the previous paragraph suggests that a superior model might nonetheless exist. Second, in the early-stopping analysis, a particular subject may achieve optimal validation performance with fewer epochs than another, leading the scientist to conclude that this subject exhibits higher behavioral noise. However, as before, this could again result from the fact that this subject's behavior is produced with little noise by a different model. The possibility of such scenarios does not mean that such scenarios are common, and the conclusions drawn in the paper are likely appropriate for the particular examples analyzed. However, it is much less obvious that the RNNs will provide optimal fits in other types of tasks, particularly those with more complex rules and long-term sequential dependencies, and in such scenarios, an ill-advised scientist might end up drawing incorrect conclusions from the application of the proposed approaches. The authors acknowledge this limitation in their discussion, but it remains a significant caveat that readers should be aware of when using the technique proposed.

In addition to this general limitation, the relationship between the number of optimal epochs and behavioral stochasticity may not hold for every task and every subject. For example, Figure 4 highlights the relationship between the optimal epochs and agent noise. Yet, it is nonetheless possible that the optimal epoch is influenced by model parameters other than inverse temperature (e.g., hyperparameters such as learning rate, etc). This could again lead to invalid conclusions, such as concluding that low-IQ is associated with optimal epoch when an alternative account might be that low-IQ is associated with low learning rate, which in turn is associated with optimal epoch. Additional factors such as the deep double-descent (Nakkiran et al., ICLR 2020) can also influence the optimal epoch value as computed by the authors. These concerns are partially addressed by the authors in the revised manuscript, where they show that the number of optimal epochs is primarily sensitive to the amount of true underlying noise, assuming the number of trials and network size are constant. The authors also acknowledge, in the discussion section, that many factors can affect the number of optimal epochs, and that inferring behavioral stochasticity from this number should be done with caution.

APPRAISAL AND DISCUSSION:

Overall, the authors propose a novel method that aims to solve an important problem, but since the evidence provided refers to a single task and to a single dataset, it is not clear that the method would be appropriate in general settings. In the future, it would be beneficial to test the proposed approach in a broader setting, including simulations of different tasks, different model classes, and different model parameters. Nonetheless, even without such additional work, the proposed methods are likely to be used by cognitive scientists and neuroscientists interested in assessing the quality and limits of their behavioral models.

Reviewer #2 (Public Review):

Summary:

The study describes differences in responses to sounds and whisker deflections as well as combinations of these stimuli in different neurochemically defined subsections of the lateral and dorsal cortex of the inferior colliculus in anesthetised and awake mice.

Strengths:

A major achievement of the work lies in obtaining the data in the first place as this required establishing and refining a challenging surgical procedure to insert a prism that enabled the authors to visualise the lateral surface of the inferior colliculus. Using this approach, the authors were then able to provide the first functional comparison of neural responses inside and outside of the GABA-rich modules of the lateral cortex. The strongest and most interesting aspects of the results, in my opinion, concern the interactions of auditory and somatosensory stimulation. For instance, the authors find that a) somatosensory-responses are strongest inside the modules and b) somatosensory-auditory suppression is stronger in the matrix than in the modules. This suggests that, while somatosensory inputs preferentially target the GABA-rich modules, they do not exclusively target GABAergic neurons within the modules (given that the authors record exclusively from excitatory neurons we wouldn't expect to see somatosensory responses if they targeted exclusively GABAergic neurons) and that the GABAergic neurons of the modules (consistent with previous work) preferentially impact neurons outside the modules, i.e. via long-range connections.

Weaknesses:

While the findings are of interest to the subfield they have only rather limited implications beyond it and the writing is not quite as precise as it could be.

Reviewer #2 (Public Review):

Summary:

This paper aimed to examine the spatial frequency selectivity of macaque inferotemporal (IT) neurons and its relation to category selectivity. The authors suggest in the present study that some IT neurons show a sensitivity for the spatial frequency of scrambled images. Their report suggests a shift in preferred spatial frequency during the response, from low to high spatial frequencies. This agrees with a coarse-to-fine processing strategy, which is in line with multiple studies in the early visual cortex. In addition, they report that the selectivity for faces and objects, relative to scrambled stimuli, depends on the spatial frequency tuning of the neurons.

Strengths:

Previous studies using human fMRI and psychophysics studied the contribution of different spatial frequency bands to object recognition, but as pointed out by the authors little is known about the spatial frequency selectivity of single IT neurons. This study addresses this gap and shows spatial frequency selectivity in IT for scrambled stimuli that drive the neurons poorly. They related this weak spatial frequency selectivity to category selectivity, but these findings are premature given the low number of stimuli they employed to assess category selectivity.

The authors revised their manuscript and provided some clarifications regarding their experimental design and data analysis. They responded to most of my comments but I find that some issues were not fully or poorly addressed. The new data they provided confirmed my concern about low responses to their scrambled stimuli. Thus, this paper shows spatial frequency selectivity in IT for scrambled stimuli that drive the neurons poorly (see main comments below). They related this (weak) spatial frequency selectivity to category selectivity, but these findings are premature given the low number of stimuli to assess category selectivity.

Main points.

(1) They have provided now the responses of their neurons in spikes/s and present a distribution of the raw responses in a new Figure. These data suggest that their scrambled stimuli were driving the neurons rather poorly and thus it is unclear how well their findings will generalize to more effective stimuli. Indeed, the mean net firing rate to their scrambled stimuli was very low: about 3 spikes/s. How much can one conclude when the stimuli are driving the recorded neurons that poorly? Also, the new Figure 2- Appendix 1 shows that the mean modulation by spatial frequency is about 2 spikes/s, which is a rather small modulation. Thus, the spatial frequency selectivity the authors describe in this paper is rather small compared to the stimulus selectivity one typically observes in IT (stimulus-driven modulations can be at least 20 spikes/s).<br /> (2) Their new Figure 2-Appendix 1 does not show net firing rates (baseline-subtracted; as I requested) and thus is not very informative. Please provide distributions of net responses so that the readers can evaluate the responses to the stimuli of the recorded neurons.<br /> (3) The poor responses might be due to the short stimulus duration. The authors report now new data using a 200 ms duration which supported their classification and latency data obtained with their brief duration. It would be very informative if the authors could also provide the mean net responses for the 200 ms durations to their stimuli. Were these responses as low as those for the brief duration? If so, the concern of generalization to effective stimuli that drive IT neurons well remains.<br /> (4) I still do not understand why the analyses of Figures 3 and 4 provide different outcomes on the relationship between spatial frequency and category selectivity. I believe they refer to this finding in the Discussion: "Our results show a direct relationship between the population's category coding capability and the SF coding capability of individual neurons. While we observed a relation between SF and category coding, we have found uncorrelated representations. Unlike category coding, SF relies more on sparse, individual neuron representations.". I believe more clarification is necessary regarding the analyses of Figures 3 and 4, and why they can show different outcomes.<br /> (5) The authors found a higher separability for faces (versus scrambled patterns) for neurons preferring high spatial frequencies. This is consistent for the two monkeys but we are dealing here with a small amount of neurons. Only 6% of their neurons (16 neurons) belonged to this high spatial frequency group when pooling the two monkeys. Thus, although both monkeys show this effect I wonder how robust it is given the small number of neurons per monkey that belong to this spatial frequency profile. Furthermore, the higher separability for faces for the low-frequency profiles is not consistent across monkeys which should be pointed out.<br /> (6) I agree that CNNs are useful models for ventral stream processing but that is not relevant to the point I was making before regarding the comparison of the classification scores between neurons and the model. Because the number of features and trial-to-trial variability differs between neural nets and neurons, the classification scores are difficult to compare. One can compare the trends but not the raw classification scores between CNN and neurons without equating these variables.

Reviewer #2 (Public Review):

The authors set out to draw further links between neural patterns observed at "rest" during fMRI, with their related thought content and personality traits. More specifically, they approached this with a "tri-partite network" view in mind, whereby the ventral attention network (VAN), the dorsal attention network (DAN) and the default mode network (DMN) are proposed to play a special role in ongoing conscious thought. They used a gradient approach to determine the low dimensional organisation of these networks. In concert, using PCA they reduced thought patterns captured at four time points during the scan, as well as traits captured from a large battery of questionnaires.

The main findings were that specific thought and trait components were related to variations in the organisation of the tri-partite networks, with respect to cortical gradients.

Strengths of the methods/results: Having a long (1 hour) resting state MRI session, which could be broken down into four separate scanning/sampling components is a strength. Importantly, the authors could show (via intra-class correlation coefficients) similarity of thoughts and connectivity gradients across the entire session. Not only did this approach increase the richness of the data available to them, it speaks in an interesting way to the stability of these measures. The inclusion of both thought patterns during scanning along with trait-level dispositional factors is most certainly a strength, as many studies will often include either/or of these, rather than trying to reconcile across. Of the two main findings, the finding that detailed self-generated thought was associated with a decoupling of regions of DAN from regions in DMN was particularly compelling, in light of mounting literature from several fields that support this.

Weaknesses of the methods/results: Considering the richness of the thought and personality data, I was a little surprised that only two main findings emerged (i.e., a relationship with trait introversion, and a relationship with the "specific internal" thought pattern). I wondered whether, at least in part and in relation to traits, this might stem from the large and varied set of questionnaires used to discern the traits. These questionnaires mostly comprised personality/mood, but some sampled things that do not fall into that category (e.g., musicality, internet addition, sleep) and some related directly to spontaneous thought properties (e.g., mind wandering, musical imagery). It would be interesting to see what relationships would emerge by being more selective in the traits measured, and in the tools to measure them.

Taken together, the main findings are interesting enough. However, the real significance of this work and its impact, lie in the richness of the approach: combing across fMRI, spontaneous thought, and trait-level factors. Triangulating across these data has important potential for furthering our understanding of brain-behaviour relationship across different levels of organisation.

Reviewer #2 (Public Review):

Summary:

The present article describes a series of experiments examining how a gradual reduction in unconditional stimulus intensity facilitates fear reduction and reduces relapse (spontaneous recovery and reinstatement) relative to a standard extinction procedure. The experiments provide compelling, if somewhat inconsistent, evidence of this effect and couch the results in a scholarly discussion surrounding how mechanisms of prediction error contribute to this effect.

Strengths:

The experiments are theoretically motivated and hypothesis-driven, well-designed, and appropriately conducted and analyzed. The results are clear and appropriately contextualized into the broader relevant literature. Further, the results are compelling and ask fundamental questions regarding how to persistently weaken fear behavior, which has both strong theoretical and real-world implications. I found the 'scrambled' experiment especially important in determining the mechanism through which this reduction in shock intensity persistently weakens fear behavior.

Weaknesses:

Overall, I found very few weaknesses with this paper. I think some might view the somewhat inconsistent effects on relapse between experiments to be a substantial weakness, I appreciate the authors directly confronting this and using it as an opportunity to aggregate data to look at general trends. Further, while Experiment 1 only used males, this was corrected in the rest of the experiments and therefore is not a substantial concern.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors set out to resolve a long-standing mystery in the field of sensory biology - how large, presynaptic bodies called "ribbon synapses" migrate to the basolateral end of hair cells. The ribbon synapse is found in sensory hair cells and photoreceptors, and is a critical structural feature of a readily-releasable pool of glutamate that excites postsynaptic afferent neurons. For decades, we have known these structures exist, but the mechanisms that control how ribbon synapses coalesce at the bottom of hair cells are not well understood. The authors addressed this question by leveraging the highly-tractable zebrafish lateral line neuromast, which exhibits a small number of visible hair cells, easily observed in time-lapse imaging. The approach combined genetics, pharmacological manipulations, high-resolution imaging, and careful quantifications. The manuscript commences with a developmental time course of ribbon synapse development, characterizing both immature and mature ribbon bodies (defined by position in the hair cell, apical vs. basal). Next, the authors show convincing (and frankly mesmerizing) imaging data of plus end-directed microtubule trafficking toward the basal end of the hair cells, and data highlighting the directed motion of ribbon bodies. The authors then use a series of pharmacological and genetic manipulations showing the role of microtubule stability and one particular kinesin (Kif1aa) in the transport and fusion of ribbon bodies, which is presumably a prerequisite for hair cell synaptic transmission. The data suggest that microtubules and their stability are necessary for normal numbers of mature ribbons and that Kif1aa is likely required for fusion events associated with ribbon maturation. Overall, the data provide a new and interesting story on ribbon synapse dynamics.

Strengths:

(1) The manuscript offers a comprehensive Introduction and Discussion sections that will inform generalists and specialists.

(2) The use of Airyscan imaging in living samples to view and measure microtubule and ribbon dynamics in vivo represents a strength. With rigorous quantification and thoughtful analyses, the authors generate datasets often only obtained in cultured cells or more diminutive animal models (e.g., C. elegans).

(3) The number of biological replicates and the statistical analyses are strong. The combination of pharmacology and genetic manipulations also represents strong rigor.

(4) One of the most important strengths is that the manuscript and data spur on other questions - namely, do (or how do) ribbon bodies attach to Kinesin proteins? Also, and as noted in the Discussion, do hair cell activity and subsequent intracellular calcium rises facilitate ribbon transport/fusion?

Weaknesses:

(1) Neither the data or the Discussion address a direct or indirect link between Kinesins and ribbon bodies. Showing Kif1aa protein in proximity to the ribbon bodies would add strength.

(2) Neither the data or Discussion address the functional consequences of loss of Kif1aa or ribbon transport. Presumably, both manipulations would reduce afferent excitation.

(3) It is unknown whether the drug treatments or genetic manipulations are specific to hair cells, so we can't know for certain whether any phenotypic defects are secondary.

Reviewer #2 (Public Review):

Summary:

This study looks at sex differences in alcohol drinking behaviour in a well-validated model of binge drinking. They provide a comprehensive analysis of drinking behaviour within and between sessions for males and females, as well as looking at the calcium dynamics in neurons projecting from the anterior insula cortex to the dorsolateral striatum.

Strengths:

Examining specific sex differences in drinking behaviour is important. This research question is currently a major focus for preclinical researchers looking at substance use. Although we have made a lot of progress over the last few years, there is still a lot that is not understood about sex-differences in alcohol consumption and the clinical implications of this.

Identifying the lateralisation of activity is novel, and has fundamental importance for researchers investigating functional anatomy underlying alcohol-driven behaviour (and other reward-driven behaviours).

Weaknesses:

Very small and unequal sample sizes, especially females (9 males, 5 females). This is probably ok for the calcium imaging, especially with the G-power figures provided, however, I would be cautious with the outcomes of the drinking behaviour, which can be quite variable.

For female drinking behaviour, rather than this being labelled "more efficient", could this just be that female mice (being substantially smaller than male mice) just don't need to consume as much liquid to reach the same g/kg. In which case, the interpretation might not be so much that females are more efficient, as that mice are very good at titrating their intake to achieve the desired dose of alcohol.

I may be mistaken, but is ANCOVA, with sex as the covariate, the appropriate way to test for sex differences? My understanding was that with an ANCOVA, the covariate is a continuous variable that you are controlling for, not looking for differences in. In that regard, given that sex is not continuous, can it be used as a covariate? I note that in the results, sex is defined as the "grouping variable" rather than the covariate. The analysis strategy should be clarified.

Reviewer #2 (Public Review):

Summary:

In this article, the authors study the function of TEDC1 and TEDC2, two proteins previously reported to interact with TUBD1 and TUBE1. Previous work by the same group had shown that TUBD1 and TUBE1 are required for centriole assembly and that human cells lacking these proteins form abnormal centrioles that only have singlet microtubules that disintegrate in mitosis. In this new work, the authors demonstrate that TEDC1 and TEDC2 depletion results in the same phenotype with abnormal centrioles that also disintegrate into mitosis. In addition, they were able to localize these proteins to the proximal end of the centriole, a result not previously achieved with TUBD1 and TUBE1, providing a better understanding of where and when the complex is involved in centriole growth.

Strengths:

The results are very convincing, particularly the phenotype, which is the same as previously observed for TUBD1 and TUBE1. The U-ExM localization is also convincing: despite a signal that's not very homogeneous, it's clear that the complex is in the proximal region of the centriole and procentriole. The phenotype observed in U-ExM on the elongation of the cartwheel is also spectacular and opens the question of the regulation of the size of this structure. The authors also report convincing results on direct interactions between TUBD1, TUBE1, TEDC1, and TEDC2, and an intriguing structural prediction suggesting that TEDC1 and TEDC2 form a heterodimer that interacts with the TUBD1- TUBE1 heterodimer.

Weaknesses:

The phenotypes observed in U-ExM on cartwheel elongation merit further quantification, enabling the field to appreciate better what is happening at the level of this structure.

Reviewer #2 (Public Review):

Summary:

Fuqua et al investigated the relationship between prokaryotic box motifs and the activation of promoter activity using a mutagenesis sequencing approach. From generating thousands of mutant daughter sequences from both active and non-active promoter sequences they were able to produce a fantastic dataset to investigate potential mechanisms for promoter activation. From these large numbers of mutated sequences, they were able to generate mutual information with gene expression to identify key mutations relating to the activation of promoter island sequences.

Strengths:

The data generated from this paper is an important resource to address this question of promoter activation. Being able to link the activation of gene expression to mutational changes in previously nonactive promoter regions is exciting and allows the potential to investigate evolutionary processes relating to gene regulation in a statistically robust manner. Alongside this, the method of identifying key mutations using mutual information in this paper is well done and should be standard in future studies for identifying regions of interest.

Weaknesses:

While the generation of the data is superb the focus only on these mutational hotspots removes a lot of the information available to the authors to generate robust conclusions. For instance.

(1) The linear regression in S5 used to demonstrate that the number of mutational hotspots correlates with the likelihood of a mutation causing promoter activation is driven by three extreme points.

(2) Many of the arguments also rely on the number of mutational hotspots being located near box motifs. The context-dependent likelihood of this occurring is not taken into account given that these sequences are inherently box motif rich. So, something like an enrichment test to identify how likely these hot spots are to form in or next to motifs.

(3) The link between changes in expression and mutations in surrounding motifs is assessed with two-sided Mann Whitney U tests. This method assumes that the sequence motifs are independent of one another, but the hotspots of interest occur either in 0, 3, 4, or 5s in sequences. There is therefore no sequence where these hotspots can be independent and the correlation causation argument for motif change on expression is weakened.

(4) The distance between -10 and -35 was mentioned briefly but not taken into account in the analysis.

The authors propose mechanisms of promoter activation based on a few observations that are treated independently but occur concurrently. To address this using complementary approaches such as analysis focusing on identifying important motifs, using something like a glm lasso regression to identify significant motifs, and then combining with mutational hotspot information would be more robust. Other elements known to be involved in promoter activation including TGn or UP elements were not investigated or discussed.

Reviewer #2 (Public Review):

Summary:

The authors assessed the conditional survival of elderly patients with non-metastatic colon cancer who had survived a certain length of time after colectomy. They used data from the Surveillance, Epidemiology, and End Results (SEER) registry to conduct a conditional survival analysis providing estimates of conditional survival rates as well as an analysis of which variables were most important for survival at baseline, one year, three years, and five years.

Strengths:

- The authors used SEER data, providing them with long-term follow-up, and thoroughly considered a wide range of variables related to cancer mortality.<br /> - The authors did a thorough job of assessing the predictive ability of their models.<br /> - The authors used conditional survival, providing estimates of survival that are meaningful for patients/physicians, making them useful for clinical practice.

Weaknesses:

- The paper would have benefited from a more thorough explanation of why the methods were improvements on existing approaches.

- This study was primarily interested in cancer mortality, and compared it to the secondary outcome of death from any cause. The study would have benefited from modeling death from non-cancer causes (the competing risk) in addition to death from colon cancer, rather than comparing only to the composite endpoint of death from any cause.

- When considering a cause-specific hazard, as done with cancer survival in this paper, it would be better to consider the cumulative incidence function rather than Kaplan Meier, since it does not assume the independence of the events like Kaplan Meier does. For this reason, the paper would benefit from focusing on the results of the adjusted cause-specific hazard models (rather than the unadjusted conditional survival estimates done using Kaplan Meier estimates shown in Figure 1 and conducting a parallel analysis for death from other causes.

- The authors mention that they consider disparities using a log-rank test. For the same reason as above, is not the best approach when dealing with competing risks as it depends on Kaplan Meier curves. The log-rank test may be fine if there is no strong dependence between the two causes of death, but the paper would benefit from some discussion of that choice, or sensitivity analysis by comparison to other approaches.

- The variables for the adjusted models were chosen with univariate Cox regression analysis, with any variables having a p-value less than 0.05 being included in the adjusted. Another approach, which may have made the models more easily comparable, would be to choose the variables that are relevant based on prior literature and include them in the multivariate model regardless of significance. The paper would benefit from a discussion of what is gained by excluding some variables from some models.

Reviewer #2 (Public Review):

Summary:

The authors investigated the roles of IncRNA Malat1 in bone homeostasis which was initially believed to be non-functional for physiology. They found that both Malat1 KO and conditional KO in osteoblast lineage exhibit significant osteoporosis due to decreased osteoblast bone formation and increased osteoclast resorption. More interestingly they found that deletion of Malat1 in osteoclast lineage cells does not affect osteoclast differentiation and function. Mechanistically, they found that Malat1 acts as a co-activator of b-Catenin directly regulating osteoblast activity and indirectly regulating osteoclast activity via mediating OPG, but not RANKL expression in osteoblast and chondrocyte. Their discoveries establish a previously unrecognized paradigm model of Malat1 function in the skeletal system, providing novel mechanistic insights into how a lncRNA integrates cellular crosstalk and molecular networks to fine-tune tissue homeostasis, and remodeling.

Strengths:

The authors generated global and conditional KO mice in osteoblast and osteoclast lineage cells and carefully analyzed the role of Matat1 with both in vivo and in vitro systems. The conclusion of this paper is mostly well supported by data.

Weaknesses:

More objective biological and biochemical analyses are required.

Reviewer #2 (Public Review):

In the manuscript entitled "VGLL2 and TEAD1 fusion proteins drive YAP/TAZ-independent transcription and tumorigenesis by engaging p300", Gu et al. studied two Hippo pathway-related gene fusion events (i.e., VGLL2-NCOA2, TEAD1-NCOA2) in spindle cell rhabdomyosarcoma (scRMS) and showed that their fusion proteins can activate Hippo downstream gene transcription independent of YAP/TAZ. Using the BioID-based mass spectrometry analysis, the authors revealed histone acetyltransferase CBP/p300 as specific binding proteins for VGLL2-NCOA2 and TEAD1-NCOA2 fusion proteins. Pharmacologically targeting p300 inhibited the fusion proteins-induced Hippo downstream gene transcription and tumorigenic events.

Overall, this study provides mechanistic insights into the scRMS-associated gene fusions in tumorigenesis and reveals potential therapeutic targets for cancer treatment. The manuscript is well-written and easy to follow.

Here, several suggestions are made for the authors to improve their study.

Main points

(1) The authors majorly focused on the Hippo downstream gene transcription in this study, while a significant portion of genes regulated by the VGLL2-NCOA2 and TEAD1-NCOA2 fusion proteins are non-Hippo downstream genes (Figure 3). The authors should investigate whether the altered Hippo pathway transcription is essential for VGLL2-NCOA2 and TEAD1-NCOA2-induced cell transformation and tumorigenesis. Specifically, they should test if treatment with the TEAD inhibitor can reverse the cell transformation and tumorigenesis caused by VGLL2-NCOA2 but not TEAD1-NCOA2. In addition, it is important to examine whether YAP-5SA expression can rescue the inhibitory effects of A485 on VGLL2-NCOA2 and TEAD1-NCOA2-induced colony formation and tumor growth. This will help clarify whether Hippo downstream gene transcription is important for the oncogenic activities of these two fusion proteins.

(2) Rationale for selecting CBP/p300 for functional studies needs to be provided. The BioID-MS experiment identified many interacting proteins for VGLL2-NCOA2 and TEAD1-NCOA2 fusion proteins (Table S4). The authors should explain the scoring system used to identify the high-interacting proteins for VGLL2-NCOA2 and TEAD1-NCOA2 fusion proteins. Was CEP/p300 the top candidates on the list? Providing this information will help justify the focus on CBP/p300 and validate their importance in this study.

(3) p300 was revealed as a key driver for the VGLL2-NCOA2 and TEAD1-NCOA2 fusion proteins-induced transcriptome alteration and tumorigenesis. To strengthen the point, the authors should identify the p300 binding region on VGLL2-NCOA2 and TEAD1-NCOA2 fusion proteins. Mutants with defects in p300 binding/recruitment should be generated and included as a control in the related q-PCR and tumorigenic studies. This work will help confirm the crucial role of p300 in mediating the oncogenic effects of these two fusion proteins.

(4) Another major issue is the overexpression system extensively used in this study. It is important to determine whether the VGLL2-NCOA2 and TEAD1-NCOA2 fusion genes are also amplified in cancer. If not, the expression levels of the VGLL2-NCOA2 and TEAD1-NCOA2 fusion proteins should be adjusted to endogenous levels to assess their oncogenic effects on gene transcription and tumorigenesis. This approach would make the study more relevant to the pathological conditions observed in scRMS cancer patients.

Reviewer #2 (Public Review):

Summary:

NPRL2 gene therapy induces effective antitumor immunity in KRAS/STK11 mutant anti-PD1 resistant metastatic non-small cell lung cancer (NSCLC) in a humanized mouse model by Meraz et al investigated the antitumor immune responses to NPRL2 gene therapy in aPD1R / KRAS/STK11mt NSCLC in a humanized mouse model, and found that NPRL2 gene therapy induces antitumor activity on KRAS/STK11mt/aPD1R tumors through DC-mediated antigen presentation and cytotoxic immune cell activation.

Strengths:

The novelty of the study.

Weaknesses:

(1) The inconsistent effect of NPRL2 combined with pembrolizumab. Figure 2I-K, showed a similar tumor intensity in the NPRL2 group and combination group. However, NPRL2 combined with pembrolizumab was synergistic in the KRASwt/aPD1S H1299 tumors in Figure 4.

(2) The authors stated that NPRL2 combined with pembrolizumab was not synergistic in the KRAS/STK11mt/aPD1R tumors but was synergistic in the KRASwt/aPD1S H1299 tumors. How did the synergistic effect defined in the study, more details need to be provided here.

(3) Nearly all of the work was performed pre-clinically. Validation in the clinical setting would provide more strong evidence for the conclusion.

(4) Figure 5 and Figure 6 have the same legend. These 2 figures could be merged as a new one.

(5) Figure 5B & C, n=9 in the Figure 5B. However, the detail number in Figure 5C was less than 9.

Reviewer #2 (Public Review):

This manuscript is motivated by the question of what mechanisms cause overyielding in mixed-species communities relative to the corresponding monocultures. This is an important and timely question, given that the ultimate biological reasons for such biodiversity effects are not fully understood.

As a starting point, the authors discuss the so-called "additive partitioning" (AP) method proposed by Loreau & Hector in 2001. The AP is the result of a mathematical rearrangement of the definition of overyielding, written in terms of relative yields (RY) of species in mixtures relative to monocultures. One term, the so-called complementarity effect (CE), is proportional to the average RY deviations from the null expectations that plants of both species "do the same" in monocultures and mixtures. The other term, the selection effect (SE), captures how these RY deviations are related to monoculture productivity. Overall, CE measures whether relative biomass gains differ from zero when averaged across all community members, and SE, whether the "relative advantage" species have in the mixture, is related to their productivity. In extreme cases, when all species benefit, CE becomes positive. When large species have large relative productivity increases, SE becomes positive. This is intuitively compatible with the idea that niche complementarity mitigates competition (CE>0), or that competitively superior species dominate mixtures and thereby driver overyielding (SE>0).

However, it is very important to understand that CE and SE capture the "statistical structure" of RY that underlies overyielding. Specifically, CE and SE are not the ultimate biological mechanisms that drive overyielding, and never were meant to be. CE also does not describe niche complementarity. Interpreting CE and SE as directly quantifying niche complementarity or resource competition, is simply wrong, although it sometimes is done. The criticism of the AP method thus in large part seems unwarranted. The alternative methods the authors discuss (lines 108-123) are based on very similar principles.

The authors now set out to develop a method that aims at linking response patterns to "more true" biological mechanisms.

Assuming that "competitive dominance" is key to understanding mixture productivity, because "competitive interactions are the predominant type of interspecific relationships in plants", the authors introduce "partial density" monocultures, i.e. monocultures that have the same planting density for a species as in a mixture. The idea is that using these partial density monocultures as a reference would allow for isolating the effect of competition by the surrounding "species matrix".

The authors argue that "To separate effects of competitive interactions from those of other species interactions, we would need the hypothesis that constituent species share an identical niche but differ in growth and competitive ability (i.e., absence of positive/negative interactions)." - I think the term interaction is not correctly used here, because clearly competition is an interaction, but the point made here is that this would be a zero-sum game.

The authors use the ratio of productivity of partial density and full-density monocultures, divided by planting density, as a measure of "competitive growth response" (abbreviated as MG). This is the extra growth a plant individual produces when intraspecific competition is reduced.

Here, I see two issues: first, this rests on the assumption that there is only "one mode" of competition if two species use the same resources, which may not be true, because intraspecific and interspecific competition may differ. Of course, one can argue that then somehow "niches" are different, but such a niche definition would be very broad and go beyond the "resource set" perspective the authors adopt. Second, this value will heavily depend on timing and the relationship between maximum initial growth rates and competitive abilities at high stand densities.

The authors then progress to define relative competitive ability (RC), and this time simply uses monoculture biomass as a measure of competitive ability. To express this biomass in a standardized way, they express it as different from the mean of the other species and then divide by the maximum monoculture biomass of all species.

I have two concerns here: first, if competitive ability is the capability of a species to preempt resources from a pool also accessed by another species, as the authors argued before, then this seems wrong because one would expect that a species can simply be more productive because it has a broader niche space that it exploits. This contradicts the very narrow perspective on competitive ability the authors have adopted. This also is difficult to reconcile with the idea that specialist species with a narrow niche would outcompete generalist species with a broad niche. Second, I am concerned by the mathematical form. Standardizing by the maximum makes the scaling dependent on a single value.

As a final step, the authors calculate a "competitive expectation" for a species' biomass in the mixture, by scaling deviations from the expected yield by the product MG ⨯ RC. This would mean a species does better in a mixture when (1) it benefits most from a conspecific density reduction, and (2) has a relatively high biomass.

Put simply, the assumption would be that if a species is productive in monoculture (high RC), it effectively does not "see" the competitors and then grows like it would be the sole species in the community, i.e. like in the partial density monoculture.

Overall, I am not very convinced by the proposed method.

(1) The proposed method seems not very systematic but rather "ad hoc". It also is much less a partitioning method than the AP method because the other term is simply the difference. It would be good if the authors investigated the mathematical form of this remainder and explored its properties.. when does complementarity occur? Would it capture complementarity and facilitation?

(2) The justification for the calculation of MG and RC does not seem to follow the very strict assumptions of what competition (in the absence of complementarity) is. See my specific comments above.

(3) Overall, the manuscript is hard to read. This is in part a problem of terminology and presentation, and it would be good to use more systematic terms for "response patterns" and "biological mechanisms".

Examples:<br /> - on line 30, the authors write that CE is used to measure "positive" interactions and SE to measure "competitive interactions", and later name "positive" and "negative" interactions "mechanisms of species interactions". Here the authors first use "positive interaction" as any type of effect that results in a community-level biomass gain, but then they use "interaction" with reference to specific biological mechanisms (e.g. one species might attract a parasite that infests another species, which in turn may cause further changes that modify the growth of the first and other species).

- on line 70, the authors state that "positive interaction" increases productivity relative to the null expectation, but it is clear that an interaction can have "negative" consequences for one interaction partner and "positive" ones for the other. Therefore, "positive" and "negative" interactions, when defined in this way, cannot be directly linked to "resource partitioning" and "facilitation", and "species interference" as the authors do. Also, these categories of mechanisms are still simple. For example, how do biotic interactions with enemies classify, see above?

- line 145: "Under the null hypothesis, species in the mixture are assumed to be competitively equivalent (i.e., absence of interspecific interactions)". This is wrong. The assumption is that there are interspecific interactions, but that these are the same as the intraspecific ones. Weirdly, what follows is a description of the AP method, which does not belong here. This paragraph would better be moved to the introduction where the AP method is mentioned. Or omitted, since it is basically a repetition of the original Loreau & Hector paper.

Other points:

- line 66: community productivity, not ecosystem productivity.<br /> - line 68: community average responses are with respect to relative yields - this is important!<br /> - line 64: what are "species effects of species interactions" ?<br /> - line 90: here "competitive" and "productive" are mixed up, and it is important to state that "suffers more" refers to relative changes, not yield changes.<br /> - line 92: "positive effect of competitive dominance": I don't understand what is meant here.

Reviewer #2 (Public Review):

Summary:

In this work, the authors propose an extension to some of the last author's previous work, where a compositional restricted Boltzmann machine was considered as a generative model of neuron-assembly interaction. They augment this model by recurrent connections between the Boltzmann machine's hidden units, which allow them to explicitly account for temporal dynamics of the assembly activity. Since their model formulation does not allow the training towards a compositional phase (as in the previous model), they employ a transfer learning approach according to which they initialise their model with a weight matrix that was pre-trained using the earlier model so as to essentially start the actually training in a compositional phase. Finally, they test this model on synthetic and actual data of whole-brain light-sheet-microscopy recordings of spontaneous activity from the brain of larval zebrafish.

Strengths:

This work introduces a new model for neural assembly activity. Importantly, being able to capture temporal assembly dynamics is an interesting feature that goes beyond many existing models. While this work clearly focuses on the method (or the model) itself, it opens up an avenue for experimental research where it will be interesting to see if one can obtain any biologically meaningful insights considering these temporal dynamics when one is able to, for instance, relate them to development or behaviour.

Weaknesses:

For most of the work, the authors present their RTRBM model as an improvement over the earlier cRBM model. Yet, when considering synthetic data, they actually seem to compare with a "standard" RBM model. This seems odd considering the overall narrative, and it is not clear why they chose to do that. Also, in that case, was the RTRBM model initialised with the cRBM weight matrix?

A few claims made throughout the work are slightly too enthusiastic and not really supported by the data shown. For instance, when the authors refer to the clusters shown in Figure 3D as "spatially localized", this seems like a stretch, specifically in view of clusters 1, 3, and 4. Moreover, when they describe the predictive performance of their model as "close to optimal" when the down-sampling factor coincided with the interaction time scale, it seems a bit exaggerated given that it was more or less as close to the upper bound as it was to the lower bound.

When discussing the data statistics, the authors quote correlation values in the main text. However, these do not match the correlation values in the figure to which they seem to belong. Now, it seems that in the main text, they consider the Pearson correlation, whereas in the corresponding figure, it is the Spearman correlation. This is very confusing, and it is not really clear as to why the authors chose to do so.

Finally, when discussing the fact that the RTRBM model outperforms the cRBM model, the authors state it does so for different moments and in different numbers of cases (fish). It would be very interesting to know whether these are the same fish or always different fish.

Reviewer #2 (Public Review):

Summary:

The paper described a behavioural characterisation of mice with presynaptically-inhibited Rac1 in the hippocampus. This is followed by a BioID and phosphoproteomic analysis of Rac1, highlighting potential downstream effectors of active or non-active Rac1 and potential downstream phosphorylated targets.

Strengths:

An original molecular approach that has been established in a previous paper by the authors (PMID 34269176) to block Rac1 function exclusively at the presynapse is now utilised to characterise a link between presynaptic dysfunction and mouse behavior. The experiments and the data well-support the conclusion that the function of Rac1 has distinct outcomes on mouse behavior, depending on its site of action.

Weaknesses:

A main limitation of the study is that it lacks physiological and biochemical analysis to follow up on hits identified in a BioID and phosphoprotemic analysis of presynaptic active and non-active Rac1 variants.

Reviewer #2 (Public Review):

This manuscript addresses an important question that has not yet been solved in the field, what is the contribution of different gamma oscillatory inputs to the development of "theta sequences" in the hippocampal CA1 region? Theta sequences have received much attention due to their proposed roles in encoding short-term behavioral predictions, mediating synaptic plasticity, and guiding flexible decision-making. Gamma oscillations in CA1 offer a readout of different inputs to this region and have been proposed to synchronize neuronal assemblies and modulate spike timing and temporal coding. However, the interactions between these two important phenomena have not been sufficiently investigated. The authors conducted place cell and local field potential (LFP) recordings in the CA1 region of rats running on a circular track. They then analyzed the phase locking of place cell spikes to slow and fast gamma rhythms, the evolution of theta sequences during behavior, and the interaction between these two phenomena. They found that place cells with the strongest modulation by fast gamma oscillations were the most important contributors to the early development of theta sequences and that they also displayed a faster form of phase precession within slow gamma cycles nested with theta. The results reported are interesting and support the main conclusions of the authors. However, the manuscript needs significant improvement in several aspects regarding data analysis, description of both experimental and analytical methods, and alternative interpretations, as I detail below.

• The experimental paradigm and recordings should be explained at the beginning of the Results section. Right now, there is no description whatsoever which makes it harder to understand the design of the study.

• An important issue that needs to be addressed is the very small fraction of CA1 cells phased-locked to slow gamma rhythms (3.7%). This fraction is much lower than in many previous studies, that typically report it in the range of 20-50 %. However, this discrepancy is not discussed by the authors. This needs to be explained and additional analysis considered. One analysis that I would suggest, although there are also other valid approaches, is to, instead of just analyzing the phase locking in two discrete frequency bands, compute the phase locking will all LFP frequencies from 25-100 Hz. This will offer a more comprehensive and unbiased view of the gamma modulation of place cell firing. Alternative metrics to mean vector length that is less sensitive to firing rates, such as pairwise phase consistency index (Vinck et a., Neuroimage, 2010), could be implemented. This may reveal whether the low fraction of phase-locked cells could be due to a low number of spikes entering the analysis.

• From the methods, it is not clear to me whether the reference LFP channel was consistently selected to be a different one that where the spikes analyzed were taken. This is the better practice to reduce the contribution of spike leakage that could substantially inflate the coupling with faster gamma frequencies. These analyses need to be described in more detail.

• The initial framework of the authors of classifying cells into fast gamma and not fast gamma modulated implies a bimodality that may be artificial. The authors should discuss the nuances and limitations of this framework. For example, several previous work has shown that the same place cell can couple to different gamma oscillations (e.g., Lastoczni et al., Neuron, 2016; Fernandez-Ruiz et al., Neuron, 2017; Sharif et al., Neuron,2021).

• It would be useful to provide a more thorough characterization of the physiological properties of FG and NFG cells, as this distinction is the basis of the paper. Only very little characterization of some place cell properties is provided in Figure 5. Important characteristics that should be very feasible to compare include average firing rate, burstiness, estimated location within the layer (i.e., deep vs superficial sublayers) and along the transverse axis (i.e., proximal vs distal), theta oscillation frequency, phase precession metrics (given their fundamental relationship with theta sequences), etc.

• It is not clear to me how the analysis in Figure 6 was performed. In Figure 6B I would think that the grey line should connect with the bottom white dot in the third panel, which would be the interpretation of the results.

Reviewer #2 (Public Review):

Summary

This manuscript makes use of live cell imaging to look at aggregates of the synaptic ribbon protein ribeye to explore synapse formation in an organotypic culture system. The authors find that microtubule disruption influences the motion of a subset of ribeye spots and changes to ribbon volume. Disruption of the microtubule motor is also found to change ribeye motion and ribbon volume, albeit in the opposite direction. Together these results support a role for microtubule-based transport in synapse assembly.

Strengths

(1) The use of the in vitro imaging approach provides a method for high-quality live cell imaging in a mammalian preparation.

(2) The data characterizing the movement of Ribeye in the cochlea is new and exciting.

(3) The role of motors in the delivery of Ribeye to the synapse had never been established. The effects of nocodozole on directional asymmetry for the subset of slow-moving particles are convincing, though it is unclear to this reviewer how frequently these objects undergo directed motion.

(4) The effect of Kif1a on ribbon size is an interesting finding that doesn't rely on overexpression and supports the importance of motors on the delivery of ribeye to the synapse.

Weaknesses

(1) The analysis leaves unclear what fraction of ribeye spots make use of active transport mechanisms. The authors make the claim that 54% underwent targeted transport because fits of their MSD vs time were best-fit by an exponent >1. This overstates the reliability of this approach. Purely diffusive motion will not always fit perfectly with an exponent of exactly 1 and one would expect roughly to have to have greater than 1 and half less than one, which is what they observe. In point of fact, truly directed transport should have an exponent near 2 (Figure 2F), which only a handful of spots seem to exhibit. I should also note that none of the examples look like those that are typically associated with directed motion.

(2) The imaging approach makes use of viral expression using a non-Ribeye promoter. This overexpression approach will likely exaggerate the number of ribeye spots and could saturate binding to other proteins or other factors. Also, the promoters aren't under the control of feedback mechanisms that would typically turn off expression at the appropriate time.

(3) The effect of Kif1A removal on the ABR threshold is very unlikely to be due to ribbon size. Complete removal of the ribbon only has a modest effect on the ABR threshold, so these modest reductions in size are unlikely to contribute much.

(4) Fusion and fission of small aggregates are difficult to resolve with light microscopy and the examples provided in Figure 3 are indistinguishable from two spots that happen to be too close to each other to resolve.

5) The "slight left shift" in the velocity distribution in Figure 5C does not look significant. Is it?

6) Nocodozole and elimination of Kif1a have opposite effects on ribbon volume, which might point to alternative roles for the microtubules.

Reviewer #2 (Public Review):

Summary:

Haupt and colleagues performed a well-designed study to test the spatial and temporal gradient of perceiving braille letters in blind individuals. Using cross-hand decoding of the read letters, and comparing it to the decoding of the read letter for each hand, they defined perceptual and sensory responses. Then they compared where (using fMRI) and when (using EEG) these were decodable. Using fMRI, they showed that low-level tactile responses specific to each hand are decodable from the primary and secondary somatosensory cortex as well as from IPS subregions, the insula, and LOC. In contrast, more abstract representations of the braille letter independent from the reading hand were decodable from several visual ROIs, LOC, VWFA, and surprisingly also EVC. Using a parallel EEG design, they showed that sensory hand-specific responses emerge in time before perceptual braille letter representations. Last, they used RSA to show that the behavioral similarity of the letter pairs correlates to the neural signal of both fMRI (for the perceptual decoding, in visual and ventral ROIs) and EEG (for both sensory and perceptual decoding).

Strengths:

This is a very well-designed study and it is analyzed well. The writing clearly describes the analyses and results. Overall, the study provides convincing evidence from EEG and fMRI that the decoding of letter identity across the reading hand occurs in the visual cortex in blindness. Further, it addresses important questions about the visual cortex hierarchy in blindness (whether it parallels that of the sighted brain or is inverted) and its link to braille reading.

Weaknesses:

Although I have some comments and requests for clarification about the details of the methods, my main comment is that the manuscript could benefit from expanding its discussion. Specifically, I'd appreciate the authors drawing clearer theoretical conclusions about what this data suggests about the direction of information flow in the reorganized visual system in blindness, the role VWFA plays in blindness (revised from the original sighted role or similar to it?), how information arrives to the visual cortex, and what the authors' predictions would be if a parallel experiment would be carried out in sighted people (is this a multisensory recruitment or reorganization?). The data has the potential to speak to a lot of questions about the scope of brain plasticity, and that would interest broad audiences.

To aid in drawing even more concrete conclusions about the flow of information, I suggest that the authors also add at least another early visual ROI to plot more clearly whether EVC's response to braille letters arrives there through an inverted cortical hierarchy, intermediate stages from VWFA, or directly, as found in the sighted brain for spoken language.

Similarly, it may be informative to look specifically at the occipital electrodes' time differences between decoding for the different parameters and their correlation to behavior.

Regarding the methods, further detail on the ability to read with both hands equally and any residual vision of the participants would be helpful.

Reviewer #2 (Public Review):

Summary:

The authors investigate the mechanisms supporting learning to suppress distractors at predictable locations, focusing on proactive suppression mechanisms manifesting before the onset of a distractor. They used EEG and inverted encoding models (IEM). The experimental paradigm alternates between a visual search task and a spatial memory task, followed by a placeholder screen acting as a 'ping' stimulus -i.e., a stimulus to reveal how learned distractor suppression affects hidden priority maps. Behaviorally, their results align with the effects of statistical learning on distractor suppression. Contrary to the proactive suppression hypothesis, which predicts reduced memory-specific tuning of neural representations at the expected distractor location, their IEM results indicate increased tuning at the high-probability distractor location following the placeholder and prior to the onset of the search display.

Strengths:

Overall, the manuscript is well-written and clear, and the research question is relevant and timely, given the ongoing debate on the roles of proactive and reactive components in distractor processing. The use of a secondary task and EEG/IEM to provide a direct assessment of hidden priority maps in anticipation of a distractor is, in principle, a clever approach. The study also provides behavioral results supporting prior literature on distractor suppression at high-probability locations.

Weaknesses:

(1) At a conceptual level, I understand the debate and opposing views, but I wonder whether it might be more comprehensive to present also the possibility that both proactive and reactive stages contribute to distractor suppression. For instance, anticipatory mechanisms (proactive) may involve expectations and signals that anticipate the expected distractor features, whereas reactive mechanisms contribute to the suppression and disengagement of attention.

(2) The authors focus on hidden priority maps in pre-distractor time windows, arguing that the results challenge a simple proactive view of distractor suppression. However, they do not provide evidence that reactive mechanisms are at play or related to the pinging effects found in the present paradigm. Is there a relationship between the tuning strength of CTF at the high-probability distractor location and the actual ability to suppress the distractor (e.g., behavioral performance)? Is there a relationship between CTF tuning and post-distractor ERP measures of distractor processing? While these may not be the original research questions, they emerge naturally and I believe should be discussed or noted as limitations.

(3) How do the authors ensure that the increased tuning (which appears more as a half-split or hemifield effect rather than gradual fine-grained tuning, as shown in Figure 5) is not a byproduct of the dual-task paradigm used, rather than a general characteristic of learned attentional suppression? For example, the additional memory task and the repeated experience with the high-probability distractor at the specific location might have led to longer-lasting and more finely-tuned traces for memory items at that location compared to others.

(4) It is unclear how IEM was performed on total vs. evoked power, compared to typical approaches of running it on single trials or pseudo-trials.

(5) Following on point 1. What is the rationale for relating decreased (but not increased) tuning of CTF to proactive suppression? Could it be that proactive suppression requires anticipatory tuning towards the expected feature to implement suppression? In other terms, better 'tuning' does not necessarily imply a higher signal amplitude and could be observable even under signal suppression. The authors should comment on this and clarify.

Minor:

(1) In the Word file I reviewed, there are minor formatting issues, such as missing spaces, which should be double-checked.

(2) Would the authors predict that proactive mechanisms are not involved in other forms of attention learning involving distractor suppression, such as habituation?

(3) A clear description in the Methods section of how individual CTFs for each location were derived would help in understanding the procedure.

(4) Why specifically 1024 resampling iterations?

Reviewer #2 (Public Review):

Summary:

In this work, Witten et al. assess visual acuity, cone density, and fixational behavior in the central foveal region in a large number of subjects.

This work elegantly presents a number of important findings, and I can see this becoming a landmark work in the field. First, it shows that acuity is determined by the cone mosaic, hence, subjects characterized by higher cone densities show higher acuity in diffraction-limited settings. Second, it shows that humans can achieve higher visual resolution than what is dictated by cone sampling, suggesting that this is likely the result of fixational drift, which constantly moves the stimuli over the cone mosaic. Third, the study reports a correlation between the amplitude of fixational motion and acuity, namely, subjects with smaller drifts have higher acuities and higher cone density. Fourth, it is shown that humans tend to move the fixated object toward the region of higher cone density in the retina, lending further support to the idea that drift is not a random process, but is likely controlled. This is a beautiful and unique work that furthers our understanding of the visuomotor system and the interplay of anatomy, oculomotor behavior, and visual acuity.

Strengths:

The work is rigorously conducted, it uses state-of-the-art technology to record fixational eye movements while imaging the central fovea at high resolution and examines exactly where the viewed stimulus falls on individuals' foveal cone mosaic with respect to different anatomical landmarks in this region. The figures are clear and nicely packaged. It is important to emphasize that this study is a real tour-de-force in which the authors collected a massive amount of data on 20 subjects. This is particularly remarkable considering how challenging it is to run psychophysics experiments using this sophisticated technology. Most of the studies using psychophysics with AO are, indeed, limited to a few subjects. Therefore, this work shows a unique set of data, filling a gap in the literature.

Weaknesses:

No major weakness was noted, but data analysis could be further improved by examining drift instantaneous direction rather than start-point-end-point direction, and by adding a statistical quantification of the difference in direction tuning between the three anatomical landmarks considered.

Reviewer #2 (Public Review):

Summary:

This paper presents miniML as a supervised method for the detection of spontaneous synaptic events. Recordings of such events are typically of low SNR, where state-of-the-art methods are prone to high false positive rates. Unlike current methods, training miniML requires neither prior knowledge of the kinetics of events nor the tuning of parameters/thresholds.

The proposed method comprises four convolutional networks, followed by a bi-directional LSTM and a final fully connected layer which outputs a decision event/no event per time window. A sliding window is used when applying miniML to a temporal signal, followed by an additional estimation of events' time stamps. miniML outperforms current methods for simulated events superimposed on real data (with no events) and presents compelling results for real data across experimental paradigms and species.

Strengths:

The authors present a pipeline for benchmarking based on simulated events superimposed on real data (with no events). Compared to five other state-of-the-art methods, miniML leads to the highest detection rates and is most robust to specific choices of threshold values for fast or slow kinetics. A major strength of miniML is the ability to use it for different datasets. For this purpose, the CNN part of the model is held fixed and the subsequent networks are trained to adapt to the new data. This Transfer Learning (TL) strategy reduces computation time significantly and more importantly, it allows for using a substantially smaller data set (compared to training a full model) which is crucial as training is supervised (i.e. uses labeled examples).

Weaknesses:

The authors do not indicate how the specific configuration of miniML was set, i.e. number of CNNs, units, LSTM, etc. Please provide further information regarding these design choices, whether they were based on similar models or if chosen based on performance.

The data for the benchmark system was augmented with equal amounts of segments with/without events. Data augmentation was undoubtedly crucial for successful training.

(1) Does a balanced dataset reflect the natural occurrence of events in real data? Could the authors provide more information regarding this matter?

(2) Please provide a more detailed description of this process as it would serve users aiming to use this method for other sub-fields.

The benchmarking pipeline is indeed valuable and the results are compelling. However, the authors do not provide comparative results for miniML for real data (Figures 4-8). TL does not apply to the other methods. In my opinion, presenting the performance of other methods, trained using the smaller dataset would be convincing of the modularity and applicability of the proposed approach.

Impact:

Accurate detection of synaptic events is crucial for the study of neural function. miniML has a great potential to become a valuable tool for this purpose as it yields highly accurate detection rates, it is robust, and is relatively easily adaptable to different experimental setups.

Additional comments:

Line 73: the authors describe miniML as "parameter-free". Indeed, miniML does not require the selection of pulse shape, rise/fall time, or tuning of a threshold value. Still, I would not call it "parameter-free" as there are many parameters to tune, starting with the number of CNNs, and number of units through the parameters of the NNs. A more accurate description would be that as an AI-based method, the parameters of miniML are learned via training rather than tuned by the user.

Line 302: the authors describe miniML as "threshold-independent". The output trace of the model has an extremely high SNR so a threshold of 0.5 typically works. Since a threshold is needed to determine the time stamps of events, I think a better description would be "robust to threshold choice".

Reviewer #2 (Public Review):

Summary:

In the manuscript by Chiu et al., "Structure and dynamics of cholesterol-mediated aquaporin-0 arrays and implications for lipid rafts," the authors address the effect of cholesterol on array formation by AQP0. Using a combination of electron crystallography and molecular dynamics simulations, the authors show binding of a "deep" cholesterol molecule between AQP0 tetramers. Each AQP0 tetramer binds four deep cholesterols to form a crystallographic array of AQP0.

Strengths:

The combined approaches of electron crystallography and MD simulations under different lipid conditions (different sphingomyelin and cholesterol concentrations) are a strength of the study. The authors provide a thorough and convincing assessment of cholesterol binding, protein-protein interactions, and array formation by AQP0. The MD simulations allow the authors to consider the propensity of cholesterol to occupy the observed binding sites in the absence of crystal contacts. The combined methods and the breadth of analyses set a high standard in the field of membrane protein structural biology.

The findings of the authors fit nicely into a growing body of literature on cholesterol binding sites that mediate membrane protein-protein interactions. Cholesterol interacts with a variety of membrane proteins via its smooth alpha face of rough beta face. AQP0 is somewhat unique in that it binds the rough face of cholesterol in a "deep" binding site that places cholesterol in the middle of the membrane bilayer. So-called "deep" cholesterol binding sites have been described for GPCRs and docking studies suggest they may exist on other ion channels and transporters. In the case of AQP0, the deep cholesterol acts as a glue that holds two tetramers together. Since each tetramer has four binding sites for deep cholesterol, the assembly and mechanical stability of an extended two-dimensional array of AQP0 tetramers is a natural consequence in lens membranes.

Weaknesses:

The authors report that the findings generally apply to raft formation in membranes. However, this point is less clear as the lens membrane in which AQP0 resides is rather unique in lipid and protein content and density. Nonetheless, the authors achieve the overall goal of evaluating cholesterol binding to AQP0, and there are many valuable and informative figures in the main manuscript and supplement that provide convincing results and interpretations.

Reviewer #2 (Public Review):

Summary:

The study focuses on the vomeronasal organ, the peripheral chemosensory organ of the accessory olfactory system, by employing single-cell transcriptomics. The author analyzed the mouse vomeronasal organ, identifying diverse cell types through their unique gene expression patterns. Developmental gene expression analysis revealed that two classes of sensory neurons diverge in their maturation from common progenitors, marked by specific transient and persistent transcription factors. A comparative study between major neuronal subtypes, which differ in their G-protein sensory receptor families and G-protein subunits (Gnai2 and Gnao1, respectively), highlighted a higher expression of endoplasmic reticulum (ER) associated genes in Gnao1 neurons. Moreover, distinct differences in ER content and ultrastructure suggest some intriguing roles of ER in Gnao1-positive vomeronasal neurons. This work is likely to provide useful data for the community and is conceptually novel with the unique role of ER in a subset of vomeronasal neurons. This reviewer has some minor concerns and some suggestions to improve the manuscript.

Strengths:

(1) The study identified diverse cell types based on unique gene expression patterns, using single-cell transcriptomic.

(2) The analysis suggests that two classes of sensory neurons diverge during maturation from common progenitors, characterized by specific transient and persistent transcription factors.

(3) A comparative study highlighted differences in Gnai2- and Gnao1-positive sensory neurons.

(4) Higher expression of endoplasmic reticulum (ER) associated genes in Gnao1 neurons.

(5) Distinct differences in ER content and ultrastructure suggest unique roles of ER in Gnao1-positive vomeronasal neurons.

(6) The research provides conceptually novel on the unique role of ER in a subset of vomeronasal neurons, offering valuable insights to the community.

Weaknesses:

(1) The connection between observations from sc RNA-seq and EM is unclear.

(2) The lack of quantification for the ER phenotype is a concern.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Shelton et al. explore the organization of the Claustrum. To do so, they focus on a specific claustrum population, the one projecting to the retrosplenial cortex (CLA-RSP neurons). Using an elegant technical approach, they first described electrophysiological properties of claustrum neurons, including the CLA-RSP ones. Further, they showed that CLA-RSP neurons (1) directly excite other CLA neurons, in a 'projection-specific' pattern, i.e. CLA-RSP neurons mainly excite claustrum neurons not projecting to the RSP and (2) received excitatory inputs from multiple cortical territories (mainly frontal ones). To confirm the 'integrative' property of claustrum networks, they then imaged claustrum axons in the cortex during single- or multi-sensory stimulations. Finally, they investigated the effect of CLA-RSP lesion on performance in a sensory detection task.

Strengths:<br /> Overall, this is a really good study, using state-of-the-art technical approaches to probe the local/global organization of the Claustrum. The in-vitro part is impressive, and the results are compelling.

Weaknesses:<br /> One noteworthy concern arises from the terminology used throughout the study. The authors claimed that the claustrum is an integrative structure. Yet, integration has a specific meaning, i.e. the production of a specific response by a single neuron (or network) in response to a specific combination of several input signals. In this study, the authors showed compelling results in favor of convergence rather than integration. On a lighter note, the in-vivo data are less convincing, and do not entirely support the claim of "integration" made by the authors.

Reviewer #2 (Public Review):

Summary:

The authors have conducted a valuable comparative analysis of perturbation responses in three nonlinear kinetic models of E. coli central carbon metabolism found in the literature. They aimed to uncover commonalities and emergent properties in the perturbation responses of bacterial metabolism. They discovered that perturbations in the initial concentrations of specific metabolites, such as adenylate cofactors and pyruvate, significantly affect the maximal deviation of the responses from steady-state values. Furthermore, they explored whether the network connectivity (sparse versus dense connections) influences these perturbation responses. The manuscript is reasonably well written.

Strengths:

Well-defined and valuable research questions.

Weaknesses:

(1) In the study on determining key metabolites affecting responses to perturbations (starting from line 171), the authors fix the values of individual concentrations to their steady-state values and observe the responses. Such a procedure adds artificial constraints to the network because, in the natural responses of cells (and models) to perturbations, it is highly unlikely that metabolites will not evolve in time. By fixing the values of specific metabolites, the authors prohibit the metabolic network from evolving in the most optimal way to compensate for the perturbation. Instead of this procedure, have the authors considered for this task applying techniques from variance-based sensitivity analysis (Sobol, global sensitivity analysis), where they can calculate the first-order sensitivity index and total effect index? Using this technique, the authors would be able to determine the key metabolites while allowing for metabolic responses to perturbations without unnatural constraints.

(2) To follow up on the previous remark, the authors state that the metabolites that augment the response coefficient when their concentration is fixed tend to be allosteric regulators. The authors should report which allosteric regulations are implemented in each of the models so that one can compare against Figure 2. Again, the effect of allosteric regulation by a specific metabolite that is quantified the way the authors did is biased by fixing the concentration value - it is true that negative feedback is broken when the metabolite concentration is fixed, however, in the rate law, there is still the fixed inhibition term with its value corresponding to the inhibition at the steady state. To see the effect of allosteric regulation by a metabolite, one can change the inhibition constants instead of constraining the responses with fixed concentrations.

(3) Given the role of ATP in metabolic processes, the authors' finding of the sensitivity of the three networks' responses to perturbations in the AXP concentrations seems reasonable. However, drawing such firm conclusions from only three models, with each of them built around one steady state and having one kinetic parameter set despite that they were built for different physiologies, raises some questions. It is well-known in studies related to basins of attraction of the steady states that the nonlinear responses also depend on the actual steady states, the values of kinetic parameters, and implemented kinetic rate law, i.e., not only on the topology of the underlying systems. In the population of only three models, we cannot exclude the possibility of overlaps and strong similarities in the values of kinetic parameters, steady states, and enzyme saturations that all affect and might bias the observed responses. Ideally, to eliminate the possibility of such biases, one should simulate responses of a large population of models for multiple physiologies (and the corresponding steady states) and multiple parameter sets per physiology. This can be a difficult task, but having more kinetic models in this work would go a long way toward more convincing results. Recently, E. coli nonlinear kinetic models from several groups appeared that might help in this task, e.g., Haiman et al., PLoS Comput Biol, 17(1): e1008208, (2021), Choudhury et al., Nat Mach Intell, 4, 710-719, (2022); Hu et al., Metab Eng, 82, 123-133 (2024), Narayanan et al., Nat Commun, 15:723, (2024).

(4) Can the authors share their insights on what could be the underlying reasons for the bimodal distribution in Figure 1E? Even after adding random reactions, the distribution still has two modes - why is that?

(5) Considering the effects of the sparsity of the networks on the perturbation responses (from line 223 onwards), when we compare the three analyzed models, it is clear that the Khodayari et al. model is a superset of the other two models. Therefore, this model can be considered as, e.g., Chassagnole model with Nadd reactions (though not randomly added). Based on Figures 1b and S2, one can observe that the responses of the Khodayari models have stronger responses, which is exactly opposite to the authors' conclusion that adding the reactions weakens the responses. The authors should comment on this.

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).